Folate receptor 1 antibodies and immunoconjugates and uses thereof

ANTIBODIES AND IMMUNOCONJUGADOS OF THE RECEIVER 1 OF

FOLATE AND ITS USES

CROSS REFERENCE WITH RELATED APPLICATIONS

This request claims the benefit of the provisionai request priority of

United States No. 61 / 307,797, filed on February 12, 2010, United States Provisional Application No. 61 / 346,595, filed on May 20, 2010, and United States Provisional Application No. 61 / 413,172, filed on November 12, 2010, each of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The field of this invention relates, in general, to antibodies and immunoconjugates that bind to human folate receptor 1, as well as to the methods of use of antibodies and immunoconjugates for the treatment of diseases such as cancer.

BACKGROUND OF THE INVENTION

Cancer is one of the leading causes of death in the developed world, with more than one million people diagnosed with cancer and 500,000 deaths per year in the United States alone. In general, it is estimated that more than 1 in 3 people will develop some type of cancer during their lifetime. There are more than 200 different types of cancer, four of which - marna, lung, colorectal and prostate - account for more than half of all new cases (Jemal et al., 2003, Cancer J. Clin. 53: 5-26).

Folate receptor 1 (FOLR1), also known as folate receptor-alpha, or folate binding protein, is an N-glycosylated protein that is expressed in the piasmatic cell membrane. FOLR1 has a high affinity for folic acid and for various reduced derivatives of folic acid. The FOLR1 mediates the delivery of the physiological folate, the

5-methyltetrahydrofolate, inside the cells.

FOLR1 is overexpressed in the vast majority of ovarian cancers, as well as in many uterine, endometrial, pancreas, renai, lung and marian cancers, while FOLR1 expression on normal tissues is limited to the apical membrane of the

<img file="AR080301A1_D0001.tif" />

epithelial cells in the proximal tubules of the kidney, alveolar lung pneumocytes, bladder, testicles, choroid and thyroid plexus (Weitman SD, et al., Cancer Res 52: 3396-3401 (1992); Anthony AC, Annu Rev Nutr 16: 501-521 (1996); Kalli KR, et al. Gynecol Oncol 108: 619-626 (2008)). This pattern of expression makes

FOLR1 a convenient goal for cancer therapy targeting FOLR1.

Because ovarian cancer is typically asymptomatic until the advanced stage, it is frequently diagnosed at a late stage and has a poor prognosis when dealing with currently available procedures, typically with chemotherapeutic drugs that are applied after cytoreductive surgery (von

Gruenigen V et al., Cancer 112: 2221-2227 (2008); Ayhan A and others, Am J Obstet Gynecol 196: 81 e81-86 (2007); Harry VN and others, Obstet Gynecol Surv 64: 548-560 (2009)). In this way, there is a clear unsatisfied medical need for more effective therapies for ovarian cancers.

Three anti-FOLRl antibodies have been tested as potent anti-cancer drugs.

The murine monoclonal antibodies Movl8 and Movl9 were isolated in the late 1980s (Miotti S et al., Int J Cancer 39: 297-303 (1987)); were confirmed for objective FOLR1 (Coney LR et al., Cancer Res 51: 6125-6132 (1991)); and were tested in pre-clinical studies for their ability to eradicate cancer cells that express the antigen when conjugated with the cytotoxic protein that inactivates the ribosome (Count FP et al., Eur JBiochem 178: 795-802 (1989)) .

Movl9 was tested as a bi-specific antibody directed to cytotoxic T cells and natural cytolytic cells (Mezzanzanica D et al., Int J Cancer 41: 609615 (1988); Ferrini S et al., Int J Cancer Suppl 4: 53-55 (1989); Ferrini S et al., Int J Cancer 48: 227-233 (1991)); and as a single chain Fv fusion protein (scFv) from Movl9 with interleukin-2 in vivo (Melani C et al., Cancer Res 58: 4146-4154 (1998)). Chimeric anti-FOLRl antibodies (murine variable chain / human constant chain) from Movl8 and Movl9 have been pre-clinically examined for their ability to mediate cytotoxic immune cell-dependent death in tumor cells expressing FOLR1 in vitro (Coney LR and others, Cancer Res 54: 2448-2455 (1994)); and a chimeric IgE-Movl8 was tested in IgE-dependent preclinical immunotherapeutic models (Karagiannis SN et al., J Immunol 179: 2832-2843 (2007); Gould HJ et al., Eur J Immunol 29: 3527-3537 (1999)).

<img file="AR080301A1_D0002.tif" />

Movi 8 was studied in the form of conjugates with different radionuclides in preclinical studies and then, in the early 1990s, in clinical trials (Zacchetti A et al., Nucl Med Biol 36: 759-770 (2009)); which ended without any drug being approved for clinical use.

MORAb003, a humanized form of the murine monoclonal antibody antiFOLR1 LK26 was pre-clinically evaluated as an unmodified antibody (Ebel W et al., Cancer Immun 7: 6 (2007)) and as a conjugate with the radionuclide In¹¹¹ (SmithJones PM and others, Nucl Med Biol 35: 343-351 (2008)); and an unmodified antibody is currently tested in clinical trials (DK Armstrong et al., J. Clin.

Oncol. 26 :, May 10, 2008, suppl; summary 5500).

SUMMARY OF THE INVENTION

The present invention provides new antibodies that bind to human folate receptor 1, immunoconjugates comprising these antibodies, and methods for their use. The present invention also provides new polypeptides, such as antibodies that bind to human folate receptor 1, fragments of such antibodies, and other polypeptides related to such antibodies. The polynucleotides comprising the nucleic acid sequences encoding the polypeptides are also provided, as are the vectors comprising the polynucleotides. Cells comprising the polypeptides and / or polynucleotides of the invention are also provided. The compositions (for example, the pharmaceutical compositions); which comprise the new antibodies or immunoconjugates for folate receptor 1 are also provided. In addition, the methods of preparing and using the new antibodies or immunoconjugates for folate receptor 1 are also provided, as well as methods of using new antibodies or immunoconjugates for folate receptor 1 to inhibit tumor growth and / or to confuse the Cancer

Thus, in one aspect, the invention provides a humanized antibody or an antigen-binding fragment thereof that specifically binds to a human folate receptor 1, wherein the antibody comprises (a) a heavy chain CDR1 that it comprises GYFMN (sec. with identification number: 1); a heavy chain CDR2 comprising RIHPYDGDTFYNQXaa] FXaa2Xaa3 (sec. with ID no .: 56); and a heavy chain CDR3 comprising YDGSRAMDY (sec. with ID No.:3) and (b) a

<img file="AR080301A1_D0003.tif" />

Light chain CDR1 comprising KASQSVSFAGTSLMH (sec. With ID no .: 7); a light chain CDR2 comprising RASNLEA (sec. with ID no .: 8); and a light chain CDR3 comprising QQSREYPYT (sec. with ID no .: 9); where Xaai is selected from K, Q, H and R; Xaa₂ is selected from Q, H, N, and R, and Xaa₃ is selected from G, E, T, S, A and V. In a certain embodiment, the humanized antibody or the antigen binding fragment thereof binds to a human folate receptor 1 with the same substantial affinity as the chimeric antibody Movl9. In a certain embodiment, the humanized antibody or the antigen binding fragment thereof comprises the CDR2 of the sequence RIHPYDGDTFYNQKFQG (sec. With identification number: 2) of a heavy chain.

In a certain mode, binding affinity is measured by flow cytometry, Biacore, or radioimmunoassay.

In another embodiment, the invention provides a humanized antibody or an antigen binding fragment thereof that specifically binds to a human folate receptor 1, wherein the antibody comprises: (a) a heavy chain CDR1 comprising GYFMN ( sec. with identification number: 1); or a variant thereof comprising 1, 2, 3, or 4 amino acid conserved substitutions; a heavy chain CDR2 comprising RIHPYDGDTFYNQKFQG (sec. with ID no. :two); or a variant thereof comprising 1, 2, 3, or 4 amino acid conserved substitutions; and a heavy chain CDR3 comprising YDGSRAMDY (sec. with identification number: 3); or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and / or (b) a light chain CDR1 comprising KASQSVSFAGTSLMH (sec. with no. from ident.:7); or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; a light chain CDR2 comprising RASNLEA (sec. with identification number: 8); or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and a light chain CDR3 comprising QQSREYPYT (sec. with ID no .: 9); or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions.

In a certain embodiment, the invention provides a humanized antibody or an antigen binding fragment thereof that specifically binds to human folate receptor 1 comprising a sec heavy chain. with no. of ident .: 6. In another embodiment, the humanized antibody or the antigen binding fragment thereof is

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encodes by plasmid DNA deposited with the ATCC on April 7, 2010 and which has the deposit numbers and the ATCC PTA-10772 and PTA-10773 or 10774.

In a certain embodiment, the invention provides a humanized antibody or an antigen binding fragment thereof that competes for binding to FOLR1 with an antibody comprising (a) a heavy chain CDR1 comprising GYFMN (sec. With no. ident .: 1); a heavy chain CDR2 comprising RIHPYDGDTFYNQXaaiFXaa2Xaa3 (sec. with ID no .: 56); and a heavy chain CD3 comprising YDGSRAMDY (sec. with no. of ident.:3); and (b) a light chain CDR1 comprises KASQSVSFAGTSLMH (sec. with ID no .: 7); a light chain CDR2 comprising RASNLEA (sec. with ID no .: 8); and a light chain CDR3 comprising QQSREYPYT (sec. with identification number: 9); where Xaai is selected from K, Q, H and R; Xaa2 is selected from Q, H, N, and R, and Xaa3 is selected from G, E, T, S, A and V. In a certain embodiment, the humanized antibody comprises the CDR2 of sequence RIHPYDGDTFYNQKFQG (sec. With no. of ident. : 2) of a heavy chain.

In a certain embodiment, the invention provides a polypeptide, a humanized antibody or an antigen binding fragment thereof comprising a heavy chain variable domain at least about 90% identical to sec. with no. Ident .: 4, and a variable light chain domain at least approximately 90% identical to sec. with no. Ident .: 10 or sec. with no. from ident.:ll. In another embodiment, the humanized antibody or antigen binding fragment comprises a heavy chain variable domain at least about 95% identical to sec. with no. of ident. : 4, and a variable domain of a light chain at least about 95% identical to sec. with no. Ident .: 10 or sec. with no. from ident.:ll. In a further embodiment, the humanized antibody comprises a heavy chain variable domain at least about 99% identical to sec. with no. of ident. : 4, and a light chain variable domain at least about 99% identical to sec. with no. Ident .: 10 or sec. with no. from ident.:ll. In a certain embodiment, the humanized antibody comprises the heavy chain variable domain of the sec. with no. of ident .: 4, and the light chain variable domain of sec. with no. Ident .: 10 or sec. with no. Ident .: 11. In certain embodiments, the invention provides a polypeptide, an antibody, or an antigen binding fragment at least about 90% identical to sec. with no. of ident.:88-l 19. In certain embodiments, the invention provides a li

<img file="AR080301A1_D0006.tif" />

polypeptide, an antibody, or an antigen binding fragment at least about 95% identical to sec. with no. ID: 88-119. In certain embodiments, the invention provides a polypeptide, an antibody, or an antigen binding fragment at least about 99% identical to sec. with mim. of ident.:88-l 19.

In a certain embodiment, the invention provides a humanized antibody or an antigen binding fragment thereof that is expressed at least ten times more than Movl9ch in eukaryotic cells. In a certain embodiment, eukaryotic cells are HEK-293T cells.

In certain embodiments, the invention provides an antibody or antigen binding fragment thereof that specifically binds to a human folate receptor 1, wherein the antibody comprises:

(a) a heavy chain CDR1 comprising SSYGMS (sec. with ID 30); a heavy chain CDR2 comprising TISSGGSYTY (sec. with ID number: 31); and / or a heavy chain CDR3 comprising DGEGGLYAMDY (sec. with ID number: 32); and / or (b) a light chain CDR1 comprising KASDHINNWLA (sec. with ID number: 27); a light chain CDR2 comprising GATSLET (sec. with ID number: 28); and a light chain CDR3 comprising QQYWSTPFT (sec. with ID number: 29). In another embodiment, the invention provides an antibody or antigen binding fragment thereof that specifically binds to a human folate receptor 1, wherein the antibody comprises: (a) a heavy chain CDR1 comprising TNYWMQ (sec with ID number: 60); a heavy chain CDR2 comprising AIYPGNGDSR (sec. with ID No. 61); and / or a heavy chain CDR3 comprising RDGNYAAY (sec. with mim. from ident.:62); and / or (b) a light chain CDR1 comprising RASENIYSNLA (sec. with ID number: 57); a light chain CDR2 comprising AATNLAD (sec. with ID number: 58); and a light chain CDR3 comprising QHFWASPYT (sec. with ID number: 59). In another embodiment, the invention provides an antibody or antigen binding fragment thereof that specifically binds to a human folate receptor 1, wherein the antibody comprises: (a) a heavy chain CDR1 comprising TNYWMY (sec with ID number: 66); a heavy chain CDR2 comprising AIYPGNSDTT (sec. with ID number: 67); and / or a heavy chain CDR3 comprising RHDYGAMDY (sec. with ID number: 68);

<img file="AR080301A1_D0007.tif" />

and / or (b) a light chain CDR1 comprising RASENIYTNLA (sec. with ID no .: 63); a light chain CDR2 comprising TASNLAD (sec. with ID no .: 64); and a light chain CDR3 comprising QHFWVSPYT (sec. with ID no .: 65). In another embodiment, the invention provides an antibody or antigen binding fragment thereof that specifically binds to a human folate receptor 1, wherein the antibody comprises: (a) a heavy chain CDR1 comprising SSFGMH (sec with identification number: 72); a heavy chain CDR2 comprising YISSGSSTIS (sec. with ID no .: 73); and / or a heavy chain CDR3 comprising EAYGSSMEY (sec. with no. from ident.:74); and / or (b) a light chain CDR1 comprising RASQNINNNLH (sec. with ID no .: 69); a light chain CDR2 comprising YVSQSVS (sec. with identification number: 70); and a light chain CDR3 comprising QQSNSWPHYT (sec. with ID No.:71). In another embodiment, the invention provides an antibody or antigen binding fragment thereof that specifically binds to a human folate receptor 1, wherein the antibody comprises: (a) a heavy chain CDR1 comprising TSYTMH (sec with identification number: 78); a heavy chain CDR2 comprising YINPISGYTN (sec. with identification no .: 79); and / or a heavy chain CDR3 comprising GGAYGRKPMDY (sec. with no. from ident.:80); and / or (b) a light chain CDR1 comprising KASQNVGPNVA (sec. with ID no .: 75); a light chain CDR2 comprising SASYRYS (sec. with ID No. 76); and a light chain CDR3 comprising QQYNSYPYT (sec. with ID no .: 77).

In certain embodiments, the polypeptides of the invention are full length antibodies or antigen binding fragments. In certain embodiments, the antibodies or antigen binding fragments are a Fab, a Fab ', a F (ab') 2, an Fd, a Fv or Fvsc single chain, a Fv linked to disulfide, a V-NAR domain , an IgNar, an intrabody, an IgG-CH2, a mini-body, an F (ab ') 3, a tetrabody, a triabody, a diabody, a single domain antibody, Ig-DVD, Fcab, mAb2, a (scFv) 2, or a scFvFc.

In certain embodiments, an antibody or polypeptide of the invention binds to a human folate receptor 1 with a Kd of about 1.0 to about 10 nM. In one embodiment, the antibody or polypeptide binds to a folate receptor 1

<img file="AR080301A1_D0008.tif" />

human with a Kd of approximately 1.0 nM or better. In a certain mode, binding affinity is measured by flow cytometry, Biacore or radioimmunoassay.

The invention also provides a method of preparing an antibody of the invention comprising culturing a cell expressing said antibody, and (b) isolating the antibody from said cell in culture. In a certain mode, the cell is a eukaryotic cell.

The invention also provides an immunoconjugate having the Formula (A) (L) - (C); wherein: (A) is an antibody fragment or an antigen binding fragment or a polypeptide of the invention; (L) is a ligand; and (C) is a cytotoxic agent, wherein said ligand (L) binds (A) to (C).

In one embodiment, the ligand is selected from the group of a cleavable ligand, a non-cleavable ligand, a hydrophilic ligand and a dicarboxylic acid based ligand. In a further embodiment, the ligand is selected from the group consisting of: Nsuccinimidyl 4- (2-pyridyldithio) pentanoate (SPP) or A-succinimidyl 4- (2-pyridyldithio) -2sulfopentanoate (sulfo-SPP); A-succinimidyl 4- (2-pyridyldithio) butanoate (SPDB) or Nsuccinimidyl 4- (2-pyridyldithio) -2-sulfobutanoate (sulfo-SPDB); A-succinimidyl 4 (maleimidamethyl) cyclohexanecarboxylate (SMCC); A-sulfosuccinimidyl 4 (maleimidamethyl) cyclohexanecarboxylate (sulfoSMCC); A-succinimidyl-4- (iodoacetyl) aminobenzoate (SLAB); and the ester of A-succinimidyl - [(N-maleimidapropionamide) tetraethylene glycol] (NHS-PEG4-maleimide). In a certain embodiment, the ligand is the ester of / V-succinimidyl - [(N-maleimidapropionamide) -tetraethylene glycol] (NHS-PEG4maleimide).

In one embodiment, the immunoconjugates comprise a cytotoxic agent selected from the group of a maitansinoid, maitansinoid analogue, benzodiazepine, taxoid, CC-1065, analogue of CC-1065, duocarmycin, duocarmycin analogue, calicheamycin, dolastatin, dolastatin analogue to dolastatin , tomaimycin derivative and leptomycin derivative or agent prodrug. In a further embodiment, the cytotoxic agent is a maitansinoid. In another embodiment, the cytotoxic agent is N (2 ') deacetyl-N (2') - (3-mercapto-l-oxopropyl-maitansine or N (2 ') - deacetyl-N2- (4-mercapto-4methyl-1 -oxopentilj-maitansin.

In one embodiment the invention provides an immunoconjugate comprising: (A) a humanized antibody comprising the variable domain of a

<img file="AR080301A1_D0009.tif" />

heavy chain of sec. with no. of ident. : 4, and the variable domain of a light chain of sec. with no. Ident .: 10 or sec. with no. from ident.:11; (L) the ester of 7V-succinimidyl - [(Nmaleimidapropionamide) -tetraethylene glycol] (SNS-PEG4-maleimide) and (C) the N (2 ') - deacetyl N2- (4-mercapto-4-methyl-l-oxopentyl) -maitansin; where (L) links (A) to (C).

In one embodiment the invention provides an immunoconjugate comprising: (A) a humanized antibody comprising the variable domain of a sec heavy chain. with no. of ident. : 4, and the variable domain of a light chain of sec. with no. Ident .: 10 or sec. with no. from ident.:ll; (L) A-succinimidyl 4- (2-pyridyldithio) butanoate (SPDB) and (C) N (2 ') - deacetyl-N2- (4-mercapto-4-methyl-l-oxopentyl) -maitansin; where (L) links (A) to (C).

In one embodiment, the invention provides an immunoconjugate comprising: (A) a humanized antibody comprising the variable domain of a sec heavy chain. with no. of ident. : 4, and the variable domain of a light chain of sec. with no. Ident .: 10 or sec. with no. from ident.:ll; (L) TV-succinimidyl 4- (2-pyridyldithium) 2sulfobutanoate (sulfo-SPDB) and (C) N (2 ') - deacetyl-N2- (4-mercapto-4-methyl-l-oxopentyl) maitansin; where (L) links (A) to (C).

In one embodiment, the invention provides an immunoconjugate comprising: (A) a humanized antibody comprising the variable domain of a sec heavy chain. with no. of ident .: 4, and the variable domain of a light chain of sec. with no. of ident.:10 or sec. with no. from ident.:ll; (L) A-succinimidyl 4- (2-pyridyldithio) -2sulfopentanoate (sulfo-SPP); and (C) N (2 ') - deacetyl-N (2') - (3-mercapto-l-oxopropyl) maitansin; where (L) links (A) to (C).

In one embodiment, the invention provides an immunoconjugate comprising: (A) a humanized antibody comprising the variable domain of a sec heavy chain. with no. of ident .: 4, and the variable domain of a light chain of sec. with no. Ident .: 10 or sec. with no. Ident .: 11; (L) A-succinimidyl 4- (2-pyridyldithio) pentanoate (SPP); and (C) N (2 ') - deacetyl-N (2') - (3-mercapto-l-oxopropyl) -maitansin; where (L) links (A) to (C).

The invention also provides a pharmaceutical composition comprising an antibody, an antigen-binding fragment, a polypeptide, or an immunoconjugate of the invention and a pharmaceutically acceptable carrier. In a certain embodiment, the pharmaceutical composition also includes a second agent against cancer.

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The invention also provides a diagnostic reagent comprising an antibody, an antigen binding fragment, a polypeptide, or an immunoconjugate of the invention that is labeled. In one embodiment, the label is selected from the group of a radiolabel, a fluorophore, a chromophore, an image agent and a metal ion.

The invention also provides a kit comprising the antibody, the antigen binding fragment, the polypeptide, or the immunoconjugate of the invention.

The invention also provides a method for inhibiting tumor growth in a subject, which comprises administering to the subject a therapeutically effective amount of the antibody, the antigen binding fragment, the polypeptide, the immunoconjugate, or the pharmaceutical composition of the invention. In a certain embodiment, the invention provides a method for inhibiting tumor growth in a subject comprising administering a therapeutically effective amount of an immunoconjugate having the Formula (A) - (L) - (C); where: (A) is an antibody or antigen binding fragment thereof that specifically binds to a human folate receptor 1; (L) is a ligand, and (C) is a cytotoxin selected from the group consisting of a maitansinoid and a maitansinoid analog; where (L) links (A) to (C) and where the immunoconjugate reduces the mean tumor volume at least twice in a xenograft model in KB. In a certain embodiment, the method comprises administering an antibody or an antigen binding fragment thereof comprising (a) a heavy chain CDR1 comprising GYFMN (sec. With ID no .: 1); a heavy chain CDR2 comprising RIHPYDGDTFYNQXaalFXaa2Xaa3 (sec. with no. from ident.:56); and a heavy chain CDR3 comprising YDGSRAMDY (sec. with ID No.: 3) and (b) a light chain CDR1 comprises KASQSVSFAGTSLMH (sec. with ID No.: 7); a light chain CDR2 comprising RASNLEA (sec. with ID no .: 8); and a light chain CDR3 comprising QQSREYPYT (sec. with identification number: 9); where Xaai is selected from K, Q, H and R; Xaa₂ is selected from Q, H, N, and R, and Xaa₃ It is selected from G, E, T, S, A and V. In a further embodiment, the antibody comprises a CDR2 comprising RIHPYDGDTFYNQKFQG (sec. with ID no .: 2) heavy chain.

In a certain embodiment, the invention provides a method for inhibiting tumor growth comprising administering an antibody or antigen binding fragment thereof encoded by plasmid DNA deposited in the ATCC on April 7

<img file="AR080301A1_D0011.tif" />

of 2010 and that have the deposit numbers of the ATCC, PTA-10772 and PTA-10773 or PTA-10774.

In another embodiment, the method provides administering an immunoconjugate comprising a humanized antibody comprising the variable domain of a sec heavy chain. with no. of ident. : 4, and the variable domain of a light chain of sec. with no. Ident .: 10 or sec. with no. from ident.:ll; (L) the ester of 7V-succinimidyl - [(Nmaleimidapropionamide) -tetraethylene glycol] (SNS-PEG4-maleimide) and (C) N (2 ') - deacetyl N2- (4-mercapto-4-methyl-1-oxopentyl) -maitansin.

In another embodiment, the method provides administering an immunoconjugate comprising a humanized antibody comprising the variable domain of a sec heavy chain. with no. of ident. : 4, and the variable domain of a light chain of sec. with no. Ident .: 10 or sec. with no. from ident.:ll; (L) TV-succinimidyl 4- (2-pyridyldithio) butanoate (SPDB); and (C) N (2 ') - deacetyl-N2- (4-mercapto-4-methyl-l-oxopentyl) maitansin; where (L) links (A) to (C).

In another embodiment, the method comprises administering an immunoconjugate comprising (A) a humanized antibody comprising the variable domain of a sec heavy chain. with no. of ident. : 4, and the variable domain of a light chain of sec. with num. Ident .: 10 or sec. with no. from ident.:ll; (L) A-succinimidyl 4- (2-pyridyldithium) 2-sulfobutanoate (sulfo-SPDB) and (C) N (2 ') - deacetyl-N2- (4-mercapto-4methyl-1- oxopentylj-maitansin, where ( L) binds (A) to (C).

In another embodiment, the method comprises administering an immunoconjugate comprising (A) a humanized antibody comprising the variable domain of a sec heavy chain. with no. of ident. : 4, and the variable domain of a light chain of sec. with no. Ident .: 10 or sec. with num. from ident.:ll; (L) A-succinimidyl 4- (2-pyridyldithio) -2-sulfopentanoate (sulfo-SPP); and (C) N (2 ') - deacetyl-N (2') - (3-mercapto-l oxopropyl-maitansin; where (L) binds (A) to (C).

In another embodiment, the method comprises administering an immunoconjugate comprising (A) a humanized antibody comprising the variable domain of a sec heavy chain. with no. of ident. : 4, and the variable domain of a light chain of sec. with no. Ident .: 10 or sec. with no. from ident.:ll; (L) N-succinimidyl 4- (2-pyridyldithio) pentanoate (SPP); and (C) N (2 ') - deacetyl-N (2') - (3-mercapto-l-oxopropyl) maitansin; where (L) links (A) to (C).

<img file="AR080301A1_D0012.tif" />

In another embodiment, the method comprises administering an immunoconjugate comprising the FR-l-21hu antibody deposited with the ATCC on April 7, 2010 and having the ATCC deposit numbers, PTA-10775 and PTA-10776. In a certain embodiment, the FRl-21hu antibody comprises (a) a heavy chain CDR1 comprising SSYGMS (sec. With ID No. 30); a heavy chain CDR2 comprising TISSGGSYTY (sec. with no. from ident.:31); and a heavy chain CDR3 comprising DGEGGLYAMDY (sec. with ID no .: 32); and (b) a light chain CDR1 comprises KASDHINNWLA (sec. with identification no .: 27); a light chain CDR2 comprising GATSLET (sec. with ID no .: 28); and a light chain CDR3 comprising QQYWSTPFT (sec. with ID No. 29). In certain embodiments, the method comprises administering an immunoconjugate comprising the antibody that is the FRl-48hu antibody comprising: (a) a heavy chain CDR1 comprising TNYWMQ (sec. With ID no .: 60); a heavy chain CDR2 comprising AIYPGNGDSR (sec. with ID no .: 61); and a heavy chain CDR3 comprising RDGNYAAY (sec. with ID No.:62); and (b) a light chain CDR1 comprises RASENIYSNLA (sec. with no. from ident.:57); a light chain CDR2 comprising AATNLAD (sec. with ID no .: 58); and a light chain CDR3 comprises QHFWASPYT (sec. with ID No.:59). In certain embodiments, the method comprises administering an immunoconjugate comprising the antibody that is the FRl-49hu antibody comprising: (a) a heavy chain CDR1 comprising TNYWMY (sec. With ID No.:66); a heavy chain CDR2 comprising AIYPGNSDTT (sec. with no. from ident.:67); and a heavy chain CDR3 comprising RHDYGAMDY (sec. with ID no .: 68); and (b) a light chain CDR1 comprising RASENIYTNLA (sec. with identification number: 63); a light chain CDR2 comprising TASNLAD (sec. with ID no .: 64); and a light chain CDR3 comprising QHFWVSPYT (sec. with ID no .: 65). In certain embodiments, the method comprises administering an immunoconjugate comprising the antibody that is the FRl-57hu antibody comprising: (a) a heavy chain CDR1 comprising SSFGMH (sec. With ID no .: 72); a heavy chain CDR2 comprising YISSGSSTIS (sec. with ID no .: 73); and a heavy chain CDR3 comprising EAYGSSMEY (sec. with ID No.:74); and (b) a light chain CDR1 comprises RASQNINNNLH (sec. with no. from ident.:69); a light chain CDR2 that

<img file="AR080301A1_D0013.tif" />

comprises YVSQSVS (sec. with ID no .: 70); and a light chain CDR3 comprising QQSNSWPHYT (see. with id. ID: 71). In certain embodiments, the method comprises administering an immunoconjugate comprising the antibody that is the FRl-65hu antibody, comprising: (a) a heavy chain CDR1 comprising TSYTMH (see, with ID no .: 78); a heavy chain CDR2 comprising YINPISGYTN (see. with no. from ident.:79); and a heavy chain CDR3 comprising GGAYGRKPMDY (sec. with identification number: 80); and (b) a light chain CDR1 comprising KASQNVGPNVA (sec. with ID no .: 75); a light chain CDR2 comprising SASYRYS (sec. with ID no .: 76); and a light chain CDR3 comprising QQYNSYPYT (sec. with ID no .: 77).

In one embodiment, the method inhibits the growth of the ovarian tumor, brain tumor, marna tumor, uterus tumor, endometrial tumor, pancreatic tumor, renai tumor, or lung tumor. In a certain mode, the method inhibits the growth of the ovarian tumor. In another embodiment, the invention inhibits lung tumor growth. In a certain modality, tumor growth inhibition is used to treat cancer. In a further embodiment, the method comprises administering to the subject a second agent against cancer. In a certain embodiment, the second agent against cancer is a chemotherapeutic agent.

The invention also provides an isolated cell that produces the antibody, the antigen binding fragment, or the polypeptide of the invention.

The invention also provides an isolated polynucleotide comprising a sequence at least 90% identical to a sequence selected from the group consisting of sec. with no. Ident .: 5, 14, 15, 37, 38, 43, 44, 47, 48 and 120-127. In a certain embodiment, the isolated polynucleotide is at least 95% identical to a sequence selected from the group consisting of sec. with no. Ident .: 5, 14, 15, 37, 38, 43, 44, 47, 48 and 120-127. In another embodiment, the isolated polynucleotide is at least 99% identical to a sequence selected from the group consisting of sec. with no. ID: 5, 14, 15,

37, 38, 43, 44, 47, 48 and 120-127. The invention also provides a vector comprising any of the polynucleotides of sec. with no. ID: 5, 14, 15, 37,

38, 43, 44, 47, 48 and 120-127. In another embodiment, the invention provides a host cell comprising a vector containing a sec polynucleotide. with no. Ident .: 5, 14, 15, 37, 38, 43, 44, 47, 48 and 120-127.

<img file="AR080301A1_D0014.tif" />

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Superficial residues of murine Movl9 (Movl9mu) and humanized (Movl9hu). (A) Superficial residues of a murine and humanized Movl9 light chain. The surface residues of the variable region framework of a murine and humanized Movl9 light chain and the number of position (Kabat system) are given. Human residues that are different from the original murine sequences are underlined. * Position 74 is not a surface position, but to eliminate a glycosylation site linked to N-consensus in version 1.00, this position was changed to a Threonine (the most common human residue in this position); resulting in version 1.60. (B) Superficial wastes from a heavy and humanized Movi 9 heavy chain. The surface residues of the variable region framework of a murine and humanized Movi 9 heavy chain and the position number (Kabat system) are given. Human residues that are different from the original murine sequences are underlined.

Figure 2. Alignments of the variable domains of a light and heavy chain of chimeric Movi9 and Movl9hu and the variable domains of a heavy and light chain of FRl-21mu and FRI 21hu. Alignment of coated sequences of the Movi 9 and Fr 1-21 variable regions with their murine counterparts. A) and C) variable domains of a light chain; B) and D) variable domain of a heavy chain. The dashes indicate the identity with the mulina sequence. The CDRs (Kabat definition) are underlined.

Figure 3. Expression of chimeric Movi9 and Movl9hu in HEK cells. The chimeric and human Movi 9 expression plasmids were transfected transiently into the HEK293 T suspension cells, harvested 7 days later, and the expressed antibody was determined by quantitative ELISA. The light chain and heavy chain plasmids were transfected to the respective molar ratios of either 3: 1 or 6: 1.

Figure 4. Specificity of binding of anti-FOLRl antibodies, as detected by their binding to 300-19 cells expressing FOLR1. The binding of Movl9hu to 300-19-FOLR1 cells by flow cytometry. Parental cells 300-19 expressing FOLR-1. Solid gray shading represents cell autofluorescence, black dotted lines represent cells incubated with the FITC-conjugated secondary anti-human antibody, solid black lines represent cells incubated with Mov-19hu antibody and secondary anti-human antibody conjugated with FITC.

<img file="AR080301A1_D0015.tif" />

Figure 5. Binding affinities and cytotoxic activity, in vitro, of anti-FOLRl and immunoconjugate antibodies. The binding affinity of Movl9hu and various murine and humanized FR-1 antibodies was measured in SKOV3 cells. The cytotoxic activity, in vitro, of the PEG4-Mal-DM4 conjugates with the aforementioned antibodies was also tested.

Figure 6. Cellular cytotoxicity dependent on the immunoconjugate antibody. The ADCC activity of Movl9hu, FRl-21hu, and Mor003 was tested against Igrovl cells. Igrov 1 were incubated at 15,000 cells / target well: NK in the cell ratio of 1: 4.

Figure 7. Cytotoxic activity by continuous exposure of FRl-21hu-PEG4mal-DM4 and Movl9hu-PEG4-mal-DM4 in KB cells. An excess of unconjugated antibodies suppressed the activity of immunoconjugates when they were co-incubated in the presence of KB cells, which indicates that cytotoxic activity is antigen dependent.

Figure 8. Efficiency, in vivo, of the conjugates directed by the Movl9hu in a xenograft model with KB. The Movl9hu-SPDB-DM4 cleavable conjugate directed to FOLR1 (B) compared to C242hu-SPDB-DM4 not directed to FOLR1 (D); and the non-cleavable Movl9hu-PEG4-Mal-DM4 (C) conjugate compared to the non-directed C242huPEG4-Mal-DM4 (E) were tested using an established xenograft model for KB cells that are implanted subcutaneously in SCID mice . Directing the Movl9hu to FOLR1 resulted in a significant decrease in the average tumor volume.

Figure 9. Efficacy, in vivo, of Movl9hu-PEG4-Mal-DM4 compared to murine anti-FOLRl antibodies FR-1 in a xenograft model with KB. The FR-1 series antibodies, either unconjugated, or conjugated to PEG4-Mal-DM4 were tested for their ability to reduce the mean tumor volume compared to Movl9hu-PEG4-Mal-DM4 in a xenograft tumor model in KB cells. (A) FR-19, (B) FR-1-13, (C) FR-1-22, and (D) FR-1-23.

Figure 10. Efficiency, in vivo, of Movl9hu-PEG4-Mal-DM4 and FRl-21hu-PEG4Mal-DM4 in a xenograft model with KB cells. Single injections with 10 mg / kg of Movl9hu-PEG4-Mal-DM4 and FRl-21hu-PEG4-Mal-DM4 were made on day 6 after inoculation. Both Movl9hu-PEG4-Mal-DM4 and FRl-21hu -PEG4-Mal-

<img file="AR080301A1_D0016.tif" />

DM4 showed a significant reduction in the mean tumor volume. Media TV refers to the average tumor volume.

Figure 11. Movl9hu-PEG4-Mal-DM4 shows a dose-dependent activity in the xenograft model with KB cells. The dose-dependent activity of the immunoconjugate was tested over the entire range of doses tested. The weekly dosage resulted in the improvement of anti-tumor activity. Alias ​​drug loads only marginally improved activity in the 10 mg / kg dose groups, with reduced activity in the lower dose groups. DAR 3.7 refers to 3.7 drug molecules per antibody.

Figure 12. Efficiency, in vivo, of Movl9hu conjugated with DM1 and DM4 with different ligands. Movl9hu was conjugated with SMCC-DM1 to 3.9 molecules of the drug per antibody; sulfo-Mal-DM4 at 3.7 drug molecules per antibody (B); and sulfo-Mal-DM4 to 8.23 ​​molecules of the drug per antibody (C) and were tested for their ability to reduce the average tumor volume at different concentrations compared to Movi9hu-PEG4-Mal-DM4. Figure 13 Efficiency, in vivo, of Movl9hu conjugated with DM1 and DM4 with different ligands. Movl9hu was conjugated with SPP-DM1 to 4.3 drug molecules per antibody; sulfo-SPDB-DM4 at 3.8 molecules of the drug by antibody, SPDB-DM4 at 3.8 molecules of the drug by antibody, and sulfo-SPDB DM4 at 6.8 molecules of the drug by antibody and were analyzed for their ability to reduce the mean of tumor volume Mice were treated with 5 mg / kg (A) and 2.5 mg / kg (B) of one of the above-mentioned conjugates or only with PBS.

Figure 14. Efficiency, in vivo, of Movl9hu-sulfo-SPDB-DM4 in the xenograft tumor model with OVCAR-3. Mice were treated with 25, 50, or 100 pg / kg of Movl9hu-sulfo-SPDB-DM4 or only with PBS.

Figure 15. Efficiency, in vivo, of Movl9hu-sulfo-SPDB-DM4 in the tumor model of xenograft with IGROV-1. Mice were treated with 25, 50, or 100 pg / kg of Movl9hu-sulfo-SPDB-DM4 or only with PBS.

Figure 16. Efficiency, in vivo, of Movl9hu-sulfo-SPDB-DM4 in the xenograft tumor model with OV-90. Mice were treated with 25, 50, or 100 pg / kg of Movl9hu-sulfo-SPDB-DM4 or only with PBS.

<img file="AR080301A1_D0017.tif" />

Figure 17. Effect of cleavable ligands and non-cleavable ligands on the efficacy of immunoconjugates in xenograft models in KB cells.

Figure 18. Effect of cleavable ligands on the efficacy of immunoconjugates in (A) the xenograft model with KB, (B) the xenograft model with KBOVCAR-3.

Figure 19. Efficacy in vitro and in vivo of FRl-48hu, FRl-49hu, FRl-57hu, and FRl-65hu-SMCC-DMl in KB and tumor models of xenografts. Mice were treated with 200 pg / kg of single doses.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides new agents, which include but are not limited to polypeptides, such as antibodies, and immunoconjugates that bind to human folate receptor 1 (FOLR1). Related polypeptides and polynucleotides, compositions comprising FOLR1 binding agents, and methods for preparing FOLR1 binding agents are provided. In addition, the methods of using the new FOLR1 binding agents are provided, as well as methods of inhibiting tumor growth and / or treating cancer.

I. Definitions

To facilitate an understanding of the present invention, a number of terms and phrases are defined below.

The terms human folate receptor 1 or FOLR1, which are used herein, refer to any native human FOLR1, unless otherwise indicated. The term FOLR1 includes unprocessed, full-length FOLR1 as well as any form of FOLR1 that resumes from processing within the cell. The term also includes FOLR1 variants of natural origin, for example, splice variants, allelic variants and isoforms. The FOLR1 polypeptides described herein can be isolated from different sources, such as human tissue types or other sources, or prepared by recombinant or synthetic methods. Examples of the FOLR1 sequences include, but are not limited to, NCBI reference numbers P15328, NP 001092242.1, AAX29268.1, AAX37119.1,

NP 057937.1, and NP 057936.1.

<img file="AR080301A1_D0018.tif" />

The term "antibody" means an immunoglobulin molecule that specifically recognizes and binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing, at least through a recognition site. of the antigen within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" includes intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (tabs as Fab, Fab ', F (ab') 2, and Fv fragments); single chain Fv mutants (scFv), multispecific antibodies such as bispecific antibodies generated at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising a part for the determination of the antigen of an antibody, and any other immunoglobulin molecule comprising an antigen recognition site so extensive that the antibodies have the appropriate biological activity. An antibody can be one of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (for example IgGl, IgG2, IgG3, IgG4, IgAl and IgA2); based on the identity of their constant domains of the heavy chains that refer to alpha, delta, epsilon, gamma, and mu, respectively. Different classes of immunoglobulins have different and well known three-dimensional subunit structures and configurations. Antibodies can be stripped or conjugated to other molecules such as toxins, radioisotopes, etc.

A blocking antibody or an antagonistic antibody is one that inhibits ο reduces the biological activity of the antigen that binds, such as FOLR1. In a certain embodiment, blocking antibodies or antagonistic antibodies substantially or completely inhibit the biological activity of the antigen. It is desirable that the biological activity be reduced by 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or even 100%.

The term "anti-FOLRl antibody" or an antibody that binds to FOLR1 refers to an antibody that is capable of binding to FOLR1 with sufficient affinity such that the antibody is useful as an agent for the diagnosis and / or therapeutic agent directed to FOLR1. The magnitude of the binding of an anti-FOLR1 antibody to an unrelated protein, a non-FOLR1 protein is approximately less than 10% of the antibody binding to FOLR1 when measured, for example, by a radioimmunoassay (RIA).

<img file="AR080301A1_D0019.tif" />

In certain embodiments, an antibody that binds to FOLR1 has a dissociation constant (Kd) of &lt;1μΜ, &lt;100 nM, &lt;10 nM, &lt;1 nM, or &lt;0.1 nM.

The term "antibody fragment" refers to a portion of an intact antibody and refers to the variable regions of antigenic determinants of an intact antibody. Examples of antibody fragments include, but are not limited to Fab, Fab fragments.¹, F (ab ') 2, and Fv, linear antibodies, single chain antibodies, and multispecific antibodies formed from antibody fragments.

A monoclonal antibody refers to a homogeneous population of antibody involved in highly specific recognition and binding of a simple antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. The term "monoclonal antibody" includes both intact monoclonal antibodies and full-length monoclonal antibodies, as well as antibody fragments (such as Fab, Fab, F (ab ') 2, Fv); single chain mutants (scFv), fusion proteins comprising an antibody part, and any other modified immunoglobulin molecule comprising an antigen recognition site. In addition, the monoclonal antibody refers to those antibodies prepared in many ways that include but are not limited to those of hybridoma, phage selection, recombinant expression, and transgenic animals.

The term "humanized antibody" refers to the forms of nonhuman antibodies (for example, murine) that are specific chains of immunoglobulins, chimeric immunoglobulins, or fragments thereof containing the minimum number of non-human sequences (for example, murine ). Typically, humanized antibodies are human immunoglobulins in which the residues of the complementarity determining region (CDR) are replaced by the CDR residues of one of the non-human species (e.g., mouse, rat, rabbit, hamster) that have the specificity, affinity, and ability suitable (Jones et al., 1986, Nature, 321: 522-525; Riechmann et al., 1988, Nature, 332: 323-327; Verhoeyen et al., 1988, Science, 239: 1534 -1536). In some examples, residues of the framework region (FR) of Fv of a human immunoglobulin are replaced by the corresponding residues of an antibody of one of the non-human species having the specificity, affinity and suitable capacity. The humanized antibody can also be modified by the substitution of residues

<img file="AR080301A1_D0020.tif" />

additional either in the PV framework region and / or within the replaced non-human residues to improve and optimize the specificity, affinity and / or capacity of the antibody. In general, the humanized antibody will comprise substantially all of at least one variable domain, and typically two or three variable domains that contain all or substantially all of the CDR regions corresponding to the non-human immunoglobulin, while in all or substantially all regions of FR are those of a consensus sequence of human immunoglobulin. The humanized antibody may also comprise at least a part of a region or constant domain of the immunoglobulin (Fc); typically of a human immunoglobulin. Examples of the methods used to generate humanized antibodies are described in US Pat. 5,225,539 or 5,639,641.

A variable region of an antibody refers to the variable region of a light chain of the antibody or the variable region of a heavy chain of the antibody, either alone or in combination. The variable regions of a heavy and light chain each consisting of four framework regions (FR) are connected by three regions determining complementarity (CDR); also known as hypervariable regions. The CDRs in each chain are held together at close range by the FRs and with the CDRs of the other chain, they contribute to the formation of the binding site of the antibodies to the antigen. There are at least two techniques for determining CDR: (1) an approach based on cross-sequence sequence variability (i.e., Kabat and others, Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health , Bethesda Md.)); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al. (1997) J. Molec. Biol. 273: 927-948)). In addition, combinations of these two approaches are sometimes used in the technique to determine the RDA.

The Kabat numbering system is generally used to refer to a residue in the variable domain (approximately 1-107 residues of a light chain and 1-113 residues of a heavy chain) (for example, Kabat and others, Sequences of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

The amino acid position numbering according to Kabat, refers to the numbering system used for the variable domains of a heavy chain or the variable domains of a light chain in the compendium of antibodies of Kabat and others, Sequences

<img file="AR080301A1_D0021.tif" />

of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). By this numbering system, the linear amino acid sequence present may contain less or more amino acids corresponding to a reduction of, or an insertion to, an FR or a CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and residues inserted (e.g., residues of 82a, 82b, and 82c, etc. according to Kabat) after residue 82 of the FR of a heavy chain. The Kabat numbering of the residues can be determined for an antibody given by the alignment of the homology regions of the antibody sequence with a standard sequence numbered by Kabat. Chothia instead refers to the location of structural bonds (Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)). The Chothia CDR-H1 loop terminal when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the inserts in H35A and H35B , if neither 35A nor 35B is present, the loop ends at 32, if only 35A is present, the loop ends at 33, if both 35A and 35B are present, the loop ends at 34). The hypervariable AbM regions represent a compromise between the Kabat CDRs and the Chothia structural bonds, and are used by the Molecular Oxford AbM antibody modeling program.

<td>Tie</td><td>Kabat</td><td>AbM</td><td>Chothia</td>

<td>LI</td><td>L24-L34</td><td>L24-L34</td><td>L24-L34</td>

<td>L2</td><td>L50-L56</td><td>L50-L56</td><td>L50-L56</td>

<td>L3</td><td>L89-L97</td><td>L89-L97</td><td>L89-L97</td>

<td>HI</td><td>H31-</td><td>H26-</td><td>H26-</td>

<td></td><td>H35B</td><td>H35B</td><td>H32..34</td>

(Kabat numbering)

<td>HI</td><td>H31-H35</td><td>H26-H35</td><td>H26-H32</td>

<td></td><td></td><td colspan="2">(Chothia numbering)</td>

<td>H2</td><td>H50-H65</td><td>H50-H58</td><td>H52-H56</td>

<td rowspan="2">H3</td><td>H95-</td><td>H95-</td><td>H95-</td>

<td>H102</td><td>H102</td><td>H102</td>

<img file="AR080301A1_D0022.tif" />

The term "human antibody" denotes an antibody produced by a human or an antibody having an amino acid sequence that corresponds to an antibody produced by a human that is prepared by any method known in the art. This definition of a human antibody includes intact or full-length antibodies, their antibodies, and / or antibodies comprising at least one heavy chain and / or human light chain polypeptide, such as, for example, an antibody that It comprises the murine light chain and human heavy chain polypeptides.

The term "chimeric antibodies" refers to antibodies where the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from one of the mammalian species (eg, mouse, rat, rabbit, etc.) with the specificity, affinity and suitable capacity , while the constant regions are homologous to the sequences of antibodies derived from another species (usually human) to avoid induction of an immune response in these species.

The terms epitope or antigenic determinant are used interchangeably herein and refer to the part of an antigen capable of being specifically recognized and bound by a particular antibody. When the antigen is a polypeptide, epitopes can be formed from both contiguous amino acids and noncontiguous amino acids juxtaposed by the tertiary folding of a protein. Epitopes formed from contiguous amino acids are normally retained with protein denaturation, while epitopes formed by tertiary folding are normally lost with protein denaturation. An epitope usually includes at least 3, and usually more, at least 5 or 8 to 10 amino acids in a single spatial conformation.

Binding affinity generally refers to the strength of the total sum of non-covalent interactions between a single binding site of a molecule (for example, an antibody) and its binding partner (for example, an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity that reflects a 1: 1 interaction between members of a binding pair (eg, antibody and antigen). ). The affinity of an X molecule for its Y partner is

<img file="AR080301A1_D0023.tif" />

It can represent in general, by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, which include those described herein. Low affinity antibodies, in general, slowly bind to the antigen and tend to dissociate easily, while high affinity antibodies generally bind rapidly to the antigen and tend to remain bound for longer. A variety of methods for measuring binding affinity are known in the art, some of which can be used for the purposes of the present invention. The specific illustrative modalities are described below.

When used herein or better to refer to the affinity of union, it refers to a stronger union between a molecule and its binding partner. When used in this document or better, it refers to a stronger union, which is represented by a smaller numerical value Kd. For example, an antibody that has an affinity for an antigen of 0.6 nM or better, the affinity of the antibody for the antigen is &lt;0.6 nM, that is, 0.59 nM, 0.58 nM, 0.57 nM etc. or any value less than 0.6 nM.

The phrase substantially similar, or substantially the same, as used herein, denotes a sufficiently high degree of similarity between two numerical values ​​(usually one is associated with an antibody of the invention and the other is associated with a reference / comparison antibody) so that a person skilled in the art might consider that the difference between the two values ​​would be of little or no biological and / or statistical significance in the context of the biological characteristics measured by said values ​​(for example, Kd values). The difference between these two values ​​is less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% depending on the value for the reference / comparison antibody.

A polypeptide, an antibody, a polynucleotide, a vector, a cell, or a composition that is isolated is a polypeptide, an antibody, a polynucleotide, a vector, a cell, or a composition that is in a form not found in nature. . Polypeptides, antibodies, polynucleotides, vectors, cells or isolated compositions include those that have been purified to a degree that are no longer in a form in which they are found in nature. In some embodiments, an antibody, a polynucleotide, a vector, a cell, or a composition that is isolated is substantially pure.

<img file="AR080301A1_D0024.tif" />

As used herein, "substantially pure" refers to a material that is at least 50% pure (ie, free of contaminants); at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

The term "immunoconjugate" or "conjugate" as used herein, refers to a compound or derivative thereof that binds to a cell binding agent (i.e., an anti-FOLRl antibody or a fragment thereof) and is defined by a generic formula: CLA, where C = cytotoxin, L = ligand, and A = cell binding agent or anti-FOLRl antibody or antibody fragment. Immunoconjugates can also be defined by the generic formula in the reverse order: LAC.

A ligand is any chemical half that is capable of binding a compound, usually a drug, such as a maitansinoid, with a cell binding agent such as an anti-FOLR1 antibody or a fragment thereof. The ligands may be susceptible or substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfuric bond cleavage, under conditions where the compound or antibody remains active. . Suitable ligands are well known in the art and include, for example, disulfuric groups, thioether groups, acid-labile groups, photolabile groups, peptidase-labile groups and esterase-labile groups. The ligands also include charged ligands, and the hydrophilic forms thereof as described herein and are known in the art.

The terms cancer and cancer refer to or describe the physiological disease in mammals in which a population of cells is characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma and leukemia. More particular examples of these cancers include squamous cell cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous lung carcinoma, peritoneum cancer, hepatocellular cancer, gastrointestinal cancer, pancreas cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, marna cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivai gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma and various types of head and neck cancers.

<img file="AR080301A1_D0025.tif" />

The tumor and the neoplasm refer to any mass of tissue that results from excess cell growth or proliferation, either benign (non-cancerous) or malignant (cancerous) that includes pre-cancerous lesions.

The terms cancer cell, tumor cell, and grammatical equivalents refer to the total population of cells derived from a tumor or a pre-cancerous lesion, which include both non-tumor cells, which comprise the removal of the tumor cell population. as the tumorigenic stem cells (cancer stem cells). As used herein, the term tumor cell will be modified by the notumorigenic term when it refers exclusively to those tumor cells that lack the ability to renew and differentiate to distinguish those tumor cells from the stem cells of cancer.

The term "subject" refers to any animal (for example, a mammal); It includes but is not limited to humans, nonhuman primates, rodents, and the like, which must be the recipient of a particular treatment. Typically, the terms subject and patient are used interchangeably in this document with respect to a human subject.

Administration in combination with one or more additional therapeutic agents includes simultaneous (concomitant) and consecutive administration in any order.

The term "pharmaceutical formulation" refers to a preparation that is in some way to allow the biological activity of the active ingredient to be effective, and which does not contain additional components that are toxic unacceptable to a subject for which the formulation could be administered. This formulation can be sterile.

The effective amount of an antibody as disclosed herein is an amount sufficient to carry out a specifically established purpose. An effective amount can be determined empirically and in a routine manner, in relation to the established purposes.

The term "therapeutically effective amount" refers to an amount of an antibody or other drug effective to treat a disease or disorder in a subject or mammal. In the case of cancer, the therapeutically effective amount of the drug can reduce the number of cancer cells, reduce the size of the tumor, inhibit (that is, slow down to some extent and in a certain mode, stop) the infiltration of cancer cells into peripheral organs, inhibit (i.e. slow down to some extent and in a

<img file="AR080301A1_D0026.tif" />

certain modality, stop) the metastasis of the tumor, inhibit, to some extent, the growth of the tumor, and / or alleviate to some extent one or more of the symptoms associated with cancer. See the definition of dealing with this document. To the extent that the drug can prevent growth and / or kill cancer cells that exist, it can be cytostatic or cytotoxic. A prophylactically effective amount refers to an effective amount, in doses and for periods of time necessary, to achieve the convenient prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in the subjects before or at an early stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

The word "brand" when used herein refers to a detectable compound or to the composition that is directly or indirectly conjugated with the antibody in order to generate a labeled antibody. The label can be detected by itself (for example, radioisotope markers or fluorescence markers) or, in the case of an enzymatic marker, which can catalyze the chemical modification of a compound or a substrate composition that is detected.

A chemotherapeutic agent is a chemical compound useful in the treatment of cancer, regardless of the mechanism of action. Classes of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, spindle venom plant alkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Chemotherapeutic agents include the compounds used in targeted therapy and conventional chemotherapy.

The terms such as trust or treatment or to treat or relieve or relieve refer both to 1) therapeutic measures that eury, decelerate, decrease symptoms, and / or the cessation of the progression of a pathological condition or disorder identified and 2) prophylactic measures or preventive measures that prevent or reduce the development of an objective disease or pathological disorder. Thus, those who need treatment include those who are already with the disorder; those prone to having the disorder, and those in which the disorder should be avoided. In certain embodiments, according to the methods of the present invention, a subject is successfully trusted for cancer, if the patient exhibits one or more of the following effects: a reduction in the number or complete absence of cancer cells, a reduction in the size of

<img file="AR080301A1_D0027.tif" />

tumor, inhibition or absence of infiltration of cancer cells in peripheral organs that include, for example, the spread of cancer in soft tissues and bones; inhibition or absence of tumor metastasis, inhibition or absence of tumor growth, attenuation of one or more of the symptoms associated with specific cancer; reduction of morbidity and mortality, improvement in quality of life, reduction in tumorigenicity, tumorigenic frequency, or tumorigenic capacity, of a tumor, reduction in the number or frequency of cancer stem cells in a tumor, the differentiation of tumorigenic cells to a notumorigenic state, or some combination of effects.

As polynucleotide, or nucleic acid, are used interchangeably herein, they refer to nucleotide polymers of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, as well as methylated nucleotides and their analogs. If presented, the modification in the nucleotide structure can occur before or after the polymer assembly. The nucleotide sequence can be interrupted by components that are not nucleotides. A polynucleotide can be modified further after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, the layers, the substitution of one or more of the nucleotides of natural origin with an analogue, the intemucleotide modifications taies as, for example, those with the no-load ligands (for example, methyl phosphonates , phosphotriesters, phosphoamidates, carbamates, etc.) and with the charged ligands (for example, phosphorothioates, phosphorodithioates, etc.); those containing pendant portions, such as, for example, proteins (for example, nucleases, toxins, antibodies, serial peptides, L-lysine layer, etc.); those with interleavers (for example, acridine, soralene, etc.); those that contain chelants (for example, metals, radioactive metals, boron, oxidizing metals, etc.); those containing alkylating agents, those with modified bonds (for example, anomeric alpha nucleic acids, etc); thus as the unmodified forms of polynucleotide (s). In addition, any of the hydroxyl groups normally present in sugars can be replaced, for example, by phosphonate groups, phosphate groups, can be protected by the protecting groups

<img file="AR080301A1_D0028.tif" />

standard, or they can be activated to prepare additional bonds to additional nucleotides, or they can be conjugated with solid supports. The 5 'and 3' OH terminal can be phosphorylated or substituted with amines or portions with organic layer groups of 1 to 20 carbon atoms. Other hydroxyls can also be referred to standard protecting groups. The polynucleotides may also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, which include, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2 '-azido-ribosa, carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars taies corno arabinosa, xyloses or lyxose, pyranous sugars, furanose sugars, sedoheptulose sugars, acyclic analogues and the corosid nucleoside analogues. One or more phosphodiester bonds can be replaced by alternative link groups. These alternative binding groups include, but are not limited to, the modalities where phosphate is replaced by P (O) S (thioate); P (S) S (dithioate); (O) NR₂ (Amidate), P (O) R, P (O) OR, CO or CH₂ (formacetal); wherein each R or R 'is alkyl (1-20 C) substituted or unsubstituted independently of H which optionally contains an ether (—O—), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl bond. Not all bonds in a polynucleotide need to be identical. The above description applies to all polynucleotides referred to herein, which include RNA and DNA.

The term vector means a construct, which is capable of delivering, and expressing, one or more genes (s) or sequence (s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensation agents, expression vectors of DNA or RNA encapsulated in liposomes, and certain eukaryotic cells, as well as producing cells.

The terms polypeptide, peptide, and protein are used interchangeably herein to refer to amino acid polymers of any length. The polymer may be linear or branched, may comprise modified amino acids, and may be interrupted by non-amino acids. The terms also include an amino acid polymer that has been modified naturally or by intervention, for example, the formation of disulfide bond, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a component

<img file="AR080301A1_D0029.tif" />

marked. Also included in the definition are, for example, polypeptides that contain one or more analogs of an amino acid (which include, for example, artificial amino acids, etc.); So as other modifications known in the technique. It is understood that, because the polypeptides of this invention are based on antibodies, in certain embodiments, the polypeptides may appear as single chains or associated chains.

The terms identical or percent identity in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or sub-sequences that are equal or have a specific percentage of nucleotides or amino acid residues that are equal, when compared and align (which introduce gaps, if necessary) for maximum correspondence, without considering any of the conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured by the sequence or algorithm comparison program or by visual inspection. Different algorithms and programs that can be used to obtain the alignments of amino acid or nucleotide sequences are known in the art. Such a non-limiting example of an algorithm for sequence alignment is the algorithm described in Karlin et al., 1990, Proc. Nati Acad. Sci., 87: 2264-2268, as modified in Karlin et al., 1993, Proc. Nati Acad. Sci., 90: 5873-5877, and incorporated into the NBLAST and XBLAST programs (Altschul et al., 1991, Nucleic Acids Res., 25: 33893402). In certain embodiments, Gapped BLAST can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25: 3389-3402. BLAST-2, WU-BLAST-2 (Altschul et al., 1996, Methods in Enzymology, 266: 460-480); ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or Megalign (DNASTAR) are additional publicly available software programs that can be used to align sequences. In certain embodiments, the percent identity between two nucleotide sequences is determined by the GAP program in the GCG software (for example, using a NWSgapdna.CMP matrix and a range weight of 40, 50, 60, 70, or 90 and a section weight of 1, 2, 3, 4, 5 or 6). In certain alternative modalities, the GAP program in the GCG program package, which incorporates the algorithm of Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) can be used to determine the percent identity between two amino acid sequences (for example, using either a Blossum 62 matrix or a PAM250 matrix, and a range weight of 16, 14, 12, 10, 8, 6, or 4 and a section weight of 1, 2, 3, 4, 5). On the other hand, in certain modalities, the percent of identity between

<img file="AR080301A1_D0030.tif" />

Nucleotide or amino acid sequences are determined by the Myers and Miller algorithm (CABIOS, 4: 11-17 (1989)). For example, the percent identity can be determined by the ALIGN program (version 2.0) and by a ΡΑΜΙ20 with residual table, a penalty in the length of the interval of 12 and a penalty of the interval of 4. The appropriate parameters for maximum alignment by a particular alignment program can be determined by a person skilled in the art. In certain embodiments, the default parameters of the alignment program are used. In certain embodiments, the percentage of identity X of a first amino acid sequence in a second amino acid sequence is calculated as 100 x (Y / Z); where Y is the number of amino acid residues noted as identical counterparts in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence If the length of a first sequence is greater than the second sequence, the percent identity of the first sequence with the second sequence will be greater than the percent identity of the second sequence with the first sequence.

As a non-limiting example, if any particular polynucleotide has a certain percentage of sequence identity (for example, being at least 80% identical, at least 85% identical, at least 90% identical and, in some modalities, at least 95 %, 96%, 97%, 98% or 99% identical) with a reference sequence can, in certain modalities, be determined using the Bestfit program (Wisconsin sequence analysis package, version 8 for Unix, Genetics Computer Group, University Research Park 575 Science Drive, Madison, WI 53711). The Bestfit uses Smith and Waterman's local homology algorithm, Advances in Applied Mathematics 2: 482 489 (1981); to find the best homology segment between two sequences. When Bestfit or any other sequence alignment program is used to determine if a particular sequence is, for example, 95% identical to a reference sequence according to the present invention, the parameters are adjusted such that the percent identity It is calculated on the complete sequence of complete reference nucleotides and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.

<img file="AR080301A1_D0031.tif" />

In some embodiments, two nucleic acids or polypeptides of the invention are substantially identical, which means that they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some modalities at least 95%, 96%, 97%, 98%, 99% identity of nucleotide or amino acid residues, when compared and aligned for maximum correspondence, when measured using a sequence comparison algorithm or by visual inspection. In certain embodiments, the identity exists on a region of the sequences that is at least approximately 10, 20, 40-60 residues in length or any integral value between them, or on a region longer than 60 to 80 residues, at least approximately 90 -100 residues, or the sequences are substantially identical over the complete sequences that are compared, such as, for example, the region encoding a nucleotide sequence.

A conservative amino acid substitution is one in which an amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which include basic side chains (eg, lysine, arginine, histidine); the side chains of acids (for example, aspic acid, glutamic acid); unloaded polar side chains (for example, asparagine, glutamine, serine, threonine, tyrosine, cistern); non-polar side chains (for example, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); beta-branched side chains (for example, threonine, valine, isoleucine) and aromatic side chains (for example, tyrosine, phenylalanine, tryptophan, histidine). For example, the substitution of a phenylalanine for a tyrosine is a conservative substitution. In certain embodiments, conservative substitutions in the sequences of the polypeptides and antibodies of the invention do not nullify the binding of the polypeptide or antibody containing the amino acid sequence to the antigen (s); that is, the FOLR1 to which the polypeptide or antibody binds. Methods of identifying conservative nucleotide and amino acid substitutions that do not eliminate antigen binding are well known in the art (see, for example, Brummell et al., Biochem. 32: 1180-1 187 (1993); Kobayashi and others Protein Eng. 12 (10): 879-884 (1999); and Burks and others Proc. Nati. Acad. Sci. United States 94: .412-417 (1997)).

<img file="AR080301A1_D0032.tif" />

As used in the present disclosure and in the claims, the singular forms one, one, and, are included by plural forms unless the context clearly indicates otherwise.

It is understood that wherever the modalities are described in this document with the language that it comprises, the opposite analogous modalities described in the terms of consisting of and / or consisting essentially of are also provided.

The term and / or horn uses this document in a phrase such as A and / or B is intended to include both A and B, A or B, A and B. Similarly, the term and / or horn is used in a horn phrase. A, B, and / or C intends to include each of the following modalities: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (only).

II. Binding agents-FOLRl

The present invention provides agents that specifically bind human FOLR1. These agents are referred to herein as binding agents to FOLR1. The full length amino acid (aa) and nucleotide (nt) sequences of FOLR1 are known in the art and are provided herein as represented by sec. with no. of ident .: 25 and 26, respectively.

In certain embodiments, the FOLR1 binding agents are antibodies, immunoconjugates or polypeptides. In some embodiments, FOLR1 binding agents are humanized antibodies. In certain embodiments, the FOLR1 binding agents are humanized versions of the Movi 9 murine antibody (variable of a heavy and light chain are shown as sections with ID no .: 17 and 18, respectively).

In certain embodiments, FOLR1 binding agents have one or more of the following 25 effects: it inhibits the proliferation of tumor cells, reduces tumorigenicity of a tumor by reducing the frequency of cancer stem cells in the tumor, inhibits tumor growth, increases survival, triggers cell death of tumor cells, differentiates tumor cells to a non-tumorigenic state, or prevents metastasis of tumor cells.

In certain embodiments, immunoconjugates or other agents that specifically bind to human FOLR1 trigger cell death through a cytotoxic agent. For example, in certain embodiments, an antibody of an FOLR1 antibody

<img file="AR080301A1_D0033.tif" />

Human is conjugated with a maitansinoid that is activated in tumor cells that express FOLR1 by the intemalization of the protein. In certain alternative modalities, the agent or antibody does not conjugate.

In certain embodiments, FOLR1 binding agents are capable of inhibiting tumor growth. In certain embodiments, FOLR1 binding agents are capable of inhibiting tumor growth in vivo (for example, in a mouse xenograft model and / or in a human having cancer). In certain embodiments, FOLR1 binding agents are capable of inhibiting tumor growth in a human.

Thus, the invention provides a humanized antibody or antigen binding fragment thereof that specifically binds to a human folate receptor 1, wherein the antibody comprises: (a) a heavy chain CDR1 comprising GYFMN (sec with identification number: 1) a heavy chain CDR2 comprising RIHPYDGDTFYNQXaaiFXaa2Xaa3 (section with identification number: 56); and a heavy chain CDR3 comprising YDGSRAMDY (sec. with no. Ident .: 3); and (b) a light chain CDR1 comprising KASQSVSFAGTSLMH (sec. with identification number: 7); a light chain CDR2 comprising RASNLEA (sec. with identification number: 8); and a light chain CDR3 comprising QQSREYPYT (sec. with identification number: 9); where Xaai is selected from K, Q, H and R; Xaa₂ is selected from Q, H, N, and R, and Xaa₃ it is selected from G, E, T, S, A and V. In certain embodiments, the antibody is the Movl9hu antibody, which is the antibody described above comprising the CDR2 RIHPYDGDTFYNQKFQG (sec. with ID no .: 2) of heavy chain

In certain embodiments, the invention provides humanized antibodies or antigen binding fragments that specifically bind to FOLR1 comprising Movl9hu CDRs with up to four (ie, 0, 1, 2, 3, or 4) conservative substitutions of amino acids by CDR. Thus, in certain embodiments the invention provides humanized antibodies or antigen binding fragments that specifically bind to a human folate receptor 1, wherein the antibody comprises: (a) a heavy chain CDR1 comprising GYFMN sec. with no. of ident. : 1), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; a heavy chain CDR2 comprising RIHPYDGDTFYNQKFQG (sec. with no. of ident .: 2), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and a heavy chain CDR3 comprising YDGSRAMDY (sec. with

<img file="AR080301A1_D0034.tif" />

no. Identification: 3), or a variant thereof that comprises 1, 2, 3, or 4 conservative amino acid substitutions and / or (b) a light chain CDR1 comprising KASQSVSFAGTSLMH (sec. with identification number: 7), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; a light chain CDR2 comprising RASNLEA (sec. with no. of ident .: 8), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and a light chain CDR3 comprising QQSREYPYT (sec. with identification no .: 9), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions.

The invention also provides a humanized antibody (FRl-21hu) or an antigen-binding fragment thereof that specifically binds to a human folate receptor 1, wherein the antibody comprises: (a) a heavy chain CDR1 comprising SSYGMS (sec. With ID No. 30); a heavy chain CDR2 comprising TISSGGSYTY (sec. with ID no .: 31); and a heavy chain CDR3 comprising DGEGGLYAMDY (sec. with no. from ident.:32); and / or (b) a light chain CDR1 comprising KASDHINNWLA (sec. with ID no .: 27); a light chain CDR2 comprising GATSLET (sec. with identification number: 28); and a light chain CDR3 comprising QQYWSTPFT (sec. with identification number: 29).

In certain embodiments, the invention provides humanized antibodies or antigen-binding fragments that specifically bind to FOLR1 comprising the CDRs of FRl-21hu with up to four (ie, 0, 1, 2, 3, or 4) substitutions. Conservatives of amino acids by CDR. Thus, in certain embodiments, the invention provides humanized antibodies or antigen binding fragments that specifically bind a human folate receptor, wherein the antibody comprises: (a) a heavy chain CDR1 comprising SSYGMS (sec. with identification number: 30) or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and / or a heavy chain CDR2 comprising TISSGGSYTY (sec. with no. of ident: 31) or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and / or a heavy chain CDR3 comprising DGEGGLYAMDY (sec. with identification number: 32) or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and / or (b) a light chain CDR1 comprising KASDHINNWLA (sec. with no. of ident: 27) or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and / or a light chain CDR2 comprising GATSLET

<img file="AR080301A1_D0035.tif" />

(sec. with identification number: 28) or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and / or a light chain CDR3 comprising QQYWSTPFT (sec. with identification number: 29) or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions.

In certain embodiments, the invention provides humanized antibodies or antigen binding ffagments that specifically bind to FOLR1 comprising the CDRs of FRl-48hu with up to four (i.e., 0, 1, 2, 3, or 4) substitutions. conservative amino acids by CDR. Thus, in certain embodiments, the invention provides humanized antibodies or antigen binding ffagments that specifically bind a human folate receptor, wherein the antibody comprises: (a) a heavy chain CDR1 comprising TNYWMQ (sec. with identification number: 60) or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and / or a heavy chain CDR2 comprising IYPGNGDSR (sec. with no. of ident: 61) or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and / or a heavy chain CDR3 comprising RDGNYAAY (sec. with identification number: 62) or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and / or (b) a light chain CDR1 comprising RASENIYSNLA (sec. with no. of ident: 57) or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and / or a light chain CDR2 comprising AATNLAD (sec. with identification number: 58) or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and / or a light chain CDR3 comprising QHFWASPYT (sec. with identification number: 59) or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions.

In certain embodiments, the invention provides humanized antibodies or antigen binding ffagments that specifically bind to FOLR1 comprising the CDRs of FRl-49hu with up to four (i.e., 0, 1, 2, 3, or 4) substitutions. conservative amino acids by CDR. Thus, in certain embodiments, the invention provides humanized antibodies or antigen binding ffagments that specifically bind to a human folate receptor, wherein the antibody comprises: (a) a heavy chain CDR1 comprising CDR1 TNYWMY ( sec. with identification number: 66) or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, and / or a heavy chain CDR2 comprising AIYPGNSDTT (sec. with identification number:

<img file="AR080301A1_D0036.tif" />

67) ο a variant of this comprising 1, 2, 3, or 4 conservative amino acid substitutions and / or a heavy chain CDR3 comprising RHDYGAMDY (see, with identification number: 68) or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, and / or (b) a light chain CDR1 comprising RASENIYTNLA (see with no. of ident: 63) or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, and / or a light chain CDR2 comprising TASNLAD (see, with identification no .: 64) or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions and / or a light chain CDR3 comprising QHFWVSPYT (see, with identification number: 65) or a variant thereof comprising 1, 2, 3 , or 4 conservative amino acid substitutions.

In certain embodiments, the invention provides humanized antibodies or antigen binding fragments that specifically bind to FOLR1 comprising the CDRs of FRl-57hu with up to four (i.e., 0, 1, 2, 3, or 4) substitutions. Conservatives of amino acids by CDR. Thus, in certain embodiments, the invention provides humanized antibodies or antigen binding fragments that specifically bind to a human folate receptor 1, wherein the antibody comprises: (a) a heavy chain CDR1 comprising SSFGMH ( see with identification number: 72) or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and / or a heavy chain CDR2 comprising YISSGSSTIS (see. with no. of ident: 73) or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and / or a heavy chain CDR3 comprising EAYGSSMEY (see. with identification number: 74) or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and / or (b) a light chain CDR1 comprising RASQNINNNLH (see with no. of ident: 69) or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and / or a light chain CDR2 comprising YVSQSVS (see. with identification number: 70) or a variant thereof comprising 1,2, 3, or 4 conservative amino acid substitutions; and / or a light chain CDR3 comprising QQSNSWPHYT (see. with identification number: 71) or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions.

In certain embodiments, the invention provides humanized antibodies or antigen-binding fragments that specifically bind to FOLR1 comprising the CDRs of FRl-65hu with up to four (ie, 0, 1, 2, 3, or 4) substitutions. conservative

<img file="AR080301A1_D0037.tif" />

of amino acids by CDR. Thus, in certain embodiments, the invention provides humanized antibodies or antigen binding fragments that specifically bind to a human folate receptor, wherein the antibody comprises: (a) a heavy chain CDR1 comprising TSYTMH (sec with identification number: 78) or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and / or a heavy chain CDR2 comprising YINPISGYTN (sec. with no. of ident: 79) or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and / or a heavy chain CDR3 comprising GGAYGRKPMDY (sec. with identification number: 80) or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and / or (b) a light chain CDR1 comprising KASQNVGPNVA (sec. with no. of ident: 75) or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and / or a light chain CDR2 comprising SASYRYS (sec. with ID no .: 76) or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and / or a light chain CDR3 comprising QQYNSYPYT (sec. with identification number: 77) or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions.

Polypeptides comprising one of the individual light chains or heavy chains described herein, as well as polypeptides (eg, antibodies) comprising both a light chain and a heavy chain are also provided. The polypeptides of sec. with no. of ident: 4 and 6 comprise the variable domain of a Movl9hu heavy chain, and a Movl9hu heavy chain, respectively. Sec polypeptides with no. of ident: 10-13 comprise version 1.00 of the light chain variable domain, version 1.60 of the light chain variable domain, version 1.00 of light chain and version 1.60 of light chain of Movl9hu, respectively. The polypeptides of sec. with no. of ident: 42 and 46 comprise the variable domain of a heavy chain of FRl-21hu, and a heavy chain of FRl-21hu, respectively. The polypeptides of sec. with no. of ident: 41 and 45 comprise the light chain variable domain and a FRl-21hu light chain, respectively. The polypeptides of sec. with no. of ident: 97 and 113 comprise the variable domain of a heavy chain of FRl-48hu, and a heavy chain of FRl-48hu, respectively. The polypeptides of sec. with no. of ident: 96 and 112 comprise the variable domain of a light chain and a light chain of FRl-48hu, respectively. Polypeptides of

<img file="AR080301A1_D0038.tif" />

the sec. with no. of ident: 99 and 115 comprise the variable domain of a heavy chain of FRl-49hu, and a heavy chain of FRl-49hu, respectively. The polypeptides of sec. with no. of ident: 98 and 114 comprise the variable domain of a light chain and a light chain of FRl-49hu, respectively. The polypeptides of sec. with no. of ident: 101 and 117 comprise the variable domain of a heavy chain of FRl-57hu, and a heavy chain of FRl-57hu, respectively. The polypeptides of sec. with no. of ident: 100 and 116 comprise the variable domain of a light chain and a light chain of FRl-57hu, respectively. The polypeptides of sec. with no. of ident: 103 and 119 comprise the variable domain of a heavy chain of FRl-65hu, and a heavy chain of FRl-65hu, respectively. The polypeptides of sec. with no. of ident: 102 and 118 comprise the variable domain of a light chain and a light chain of FRI 65hu, respectively.

Polypeptides comprising: (a) a polypeptide having at least about 90% sequence identity with sec. with no. of ident: 4 or 6; and / or (b) a polypeptide that has at least about 90% sequence identity with sec. with no. of ident: 10-13. Polypeptides comprising: (a) a polypeptide having approximately 90% sequence identity with sec. with no. of ident: 42 or 46; and / or (b) a polypeptide that has at least about 90% sequence identity with sec. with no. of ident: 41 and 45. Polypeptides comprising: (a) a polypeptide having at least about 90% sequence identity with sec. with no. of ident: 97 or 113; I (b) a polypeptide that has at least about 90% sequence identity with sec. with no. of ident: 96 or 112. Polypeptides comprising: (a) a polypeptide having at least about 90% sequence identity with sec. with no. of ident: 99 or 115; and / or (b) a polypeptide that has at least about 90% sequence identity with sec. with no. of ident: 98 or 114. Polypeptides comprising: (a) a polypeptide having at least about 90% sequence identity with sec. with no. of ident: 101 or 117; and / or (b) a polypeptide that has at least about 90% sequence identity with sec. with no. of ident: 100 or 116. Polypeptides are also provided comprising: (a) a polypeptide having at least about

<img file="AR080301A1_D0039.tif" />

<img file="AR080301A1_D0040.tif" />

90% sequence identity with sec. with no. of ident: 103 or 119 and / or (b) a polypeptide having at least about 90% sequence identity with sec. with no. of ident: 102 or 118. In certain embodiments, the polypeptide comprises a polypeptide having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the sequence identity with sec. with no. of ident: 6, 4, 10-13, 41, 42, 45 or 46. Thus, in certain embodiments, the polypeptide comprises (a) a polypeptide that has at least about 95% sequence identity with the sec . with no. of ident: 4 6 6, and / or (b) a polypeptide having at least about 95% sequence identity with sec. with no. of ident: 10-13. In certain embodiments, the polypeptide comprises (a) a polypeptide that has at least about 95% sequence identity with sec. with no. of ident: 42 or 46, and / or (b) a polypeptide having at least about 95% sequence identity with sec. with no. of ident: 41 or 45. Polypeptides are also provided comprising: (a) a polypeptide having at least about 95% sequence identity with sec. with no. of ident: 97 or 113 and / or (b) a polypeptide having at least about 95% sequence identity with sec. with no. of ident: 96 or 112. Polypeptides comprising: (a) a polypeptide having at least about 95% sequence identity with sec. with no. of ident: 99 or 115; and / or (b) a polypeptide that has at least about 95% sequence identity with sec. with no. of ident: 98 or 114. Polypeptides comprising: (a) a polypeptide having at least about 95% sequence identity with sec. with no. of ident: 101 or 117; and / or (b) a polypeptide that has at least about 95% sequence identity with sec. with no. of ident: 100 or 116. Polypeptides comprising: (a) a polypeptide having at least about 95% sequence identity with sec. with no. of ident: 103 or 119; and / or (b) a polypeptide that has at least about 95% sequence identity with sec. with no. of ident: 102 or 118. In certain embodiments, the polypeptide comprises (a) a polypeptide having the amino acid sequence of sec. with no. of ident: 4; and / or (b) a polypeptide having the amino acid sequence of sec. with no. of ident: 10 or sec. with no. of ident: 11. In certain modalities, the

<img file="AR080301A1_D0041.tif" />

<img file="AR080301A1_D0042.tif" />

polypeptide comprises (a) a polypeptide having the amino acid sequence of sec. with no. of ident: 45; and / or (b) a polypeptide having the amino acid sequence of sec. with no. of ident: 46. In certain embodiments, the polypeptide comprises (a) a polypeptide having the amino acid sequence of sec. with no. of ident: 6; and / or (b) a polypeptide having the amino acid sequence of sec. with no. of ident: 12 ο sec. with no. of ident: 13. In certain embodiments, the polypeptide is an antibody and / or the polypeptide specifically binds to human folate receptor 1. In certain embodiments, the polypeptide is a humanized antibody that specifically binds to human folate receptor 1. For example, the invention provides a humanized antibody or antibody that specifically binds to a human FOLR1 comprising (a) a polypeptide having the amino acid sequence of sec. with no. of ident: 4; and (b) a polypeptide having the amino acid sequence of sec. with no. of ident: 10 or sec. with no. of ident: 11. In certain embodiments of the polypeptide comprising sec. with no. of ident: 4 is a heavy chain variable region. In certain embodiments, the polypeptide comprising sec. with no. of ident: 10 or 11 is a variable light chain region. The invention also provides a humanized antibody or antibody that specifically binds to a human FOLR1 comprising (a) a polypeptide having the amino acid sequence of sec. with no. of ident: 6; and (b) a polypeptide having the amino acid sequence of sec. with no. of ident: 12 or sec. with no. of ident: 13. The invention also provides a humanized antibody or antibody that specifically binds to a human FOLR1 comprising (a) a polypeptide having the amino acid sequence of sec. with no. of ident: 45, and (b) a polypeptide having the amino acid sequence of sec. with no. of ident: 46. The invention also provides a humanized antibody or antibody that specifically binds to a human FOLR1 comprising (a) a polypeptide having the amino acid sequence of sec. with no. of ident: 112; and φ) a polypeptide having the amino acid sequence of sec. with no. of ident: 113. The invention also provides a humanized antibody or antibody that specifically binds to a human FOLR1 comprising (a) a polypeptide having the amino acid sequence of sec. with no. of ident: 114; and (b) a polypeptide having the amino acid sequence of sec. with no. of ident: 115. The invention also provides a humanized antibody or antibody that specifically binds to a

<img file="AR080301A1_D0043.tif" />

Human FOLR1 comprising (a) a polypeptide having the amino acid sequence of sec. with no. of ident: 116; and (b) a polypeptide having the amino acid sequence of sec. with no. of ident: 117. The invention also provides a humanized antibody or antibody that specifically binds to a human FOLR1 comprising (a) a polypeptide having the amino acid sequence of sec. with no. of ident: 118; and (b) a polypeptide having the amino acid sequence of sec. with no. of ident: 119. In certain embodiments, the polypeptide having a certain percentage of the sequence identity with sec. with no. Ident: 4, 6, 10-13, 41, 42, 45, 46, 96-103 and 112-119 differs from sec. with no. Ident: 4, 6, 10-13, 41, 42, 45, 46, 96-103 and 112-119 for conservative amino acid substitutions only

In certain embodiments, the FOLR1 binding agent comprises, consists essentially of, or consists of an anti-FOLRl antibody selected from the group consisting of Movl9hu, FR-1-21, FR1-48, FR1-49, FR1- antibodies. 57, and FR1-65.

In certain embodiments, the Movl9hu antibody is encoded by plasmids deposited in the American Type Culture Collection (ATCC) on April 7, 2010 and having the ATCC deposit numbers PTA-10772 and PTA-10773 or 10774.

In certain embodiments, the FR-1-21 antibody is encoded by the ATCC plasmids deposited on April 7, 2010, and the deposit denomination numbers PTA-10775 and 10776 are assigned.

In certain embodiments, humanized antibodies bind to FOLR1 with substantially the same affinity as the Movi9 chimeric antibody. The affinity or greediness of an antibody for an antigen can be determined experimentally by any suitable method well known in the art, for example, flow cytometry, enzyme linked immunosorbent assay (ELISA), or radioimmunoassay (RIA), or kinetics (for example, BIACORE ™ analysis). Direct binding assays, as well as competitive binding assay formats can easily be used. (See, for example, Berzofsky, et al., Antibody-Antigen Interactions, in Fundamental Immunology, Paul, WE, Ed., Raven Press: New York, NY (1984); Kuby, Janis Immunology, WH Freeman and Company: New York , NY (1992); and the methods described in this document. The measured affinity of a particular antigen-antibody interaction may vary if measured under different conditions (eg, sai concentration, pH, temperature). In this way, the measurements of the

<img file="AR080301A1_D0044.tif" />

affinity and other antigen binding parameters (for example, KD or Kd, Κ, η, Kff) are prepared with standardized antibody and antigen solutions, and a standardized buffer, as is known in the art and such as the buffer is described in this document.

In one aspect, binding assays can be performed by flow cytometry in cells expressing the FOLR1 antigen on the surface. For example, FOLR1 positive cells such as SKOV3 horn were incubated with different concentrations of anti-FOLRl antibodies using 1 x 10 cells per sample in 100μ1 of FACS buffer (RPMI-1640 medium supplemented with 2% of normal camera sera). Then, the cells were sedimented, washed, and incubated for 1 h with 100 µΐ of anti-mouse in FITC-conjugated chamber or the anti-human IgG antibody in chamber (such as is obtained from, for example, the Jackson Laboratory, 6 pg / ml in FACS buffer). The cells were sedimented again, washed with FACS buffer and resuspended in 200 μΐ of PBS with 1% formaldehyde. Samples were acquired, for example, by a FACSCalibur flow cytometer with the HTS multi-well dispenser and analyzed by CellQuest Pro (all from BD Biosciences, San Diego, USA). For each sample, the average fluorescence intensity for FL1 (MFI) was exported and plotted against the antibody concentration in a semi-log representation to generate a binding curve. A sigmoidal dose-response curve was adjusted for the binding curves and EC50 values ​​are calculated using Taies programs such as GraphPad Prism v4 with the default parameters (GraphPad software, San Diego, CA). EC50 values ​​can be used as a measure for the apparent dissociation constant Kd or KD for each antibody.

Monoclonal antibodies can be prepared by hybridoma methods, such as those described by Köhler and Milstein (1975) Nature 256: 495. Through the hybridoma method, a mouse, hamster, or other appropriate host animal, is immunized as described above to induce the production of antibodies by lymphocytes that can be specifically bound to an immunizing antigen. Lymphocytes can also be immunized in vitro. Following immunization, lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol, to form hybridoma cells that can be selected after lymphocytes and non-fused myeloma cells. The hybridomas that produce the

<img file="AR080301A1_D0045.tif" />

Monoclonal antibodies specifically directed against a selected antigen as determined by immunoprecipitation, immunoblotting, or by an in vitro binding assay (eg, radioimmunoassay (RIA); enzyme linked immunosorbent assay (ELISA)), can then be propagated either in vitro culture using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academie Press, 1986) or live as ascites tumors in an animal. Monoclonal antibodies can be purified after the culture medium or asettic liquid as described above for polyclonal antibodies.

On the other hand, monoclonal antibodies can also be prepared by recombinant DNA methods as described in US Pat. 4,816,567. Polynucleotides encoding a monoclonal antibody are isolated from mature B cells or hybridoma cells, such as by RT-PCR using specifically oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody. , and its sequence is determined by conventional procedures. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors, which, when transfected into host cells, such as E. coli cells, simian COS cells, Chinese hamster ovary cells ( CHO), or myeloma cells that otherwise do not produce immunoglobulin protein monoclonal antibodies are generated by host cells. Also, recombinant monoclonal antibodies or fragments thereof of the suitable species can be isolated from the phage display libraries that express the CDRs of the suitable species as described (McCafferty et al., 1990, Nature, 348: 552-554; Clackson et al., 1991, Nature, 352: 624-628; and Marks et al., 1991, J. Mol. Biol., 222: 581-597).

The polynucleotide (s) encoding a monoclonal antibody can also be modified in a number of different ways by recombinant DNA technology to generate alterative antibodies. In some embodiments, the constant domains of the light and heavy chains of, for example, a mouse monoclonal antibody can be substituted 1) by those regions of, for example, a human antibody to generate a chimeric antibody or 2) by a non-immunoglobulin polypeptide to generate a fusion antibody. In some embodiments, constant regions are truncated or deleted to generate the convenient antibody fragment of a

Îw \ &lt;Af. Ρ · 2χ monoclonal antibody. Targeted or high density site mutagenesis of the variable region can be used to optimize specificity, affinity, etc. of a monoclonal antibody.

In some embodiments, the monoclonal antibody against human FOLR1 is a humanized antibody. In certain embodiments, such antibodies are used therapeutically to reduce antigenicity and HAMA (human anti-mouse antibody) responses when administered to a human subject.

Methods for modifying, humanizing or coating nonhuman or human antibodies can also be used and are well known in the art. A humanized, coated or similarly modified antibody may have one or more amino acid residues from a source that is not human, for example, but is not limited to mouse, rat, rabbit, non-human primate or other mammal. These non-human amino acid residues are replaced by residues that are often referred to as import residues, which are normally taken from an import, constant or other variable domain of a known human sequence.

Such imported sequences can be used to reduce immunogenicity or reduce, improve or modify binding, affinity, in the rate, out of the rate, greediness, specificity, half-life, or any other suitable characteristic, as is known in The technique. In general, CDR residues are directly and more substantially involved in influencing binding to FOLR1. Therefore, part or all of the non-human or human CDR sequences are maintained while the nonhuman sequences of the variable and constant regions can be replaced with human or other amino acids.

The antibodies can optionally be humanized, coated, modified or also the human antibodies are modified with high affinity retention for the FOLR1 antigen and other favorable biological properties. To achieve this goal, humanized (or human) or modified anti-FOLRl antibodies and antibodies coated by an optional process of parental sequence analysis and various humanized and modified conceptual products can be prepared using three-dimensional models of the parental sequences, modified, and humanized Three-dimensional models of immunoglobulin are commonly available and are familiar to those with knowledge of the technique. The computer programs that illustrate and show the

<img file="AR080301A1_D0046.tif" />

Probable three-dimensional conformation structures of the selected sequences of the candidate immunoglobulin are available. The inspection of these samples allows the analysis of the probable role of the residues in the functioning of the candidate immunoglobulin sequence, that is, the analysis of the residues that influence the ability of the candidate immunoglobulin to bind its antigen, such as FOLR1. . In this way, frame residues (FR) can be selected and combined from the imported and consensus sequences so that the convenient characteristic of the antibody is achieved, such as the increased affinity for the target antigen (s).

Humanization, coating or modification of the antibodies of the present invention can be performed using any known method, such as but not limited to those described in Winter (Jones et al., Nature 321: 522 (1986); Riechmann et al. , Nature 332: 323 (1988); Verhoeyen et al., Science 239: 1534 (1988)), Sims et al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196: 901 (1987), Carter et al., Proc. Nati Acad. Sci. United States. 89: 4285 (1992); Presta et al., J. Immunol. 151: 2623 (1993), U.S. Pat. with no. 5,639,641; 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; 4,816,567; PCT /: US98 / 16280; US96 / 18978; US91 / 09630; US91 / 05939; US94 / 01234; GB89 / 01334; GB91 / 01134; GB92 / 01755; WO90 / 14443; WO90 / 14424; W090 / 14430; EP 229246; 7,557,189; 7,538,195; and 7,342,110, each of which is incorporated herein in its entirety as a reference, which includes the references cited herein.

In certain alternative modalities, the antibody to FOLR1 is a human antibody. Human antibodies can be prepared directly by various techniques known in the art. Immortalized human B lymphocytes that are immunized in vitro or isolated from an immunized individual that produces an antibody directed against a target antigen can be generated (See, for example, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., 1991, J. Immunol, 147 (1): 86-95; and U.S. Pat. 5,750,373). Also, the human antibody can be selected from a phage library, where that phage library expresses human antibodies, such as described, for example, in Vaughan et al., 1996, Nat. Biotech., 14: 309-314, Sheets and others, 1998, Proc. Nati Acad. Sci., 95: 6157-6162, Hoogenboom and Winter, 1991, J. Mol. Biol., 227: 381, and Marks and others,

<img file="AR080301A1_D0047.tif" />

1991, J. Mol. Biol., 222: 581). Techniques for the generation and use of phage antibody libraries are also described in US Pat. nums 5,969,108; 6,172,197; 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915; 6,593,081; 6,300,064; 6,653,068; 6,706,484; and 7,264,963; and Rothe and others, 2007. 2007, J. Mol. Bio., Doi: 10.1016 / j.jmb.2007.12.018 (which are incorporated in their entirety as reference). Affinity maturation strategies and chain exchange strategies (Marks et al., 1992, Bio / Technology 10: 779-783, fully incorporated as reference) are known in the art and can be used to generate antibodies High affinity humans.

Humanized antibodies can also be prepared in transgenic mice that contain human immunoglobulin loci that are capable of immunizing the entire repertoire of human antibodies in the absence of endogenous immunoglobulin production. This approach is described in US Pat. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.

This invention also includes bispecific antibodies that specifically recognize a human folate receptor 1. Bispecific antibodies are antibodies that are capable of specifically recognizing and binding at least two different epitopes. The different epitopes can be either within the same molecule (for example, the same human folate receptor 1) or in different molecules such that, for example, both antibodies can specifically recognize and bind a human folate receptor 1, as well as , for example, 1) a leukocyte effector molecule, such as a T cell receptor (for example CD3) or the Fc receptor (for example, CD64, CD32, or CD 16) or 2) a cytotoxic agent as described in detail below.

Illustrative bispecific antibodies can bind to two different epitopes, at least one of these originates from a polypeptide of the invention. On the other hand, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm that binds to a leukocyte activation molecule such as a T cell receptor molecule (e.g., CD2, CD3, CD28, or B7), or Fc receptors for IgG so as to concentrate the cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells that express a particular antigen. These antibodies have an antigen-binding arm and an arm that binds a cytotoxic agent or radionuclide.

<img file="AR080301A1_D0048.tif" />

chelator, such as EOTUBE, DTPA, DOTA, or TETA. Techniques for preparing bispecific antibodies are common in the art (Millstein et al., 1983, Nature 305: 537-539; Brennan et al., 1985, Science 229: 81; Suresh et al., 1986, Methods in Enzymol. 121: 120 ; Traunecker et al., 1991, EMBO J. 10: 3655-3659; Shalaby et al., 1992, J. Exp. Med. 175: 217-225; Kostelny et al., 1992, J. Immunol. 148: 1547-1553; Gruber et al., 1994, J. Immunol. 152: 5368; and U.S. Patent 5,731,168). Antibodies with more than two valences are also contemplated. For example, trypspecific antibodies can be prepared (Tufi et al., J. Immunol. 147: 60 (1991)). Thus, in certain embodiments the antibodies to FOLR1 are multispecific.

In certain embodiments, an antibody fragment is provided to, for example, increase tumor penetration. Different techniques are known for the production of antibody fragments. Traditionally, these fragments are obtained through proteolytic digestion of intact antibodies (eg, Morimoto et al., 1993, Journal of Biochemical and Biophysical Methods 24: 107-117; Brennan et al., 1985, Science, 229: 81) . In certain embodiments, antibody fragments are produced by recombination. Fab, Fv, and scFv antibody fragments can be expressed and secreted from E. coli cells or other host cells, thereby allowing the production of large amounts of these fragments. Such antibody fragments can also be isolated from antibody libraries in phages discussed above. The antibody fragment may also be of the linear antibodies as described in US Pat. 5,641,870, for example, and may be monospecific or bispecific. Other techniques for the production of antibody fragments will be apparent to the experienced professional.

In accordance with the present invention, the techniques can be adapted for the production of single chain antibodies specific for human folate receptor 1 (see US Patent No. 4,946,778). In addition, the methods can be adapted for the construction of Fab expression libraries (Huse, et al., Science 246: 1275-1281 (1989)) to allow rapid and efficient identification of the Fab fragments of the monoclonal with the specificity suitable for a folate receptor 1, or derivatives, fragments, analogs or homologs thereof. Antibody fragments can be produced by methods in the art that include but are not limited to: (a) an F (ab ') 2 fragment produced by the digestion of the pepsin from an antibody molecule;

<img file="AR080301A1_D0049.tif" />

(b) a Fab fragment generated by the reduction of the disulfide bridges of an F (ab ') 2 fragment, (c) a Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent, and (d) Fv fragments.

This may be convenient, in addition, especially in the case of antibody fragments, to modify an antibody in order to increase its serum half-life. This can be achieved, for example, by the incorporation of a salvage receptor binding epitope into the antibody fragment by the mutation of the appropriate region in the antibody fragment or by the incorporation of the epitope into a peptide tag that is then fused with the antibody fragment at each end or in the medium (for example, by synthesis of DNA or peptides).

Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently bound antibodies. Such antibodies have been proposed, for example, to direct immune cells to unwanted cells (US Patent No. 4,676,980). It is contemplated that the antibodies can be prepared in vitro by methods known in the chemistry of synthetic proteins, including those involving crosslinking agents. For example, immunotoxins can be constructed by a disulfide exchange reaction or by the formation of a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

For the purposes of the present invention, it will be appreciated that the modified antibodies may comprise any type of variable region that is provided for the association of the antibody with the polypeptides of a human FOLR1. In this sense, the variable region can comprise or be derived from any type of mammal that can be induced to accumulate a humoral response and generate immunoglobulins against the convenient antigen associated with the tumor. As such, the variable region of the modified antibodies can be, for example, of human, murine origin, non-human primates (eg, cynomolgus monkeys, macaques, etc.) or lupine. In some embodiments, both the variable regions and the constants of the modified immunoglobulins are human. In other embodiments, the variable regions of compatible antibodies (usually derived from a non-human source) can be modified or adapted specifically to improve binding properties or reduce the immunogenicity of the molecule. With

<img file="AR080301A1_D0050.tif" />

In this regard, the variable regions useful in the present invention can be humanized or otherwise altered through the inclusion of imported amino acid sequences.

In certain modalities, the variable domains, both in heavy and light chains, are altered at least by the partial replacement of one or more of the CDRs and, if necessary, by the partial replacement of the framework region and sequence change. Although the CDRs may be derived from an antibody of the same class or even subclass of the antibody from which the framework regions are derived, it is anticipated that the CDRs may be derived from an antibody of different class and, in certain embodiments of an antibody of A different species. It may not be necessary to replace all the CDRs with the complete CDRs of the donor variable region to transfer the antigen binding capacity from one variable domain to another. Rather, it would only be necessary to transfer those wastes that are necessary to maintain the activity of the antigen binding site. Temendo account the explanations set forth in US patents. No. 5,585,089, 5,693,761 and 5,693762, will be well within the competence of people with knowledge of the technique, whether it is to perform the ratine experimentation as obtaining by trial and error of praebas a functional antibody with reduced immunogenicity.

Despite alterations in the variable region, people skilled in the art will appreciate that the modified antibodies of this invention may comprise the antibodies (for example, full-length antibodies or immunoreactive fragments thereof) in which at least one fffaction of one or more of the domains of the constant region has been removed or otherwise altered to provide convenient biochemical characteristics such as the increased location of the tumor or to reduce serum half-life when compared to an antibody of approximately the same immunogenicity comprising a constant native region or without alteration. In some embodiments, the constant region of the modified antibodies may comprise a constant human region. The modifications of the constant region compatible with this invention include the additions, deletions or substitutions of one or more amino acids in one or more domains. That is, the modified antibodies described herein may comprise alterations or modifications in one or more of the three heavy chain constant domains (CHI, CH2 or CH3) and / or in the light chain constant domain (CL). In some modalities, modified constant regions are contemplated where one or more domains are partially or totally

<img file="AR080301A1_D0051.tif" />

removed. In some embodiments, the modified antibodies may comprise deleted domain constructs or variants where the entire CH2 domain has been removed (ACH2 constructs). In some embodiments, the omitted domain of the constant region will be replaced by a short amino acid spacer (eg, 10 residues) that provides some of the molecular flexibility normally given by the absent constant region.

In addition to its configuration, it is known in the art that the constant region mediates several effector functions. For example, the binding of the complement component CI to the antibodies activates the complement system. Complement activation is important in the opsonization and lysis of cell pathogens. Complement activation also stimulates the inflammatory response and may also be involved in autoimmune hypersensitivity. In addition, antibodies bind to cells through the Fc region, with an Fc receptor site in the Fc region of antibody that binds to an Fc receptor (FcR) in a cell. There are a number of Fc receptors that are specific for different classes of antibodies, including IgG (gamma receptors), IgE (età receptors), IgA (alpha receptors) and IgM (mu receptors). The binding of the antibodies to the Fc receptors on the cell surface triggers a number of important and diverse biological responses that include encompassing and destroying the particles coated with antibodies, eliminating immune complexes, lysis of the coated target cells with antibodies by cytolytic cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), the release of inflammatory mediators, transfer to the placenta and control the production of immunoglobulins.

In certain embodiments, FOLR1 binding antibodies provide altered effector functions that, in turn, affect the biological profile of the administered antibody. For example, the elimination or inactivation (through point mutations or other method) of a constant region domain can reduce the binding of the Fc receptor of the circulating modified antibodies thereby increasing the location of the tumor. In other cases, it could be that the modifications of the constant region, consistent with this invention, moderate complement binding and thus reduce life, however, other modifications of the constant region can be used to eliminate disulfide bonds or the portions of oligosaccharides that allow localization to be improved

<img file="AR080301A1_D0052.tif" />

due to the increased specificity of the antigen or the flexibility of the antibody. Similarly, modifications of the constant region according to this invention can be easily prepared by well-known molecular or biochemical engineering techniques within the scope of the person with knowledge in the art.

In certain embodiments, a FOLR1 binding agent that is an antibody does not have one or more effector functions. For example, in some embodiments, the antibody has no antibody-dependent cellular cytotoxicity (ADCC) activity and / or has no complement-dependent cytotoxicity (CDC) activity. In certain embodiments, the antibody does not bind to an Fc receptor and / or complement factors. In certain embodiments, the antibody has no effector function.

It may be noted that, in certain embodiments, the modified antibodies can be modified to fuse the CH3 domain directly to the hinge region of the respective modified antibodies. In other constructs it would be convenient to provide a spacer peptide between the hinge region and the modified CH2 and / or the CH3 domains. For example, compatible constructs could be expressed where the CH2 domain is deleted and the remaining CH3 domain (modified or unmodified) joins the hinge region with a 5-20 amino acid spacer. Such a separator can be added, for example, to ensure that the regulatory elements of the constant domain remain free and accessible or that the hinge region remains flexible. However, it should be noted that amino acid spacers may, in some cases, prove to be immunogenic and induce an unwanted immune response against the construct. Consequently, in certain embodiments, any spacer added to the construct will be relatively non-immunogenic, or even omitted altogether, to maintain the convenient biochemical qualities of the modified antibodies.

In addition to the elimination of the entire domains of the constant region, it will be appreciated that the antibodies of the present invention can be provided by partial removal or substitution of a few or even a single amino acid. For example, the mutation of a single amino acid in certain areas of the CH2 domain could be sufficient to substantially reduce the binding of Fc and thereby increase the location of the tumor. Similarly, it might be convenient to simply remove that part of one or more domains of the constant region that control the effector function (for example, the C1Q binding of the complement) to be modulated. Such eliminations

<img file="AR080301A1_D0053.tif" />

Partial constant regions may improve the selected characteristics of the antibody (serum half-life), while leaving other convenient functions associated with the domain of the individual's constant region intact. In addition, as mentioned above, the constant regions of the described antibodies can be modified through mutation or substitution of one or more amino acids that improve the profile of the resulting construct. In this sense it would be possible to interrupt the activity that is provided by a conserved binding site (eg, Fc binding), while substantially maintaining the configuration and immunogenic profile of the modified antibody. The determined modalities may comprise the addition of one or more amino acids of the constant region to improve the convenient characteristics, as well as decrease or increase the effector function or provide the link for more cytotoxin or carbohydrate. In such modalities it may be convenient to insert or reproduce specific sequences derived from the selected domains of the constant region.

The present invention further encompasses variants and equivalents that are substantially homologous to the chimeric, humanized and human antibodies, or antibody fragments thereof, set forth herein. These may contain, for example, mutations with conservative substitution, that is, the substitution of one or more amino acids with similar amino acids. For example, conservative substitution refers to the substitution of an amino acid with another within the same general class as horn, for example, an acidic acid with another acidic acid, a basic amino acid with another basic amino acid or a neutral amino acid with another amino acid neutral. What is intended by a conservative amino acid substitution is well known in the art.

The polypeptides of the present invention may be recombinant polypeptides, natural polypeptides, or synthetic polypeptides comprising an antibody, or fragment thereof, against a human FOLR1. It will be recognized in the art that some amino acid sequences of the invention can be varied without significant effect on the structure or function of the protein. Thus, the invention further includes variations of polypeptides that show substantial activity or that include regions of an antibody or fragment thereof, against a human folate receptor protein. Mutant taies include eliminations, insertions, inversions, repetitions and substitutions types.

<img file="AR080301A1_D0054.tif" />

Polypeptides and analogs can also be modified to contain additional chemical portions that are not normally part of the protein. Those derivative portions can improve solubility, biological half-life or protein absorption. The portions may also reduce or eliminate any convenient side effects of the proteins and the like. An overview of those portions can be found in REMINGTON'S PHARMACEUTICAL SCIENCES, 20th ed., Mack Publishing Co., Easton, PA (2000).

The isolated polypeptides described herein can be produced by any suitable method known in the art. Such methods range from direct synthetic methods for the protein to the construction of the DNA sequence encoding the isolated polypeptide sequences and the expression of those sequences in a suitable transformed host. In some embodiments, a DNA sequence is constructed by recombinant technology by the isolation or synthesis of a DNA sequence encoding a wild-type protein of interest. Optionally, the sequence can be mutated by site-specific mutagenesis to provide functional analogs thereof. See, for example, Zoeller et al., Proc. Nati Acad. Sci. United States 81: 5662-5066 (1984) and US Pat. no. 4,588,585.

In some embodiments, a DNA sequence encoding a polypeptide of interest may be constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the convenient polypeptide and select those codons that are favored in the host cell in which the recombinant polypeptide of interest may be produced. Standard methods can be applied to synthesize an isolated polynucleotide sequence encoding an isolated polypeptide of interest. For example, a complete amino acid sequence can be used to construct a post-translated gene. In addition, a DNA oligomer containing a nucleotide sequence encoding the particular isolated polypeptide can be synthesized. For example, several small oligonucleotides encoding portions of the convenient polypeptide can then be synthesized and ligated. Individual oligonucleotides usually contain 5 'or 3' protruding ends for complementary assembly.

Once assembled (by synthesis, site-directed mutagenesis or other method), the polynucleotide sequences encoding a particular isolated polypeptide of interest

<img file="AR080301A1_D0055.tif" />

they may be inserted into an expression vector and operably linked to an appropriate expression control sequence for protein expression in a convenient host. Correct assembly can be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host. As is well known in the art, to obtain high levels of expression of a transfected gene in a host, the gene must be operably linked to the transcription control and translation expression sequences that are functional in the chosen host. expression.

In certain embodiments, recombinant expression vectors are used to amplify and express the antibodies encoding the DNA, or fragments thereof, against human FOLR1. Recombinant expression vectors are replicable DNA constructs that have fragments of synthetic DNA or cDNA derivatives that encode a polypeptide chain of an anti-FOLRl antibody, or fragment thereof, operably linked to suitable transcription or translation regulatory elements derived from genes of mammals, microbes, viruses or insects. A transcriptional unit in general comprises an assembly of (1) a genetic element or elements that have a regulatory role in gene expression, for example, transcriptional promoters or enhancers, (2) a structural or coding sequence, which is transcribed to MRNA and translates into protein, and (3) appropriate transcription and initiation of translation and termination sequences, as described in detail below. Such regulatory elements may include an operator sequence to control transcription. The ability to replicate in a host is usually conferred by an origin of replication, and a selection gene can also be incorporated to facilitate the recognition of transformants. DNA regions are operatively linked when they are functionally related to each other. For example, the DNA of a signal peptide (secretion leader) is operably linked to the DNA for a polypeptide if it is expressed as a precursor that participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is placed to allow translation. Structural elements intended for use in yeast expression systems include a leader sequence that allows extracellular secretion of the protein translated by a cell

<img file="AR080301A1_D0056.tif" />

Guest. On the other hand, where the recombinant protein is expressed without a leader or transport sequence, it can include an N-terminal methionine residue. This residue may optionally be cleaved from the expressed recombinant protein to provide a final product.

The choice of the expression control sequence and the expression vector may depend on the host choice. A wide variety of host / expression vector combinations can be employed. Useful expression vectors for eukaryotic hosts, include, for example, vectors comprising the control sequences of the SV40 expression, bovine papillomavirus, adenovirus and cytomegalovirus. Useful expression vectors for bacterial hosts include the known bacterial plasmids, such as plasmids from Escherichia coli, including pCR 1, pBR322, pMB9 and derivatives thereof, plasmids from the widest range of hosts, taies as Ml3 and phage-like phage DNA. single chain

Suitable host cells for the expression of a FOLR1 binding polypeptide or antibody (or a FOLR1 protein to use as an antigen) include prokaryotes, yeasts, insects or eukaryotic upper cells under the control of appropriate promoters. Prokaryotes include gram negative or gram positive organisms, for example, E. coli or bacilli. Upper eukaryotic cells include established cell lines of mammalian origin, as described below. Cell-free translation systems could also be used. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast and mammalian cell hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985), the relevant description is incorporated herein. as reference. Additional information on protein production methods, which includes antibody production, can be found, for example, in US Pat. No. 2008/0187954, U.S. Pat. no. 6,413,746 and 6,660,501, and the international patent publication WO No. 04009823, which are incorporated herein in its entirety as reference.

Different mammalian or insect cell culture systems are also advantageously used to express the recombinant protein. The expression of recombinant proteins in mammalian cells can be done because such proteins are generally correctly folded, properly modified and

<img file="AR080301A1_D0057.tif" />

fully functional Examples of suitable mammalian host cell lines include HEK-293 and HEK-293T, COS-7 lines of monkey kidney cells, described by Gluzman (cell 23: 175, 1981), and other cell lines that include , for example, L, C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHK cell lines. Mammalian expression vectors may comprise non-transcribed elements such as an origin of replication, a suitable promoter and enhancer bound to the gene to be expressed, and other non-transcribed sequences flanking 5 'or 3', and untranslated sequences. 'or 3', ribosome binding sites, as necessary, a polyadenylation site, donor site and acceptor sites, and transcriptional termination sequences. Baculovirus systems for the production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio / Technology 6:47 (1988).

Proteins produced by a transformed host can be purified according to any suitable method. Such standard methods include chromatography (for example, single-column column chromatography, affinity and size), centrifugation, differential solubility, or by any other standard technique for protein purification. The affinity tags such as hexahistidine, binding domain to maliosa, the influenza coating sequence and glutathione-Stransferase can be attached to the protein to allow easy purification by passing through a suitable affinity column. Isolated proteins can also be physically characterized by techniques such as proteolysis, nuclear magnetic resonance and X-ray crystallography.

For example, the supernatants of the systems that secrete the recombinant protein in the culture media can first be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a suitable purification matrix. On the other hand, an anion exchange resin can be used, for example, a matrix or substrate having diethylaminoethyl pendant groups (DEAE). The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly used in protein purification. On the other hand, a cation exchange stage can be used. Suitable cation exchangers include various insoluble matrices comprising the sulfopropyl or carboxymethyl cellulose groups. Finally, one or more stages of

<img file="AR080301A1_D0058.tif" />

High performance reverse phase liquid chromatography (RP-HPLC) using RP-HPLC hydrophobic media, for example, silica gel having methyl or other aliphatic groups hanging, can also be used to purify a FOLR1 binding agent. Some or all of the preceding purification steps, in various combinations, can also be used to provide a homogeneous recombinant protein.

The recombinant protein produced in the bacterial culture can be isolated, for example, by initial extraction from the cell sediments, followed by one or more concentration steps, salt precipitation, aqueous ion exchange chromatography or size exclusion . High performance liquid chromatography (HPLC) can be used for the final stages of purification. The microbial cells employed in the expression of a recombinant protein can be broken by any convenient method, including freeze-thaw cycle, sonication, mechanical breakage, or the use of cell lysing agents.

Methods known in the art for purifying antibodies and other proteins also include, for example, those described in the publication of US Pat. no. 2008/0312425, 2008/0177048 and 2009/0187005, which are incorporated herein in their entirety as reference.

In certain embodiments, the FOLR1 binding agent is a polypeptide that is not an antibody. A variety of methods for identifying and producing polypeptides that are not antibodies that bind with high affinity to a protein target are known in the art. See, for example, Skerra, Curr. Opin. Biotechnol., 18: 295-304 (2007), Hosse et al., Protein Science, 15: 14-27 (2006), Gill et al., Curr. Opin. Biotechnol., 17: 653-658 (2006), Nygren, FEBS J., 275: 2668-76 (2008), and Skerra, FEBS J., 275: 2677-83 (2008), which are incorporated into this document in full as reference. In certain embodiments, phage display technology has been used to identify / produce the FOLR1 binding polypeptide. In certain embodiments, the polypeptide comprises a protein scaffold of a type selected from the group consisting of protein A, a lipocalin, a fribronectin domain, a consensus repetitive domain of ankirine, and thioredoxin.

In some embodiments, the agent is a non-protein molecule. In certain embodiments, the agent is a small molecule. Combinatorial chemistry libraries and useful techniques in the identification of non-protein binding agents to FOLR1 are

<img file="AR080301A1_D0059.tif" />

known by those people with knowledge in the technique. See, for example, Kennedy et al., J. Comb. Chem, 10: 345-354 (2008), Dolle et al., J. Comb. Chem., 9: 855-902 (2007), and Bhattacharyya, Curr. Med. Chem., 8: 1383-404 (2001), which are incorporated herein in their entirety as reference. In certain embodiments, in addition, the agent is a carbohydrate, a glycosaminoglycan, a glycoprotein, or a proteoglycan.

In certain embodiments, the agent is an aptamer nucleic acid. Aptamers are polynucleotide molecules that have been selected (for example, from randomized or mutagenized mixtures) based on their ability to bind to another molecule. In some embodiments, the aptamer comprises a DNA polynucleotide. In certain alternative modalities, the aptamer comprises an RNA polynucleotide. In certain embodiments, the aptamer comprises one or more modified nucleic acid residues. Methods of generation and detection of nucleic acids suitable for binding proteins are well known in the art. See, for example, US Pat. no. 5,270,163, U.S. Pat. no. 5,683,867, U.S. Pat. no. 5,763,595, U.S. Pat. no. 6,344,321, U.S. Pat. no. 7,368,236, U.S. Pat. no. 5,582,981, U.S. Pat. no. 5,756,291, U.S. Pat. no. 5,840,867, U.S. Pat. No. 7,312,325, U.S. Pat. no. 7,329,742, international publication of patent no. WO 02/077262, International Patent Publication No. WO 03/070984, publication of US patent application no. 2005/0239134, publication of US patent application no. 2005/0124565, and publication of US patent application no. 2008/0227735, which are incorporated in this document as a reference in its entirety.

III. Immunoconjugates

The present invention is also directed to the conjugates (also referred to herein as immunoconjugates), which comprises anti-FOLR1 antibodies, antibody fragments, functional equivalents, improved antibodies and their aspects as described herein, attached or conjugated with a cytotoxin (drug) or profàrmaco. Thus, in a certain embodiment, the invention provides an immunoconjugate comprising a humanized antibody or an antigen-binding fragment thereof that specifically binds to a human folate receptor 1, wherein

<img file="AR080301A1_D0060.tif" />

the antibody comprises: (a) a heavy chain CDR1 comprising GYFMN (sec. with identification number: 1); a heavy chain CDR2 comprising RIHPYDGDTFYNQXaaiFXaa2Xaa3 (sec. with ID number: 56); and a heavy chain CDR3 comprising YDGSRAMDY (sec. with identification number: 3); and (b) a light chain CDR1 comprising KASQSVSFAGTSLMH (sec. with ID no .: 7); a light chain CDR2 comprising RASNLEA (sec. with no. of ident: 8); and a light chain CDR3 comprises QQSREYPYT (sec. with identification number: 9); where Xaai is selected from K, Q, H and R; Xaa₂ is selected from Q, H, N, and R, and Xaa3 is selected from G, E, T, S, A and V. In certain embodiments, the antibody is the Movl9hu antibody, which is the antibody described above comprising the CDR2 RIHPYDGDTFYNQKFQG (sec. With identification number: 2) heavy chain. In other embodiments, the antibody is FR121 and comprises (a) a heavy chain CDR1 comprising SSYGMS (sec. With ID no .: 30); a heavy chain CDR2 comprising TISSGGSYTY (sec. with no. of ident: 31); and / or a heavy chain CDR3 comprising DGEGGLYAMDY (sec. with identification number: 32); and (b) a light chain CDR1 comprising KASDHINNWLA (sec. with identification number: 27); a light chain CDR2 comprising GATSLET (sec. with identification number: 28); and a light chain CDR3 comprising QQYWSTPFT (sec. with identification number: 29). In other embodiments, the antibody is FRI-48 and comprises: (a) a heavy chain CDR1 comprising TNYWMQ (sec. With no. of ident: 60); a heavy chain CDR2 comprising AIYPGNGDSR (sec. with identification no .: 61); and / or a heavy chain CDR3 comprises RDGNYAAY (sec. with identification number: 62), and / or (b) a light chain CDR1 comprises RASENIYSNLA (sec. with identification number: 57); a light chain CDR2 comprising AATNLAD (sec. with identification number: 58); and a light chain CDR3 comprising QHFWASPYT (sec. with identification number: 59). In other embodiments, the antibody is FR149 and comprises: (a) a heavy chain CDR1 comprising TNYWMY (sec. With ID no .: 66); a heavy chain CDR2 comprising AIYPGNSDTT (sec. with identification number: 67); and / or a heavy chain CDR3 comprising RHDYGAMDY (sec. with identification number: 68); and / or (b) a light chain CDR1 comprising RASENIYTNLA (sec. with identification number: 63); a light chain CDR2 comprising TASNLAD (sec. with no. of ident: 64); and a light chain CDR3 comprising QHFWVSPYT (sec. with ID no .: 65). In other embodiments, the antibody is FRI60

<img file="AR080301A1_D0061.tif" />

and comprises: (a) a heavy chain CDR1 comprising SSFGMH (sec. with identification no .: 72); a heavy chain CDR2 comprising YISSGSSTIS (sec. with identification number: 73); and / or a heavy chain CDR3 comprises EAYGSSMEY (sec. with identification no .: 74); and / or (b) a light chain CDR1 comprising RASQNINNNLH (sec. with identification number: 69); a light chain CDR2 comprising YVSQSVS (sec. with identification number: 70); and a light chain CDR3 comprising QQSNSWPHYT (sec. with no. of ident: 71). In still another embodiment, the antibody is FR1-65 and comprises: (a) a heavy chain CDR1 comprising TSYTMH (sec. With ID no .: 78); a heavy chain CDR2 comprising YINPISGYTN (sec. with identification no .: 79); and / or a heavy chain CDR3 comprising GGAYGRKPMDY (sec. with identification number: 80); and / or (b) a light chain CDR1 comprising KASQNVGPNVA (sec. with identification number: 75); a light chain CDR2 comprising SASYRYS (sec. with no. of ident: 76); and a light chain CDR3 comprises QQYNSYPYT (sec. with ID no .: 77).

Suitable drugs or pro-drugs are known in the art. In certain embodiments, drugs or pro-drugs are cytotoxic agents. The cytotoxic agent used in the cytotoxic conjugate of the present invention can be any compound that results in the death of a cell, or induces cell death, or in some way decreases cell viability, and includes, for example, maitansinoids and analogues of maitansinoids, benzodiazepines, taxoids, CC-1065 and analogues of CC-1065, duocarmycins and duocarmycin analogs, enediines, taies as calicheamycin analogues, Dolastatin and dolastatin analogues that include auristatins, tomaymycin derivatives, leptomycin derivatives, methotrexate, cisplatin, carboplatin, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chloraubuin and doxorphinocholine. In certain embodiments, cytotoxic agents are maitansinoids and maitansinoid analogues.

Conjugated taies can be prepared by the use of a ligand group to bind a drug or prodrug to the antibody or its functional equivalent. Suitable ligand groups are well known in the art and include, for example, disulfuric groups, thioether groups, weak acid groups, photolibile groups, weak peptidase groups, and weak esterase groups.

<img file="AR080301A1_D0062.tif" />

The drug or prodrug can, for example, be linked to the anti-FOLRl antibody or fragment thereof through a disulfide bond. The ligand molecule or cross-linking agent comprises a chemical reactive group that can react with the anti-FOLRl antibody or fragment thereof. In certain embodiments, the chemical reactive groups for reaction with the cell-binding agent are the? / - succinimidyl esters and / V-sulfosuccinimidyl esters. In addition, the ligand molecule comprises a reactive chemical group, in certain embodiments a dithiopyridyl group that can react with the drug to form a disulfide bond. In certain embodiments, ligand molecules include, for example, TV-succinimidyl 3- (2-pyridyldithio) propionate (SPDP) (see, for example, Carlsson et al., Biochem J., 173: Ί23-Τ3Ί (1978)), A-succinimidyl 4- (2-pyridyldithio) butanoate (SPDB) (see, for example, US Patent No. 4,563,304), TV-succinimidyl 4- (2-pyridyldithio) 2-sulfobutanoate (sulfo-SPDB) (see US publication No. 20090274713), A-succinimidyl 4- (2-pyridyldithio) pentanoate (SPP) ( see, for example, CAS registry number 341498-08-6), 2-iminothiolane, or acetylsuccinic anhydride. For example, the antibody or cell binding agent can be modified with the crosslinking reagents and the antibody or cell binding agent containing free thiol groups or thus derived derivatives is then reacted with thiol or disulfide containing maitansinoid. Produce the conjugates. The conjugates can be purified by chromatography, which include but are not limited to, HPLC, size exclusion, adsorption, ion exchange and affinity capture, dialysis or tangential flow filtration. In certain embodiments, the anti-FOLRl antibody binds to the cytoxin through an SPDB or sulfo-SPDB ligand. In a certain embodiment, the Movl9hu antibody binds to a cytotoxin through a SPDB or sulfoSPDB ligand.

In another aspect of the invention, the anti-FOLRl antibody binds to cytotoxic drugs through disulfide bonds and a polyethylene glycol spacer to increase potency, solubility or efficacy of the immunoconjugate. Such hydrophilic cleavage ligands are described in W02009 / 0134976. The additional benefit of this ligand design is the convenient high monomer ratio and the minimum aggregation of the drug-antibody conjugate. Specifically, the conjugates of the cell binding agents and the drugs linked through the disulfide groups (-SS-) carrying the polyethylene glycol spacers ((CH2CH2O) are contemplated in this regard._n = 1-14)

<img file="AR080301A1_D0063.tif" />

With a narrow range of the drug load of 2-8 it is described that they show relatively high potent biological activity towards cancer cells and have the convenient biochemical properties of high conjugation yield and high proportion of monomer with minimal protein aggregation.

Specifically, a conjugate of the anti-FOLRl antibody drug of Formula (I) or a conjugate of Formula (T) is contemplated in this regard:

A- [X_r(-CH₂-CH₂OR-)_n-YC]_m (I)

A- [Xl - (- CH2-CH2O-) nYC] m (I) [CY - (- CH₂-CH₂OR-)_n-X,]_m-A (Γ) where:

A represents an anti-FOLRl antibody or fragment;

C represents a cytotoxin or drug;

X represents an aliphatic, aromatic or heterocyclic unit linked to the cell binding agent through a thioether bond, an amide bond, a carbamate bond, or an ether bond;

Y represents an aliphatic, aromatic or heterocyclic unit linked to the drug through a disulfide bond;

it is 0 or 1;

m is an integer from 2 to 8, and n is an integer from 1 to 24.

In certain modalities, m is an integer from 2 to 6.

In certain modalities, m is an integer from 3 to 5.

Also, in certain embodiments, n is an integer from 2 to 8. On the other hand, as described in, for example, US Pat. No. 6,441,163 and 7,368,565, the drug may first be modified to introduce a reactive ester suitable for reacting with a cell binding agent. The reaction of these drugs containing an activated ligand region with a cell binding agent provides another method to produce a drug conjugate with the cell binding agent. The maitansinoids can also bind to the anti FOLR1 antibody or the fragment by means of PEG ligand groups, as set forth for example in US Pat. 6,716,821. These non-cleavable groups that bind PEG are soluble in both water and non-aqueous solvents, and can be used to bind one or more cytotoxic agents to a cell binding agent. Illustrative groups that link to PEG include ligands

<img file="AR080301A1_D0064.tif" />

of heterobifunctional PEGs that react with cytotoxic agents and cell binding agents at opposite ends of the ligands through a sulfhydryl or disulfide functional group at one end, and an active ester at the other end. As a general example of the synthesis of a cytotoxic conjugate by a group linking to PEG, the reference for US Pat. 6,716,821 which is incorporated herein in its entirety as a reference. The synthesis begins with the reaction of one or more cytotoxic agents carrying a reactive PEG region with a cell binding agent, resulting in the displacement of the terminal active ester of each reactive PEG region by an amino acid residue of the cell binding agent, to yield a cytotoxic conjugate comprising one or more cytotoxic agents covalently linked to a cell binding agent through a group that binds PEG. On the other hand, cell binding can be modified with the bifunctional PEG crosslinker to introduce a reactive disulfide region (such as a pyridyldisulfide), which can then be treated with a thiol containing maitansinoid to provide a conjugate. In another method, the junction cell can be modified with the bifunctional PEG crosslinker to introduce a thiol region that can then be treated with a maitansinoid reagent containing disulfide (such as a pyridyldisulfide), to provide a conjugate.

Antibody-maitansinoid conjugates with non-cleavable ligands can also be prepared. Cross-linking taies are described in the art (see ThermoScientific Pierce Crosslinking Technical Handbook and the publication of US patent application no. 2005/0169933) and include, but are not limited to, / V-succinimidyl 4 (maleimidomethyl) cyclohexanecarboxylate (SMCC), 7V-succinimidyl-4 - (/ V-maleimidomethyl) cyclohexane-l-carboxy- (6-amidocaproate), which is SMCC long chain analogue (LC-SMCC), κ-maleimidoundecanoic acid / V-succinimidil ester (KMUA), ß-maleimidopropanoic acid / V-succinimidil ester (BMPS), / V-succinimidil ester of γ-maleimidobutyric acid (GMBS), εmaleimidocaproic acid / V-hydroxysuccinimide ester (EMCS), N-hydroxysuccinimide-m-maleimidobenzoyl ester (MBS), A- (a-maleimidoacetoxy) succinimide ester (AMAS), succinimidyl-6- (ßmaleimidopropionamido) hexamide ), A-succinimidyl 4- (p-maleimidophenyl) butyrate (SMPB), and A- (p-maleimidophenyl) isocyanate (PMPI), A-succinimidyl-4 (iodoacetyl) -aminobenzoate (SIAB), / V-succinimidyl iodoacetate ( SIA), / V-succinimidyl bromoacetate (SBA), and / V-succinimidyl 3- (bromoacetamido) propionate (SBAP). In certain

<img file="AR080301A1_D0065.tif" />

modalities, the antibody is modified with cross-linking reagents, taies as succinimidyl 4- (N-maleimidomethyl) -cyclohexane-l-carboxylate (SMCC), sulfo-SMCC, maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfo-MBS or succinimidyldoacetate, as described in the literature, to introduce 1-10 reactive groups (Yoshitake et al., Eur. J. Biochem, 101: 395-399 (1979); Hashida et al., J. Applied Biochem., 56-63 (1984); and Liu et al., Biochem., 18: 690-697 (1979)). The modified antibody is then reacted with the thiol containing maitansinoid derivative to produce a conjugate. The conjugate can be purified by gel filtration through a Sephadex G25 column or by dialysis or tangential flow filtration. The modified antibodies are treated with the thiol containing maitansinoid (1 to 2 molar equivalent / maleimide group) and the maitansinoid / anti body conjugates are purified by gel filtration through a Sephadex G-25 column, chromatography on a column of hydroxyapatite ceramic, tangential flow dialysis or filtration or a combination of these methods. Normally, an average of 1-10 maitansinoids are bound by antibodies. One method is to modify the antibodies with succinimidyl 4- (Nmaleimidomethyl) -cyclohexane-l-carboxylate (SMCC) to introduce the maleimide groups followed by the reaction of the modified antibody with a thiol-containing maitansinoid to give a thioether-linked conjugate. Again the conjugates with 1 to 10 drug molecules per antibody molecule result. Maitansinoid antibody conjugates, antibody fragments, protein hormones, protein growth factors and other proteins are prepared in the same way.

In another aspect of the invention, the FOLR1 antibody (for example, Movl9hu, FR1-21, FR1-48, FR1-49, FR1-57, or 65-FR1) binds to the drug via a non-cleavable link through the intermediate of a PEG spacer. Suitable cross-linking reagents comprising the hydrophilic PEG chains that form ligands between a drug and the anti-FOLR1 antibody or fragment are also well known in the art, or are commercially available (eg, from Quanta Biodesign, Powell, Ohio ). Suitable crosslinkers containing PEG can also be synthesized from the same PEGs available on the market by standard techniques of synthetic chemistry known to people with knowledge in the art. The drugs can be reacted with cross-linked bifunctional ligands containing PEG to give compounds of the following Formula, Z-Xi-frCfU-CFf-O-jn-Yp-D, by the methods

<img file="AR080301A1_D0066.tif" />

described in detail in the publication of US Pat. 20090274713 and in W02009 / 0134976, which can react with the cell binding agent to provide a conjugate. On the other hand, cell binding can be modified with the PEG bifunctional cross-linking ligand to introduce a thiol reactive group (such as a maleimide or a haloacetamide) which can then be treated with a thiol containing maitansinoid to provide a conjugate. In another method, cell binding can be modified with the PEG bifunctional crosslinker to introduce a thiol region that can then be treated with a maitansinoid with a reactive thiol (such as a maitansinoid carrying a maleimide or a haloacetamide) to provide a conjugate

Accordingly, another aspect of the present invention is a drug conjugate of the anti-FOLRl antibody of Formula (II) or Formula (II '):

A- [X, - (- CH₂-CH₂-OR-)_n-AND_p-C]_m (II) [CY_p- (- CH₂-CH₂-OR-)_n-Xi]_m-A (II ') where, A represents an antibody or an anti-FOLRl fragment;

C represents a cytotoxin or drug;

X represents an aliphatic, aromatic or heterocyclic unit linked to the cell binding agent through a thioether bond, an amide bond, a carbamate bond or an ether bond.

And it represents an aliphatic, aromatic or heterocyclic unit linked to the drug through a covalent bond selected from the group consisting of a thioether bond, an amide bond, a carbamate bond, an ether bond, an amide bond, a carbon-carbon bond and a hydrazone bond;

it is 0 or 1; p is 0 or 1;

m is an integer from 2 to 15; and n is an integer from 1 to 2000.

In a certain mode, m is an integer from 2 to 8; and n is an integer from 1 to 24.

In a certain mode, m is an integer from 2 to 6.

In a certain mode, n is an integer from 2 to 8.

<img file="AR080301A1_D0067.tif" />

In a certain modality, m is an integer number from 3 to 5. In a certain modality, the antibody is Movl9hu. In another embodiment, the antibody is FR-1-21. In another embodiment, the antibody is FR-1-48. In another embodiment the antibody is FR-1-49. In another embodiment, the antibody is FR-1-57. In another embodiment, the antibody is FR-1-65.

Examples of suitable PEG-containing ligands include ligands having an A-succinimidyl ester or / V-sulfosucinimidyl ester region to react with the anti-FOLRl antibody or fragment thereof, as well as a maleimide or haloacetyl-based region, for the reaction with the compound. A PEG spacer can be incorporated into any crosslinker known in the art by the methods described herein.

Many of the ligands disclosed in this document are described in detail in U.S. Patent Publications. No. 20050169933 and 20090274713 and in W002009 / 0134976, whose contents are incorporated in this document in their entirety as reference.

The present invention that includes aspects where approximately 2 to 8 drug molecules ("drug load"), for example, the maitansinoid, bind to an anti-FOLR1 antibody or a fragment thereof, the antitumor effect of the conjugate is very high. more effective when compared to a drug load of a smaller or larger number of drugs bound to the same cell binding agent. The "drug load" as used herein refers to the number of drug molecules (for example a maitansinoid), which can be attached to a cell binding agent (for example an anti-FOLRl antibody or a fragment of this one). In one aspect the number of drug molecules that can bind to the cell binding agent can average about 2 to about 8 (for example 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5,

2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9,4.0, 4.1, 4.2, 4.3, 4.4,4.5, 4.6,

4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7,

6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1). In certain embodiments, the drug is A ^ '- deacetyl-TV ^' - Q-mercapto-l-oxopropyl-maitansin (DM1) or TV²-deacetylN²- (4-mercapto-4-methyl-l-oxopentyl) maitansin (DM4). Thus, in a certain mode, the Movl9hu antibody is conjugated to DM1 or DM4. In another embodiment, the FR-1-21 antibody is conjugated to DM1 or DM4. In another embodiment, the FR-1-48 antibody is conjugated to DM1 or DM4. In another embodiment, the FR-1-49 antibody is conjugated to DM1 or

<img file="AR080301A1_D0068.tif" />

DM4 In another embodiment, the FR-1-57 antibody is conjugated to DM1 or DM4. In another embodiment, the FR-1-65 antibody is conjugated to DM1 or DM4.

Thus, in one aspect, an immunoconjugate comprises 1 maitansinoid per antibody. In another aspect, an immunoconjugate comprises 2 maitansinoids per antibody. In another aspect an immunoconjugate comprises 3 maitansinoids per antibody. In another aspect, an immunoconjugate comprises 4 maitansinoids per antibody. In another aspect, an immunoconjugate comprises 5 maitansinoids per antibody. In another aspect, an immunoconjugate comprises 6 maitansinoids per antibody. In another aspect, an immunoconjugate comprises 7 maitansinoids per antibody. In another aspect, an immunoconjugate comprises 8 maitansinoids per antibody.

In one aspect an immunoconjugate comprises about 1 to about 8 maitansinoids per antibody. In another aspect an immunoconjugate comprises about 2 to about 8 maitansinoids per antibody. In another aspect an immunoconjugate comprises about 2 to about 6 maitansinoids per antibody. In another aspect an immunoconjugate comprises about 2 to about 5 maitansinoids per antibody. In another aspect an immunoconjugate comprises about 3 to about 5 maitansinoids per antibody. In another aspect an immunoconjugate comprises about 3 to about 4 maitansinoids per antibody.

In one aspect, a composition comprising immunoconjugates has an average of about 2 to about 8 (for example, 1.9, 2.0, 2.1, 2.2,

2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1,4.2, 4.3,

4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,

6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1) drug molecules (eg. Maitansinoids) bound by antibody. In one aspect, a composition comprising immunoconjugates has an average of about 1 to about 8 drug molecules (eg, maitansinoids) per antibody. In one aspect, a composition comprising immunoconjugates has an average of about 2 to about 7 molecules (for example maitansinoids) per antibody. In one aspect a composition comprising immunoconjugates has an average of about 2 to about 6 drug molecules (for example π

<img file="AR080301A1_D0069.tif" />

maitansinoids) by antibody. In one aspect a composition comprising immunoconjugates has an average of about 2 to about 5 drug molecules (for example maitansinoids) per antibody. In one aspect a composition comprising immunoconjugates has an average of about 3 to about 5 drug molecules (for example maitansinoids) per antibody. In one aspect a composition comprising immunoconjugates has an average of about 3 to about 4 drug molecules (eg, maitansinoids) per antibody. In one aspect a composition comprising immunoconjugates has an average of about 3.5 to about 4 drug molecules (for example maitansinoids) per antibody.

In one aspect, a composition comprising immunoconjugates has an average of approximately 2 ± 0.5, approximately 2.5 ± 0.5, approximately 3 ± 0.5, approximately 3.5 ± 0.5 approximately 4 ± 0.5, approximately 4.5 ± 0.5, approximately 5 ± 0.5 approximately 5.5 ± 0.5, approximately 6 ± 0.5, approximately 6.5 ± 0.5, approximately 7 ± 0.5, approximately 7.5 ± 0.5, approximately 8 ± 0.5 drug molecules (eg, maitansinoids) bound by antibody. In one aspect a composition comprising immunoconjugates, has an average of approximately 3.5 ± 0.5, drug molecules (for example maitansinoids) per antibody.

The anti-FOLRl antibody or fragment thereof can be modified by reacting a cross-functional bifunctional reagent with the anti-FOLRl antibody or fragment thereof, thereby resulting in the covalent bond of a molecule binding to the anti-FOLRl antibody or fragment thereof. . As used herein, a bifunctional cross-linking reagent is any chemical region that covalently binds a cell binding agent with a drug, such as the drugs described herein. In another method a part of the link region is provided by the drug. In this regard, the drug comprises a binding region that is part of a major ligand molecule that is used to bind the cell binding agent with the drug. For example, to form the DM1 maitansinoid, the side chain of the C-3 hydroxy group of maitansin is modified to have a free sulfhydryl (SH) group. This thiol form of maitansin can react with a modified cell binding agent to form a conjugate. Therefore, the final ligand is assembled from two components, one of which is

I

<img file="AR080301A1_D0070.tif" />

provided by the crosslinking reagent, while the other is provided by a DM1 side chain.

Drug molecules can also bind to antibody molecules through an intermediate transporter molecule such as serum albumin.

As used herein, the term "binding to a cell binding agent" or "binding to an anti-FOLRl antibody or fragment" refers to the conjugated molecule comprising at least one drug derivative bound to an agent. binding to the cell, the anti-FOLRl antibody or fragment, through a suitable binding group, or a precursor thereof. In certain modalities, the link group is SMCC.

In certain embodiments, the cytotoxic agents useful in the present invention are maitansinoids and analogues of the maitansinoids. Examples of suitable maitansinoids include maitansinol esters and maitansinol analogs. Some drugs that inhibit microtubule formation and that are highly toxic to mammalian cells are included, such as maitanasinol and maitanasinol analogues.

Examples of functional maitansinol esters include those that have a modified aromatic ring and those that have modifications in other positions. Such suitable maitansinoids are described in US Pat. No. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533; 5,208,020; 5,416,064; 5,475,092; 5,585,499; 5,846,545; 6,333,410; 7,276,497 and 7,473,796.

In a certain embodiment, the immunoconjugates of the invention use the

2 '· 2' maitansinoid containing the thiol (DM1) formally referred to as N '-deacetyl-vV - (3mercapto-l-oxopropyl) -maitansin, as the cytotoxic agent. The DM1 is represented by the following structural formula (III)

<img file="AR080301A1_D0071.tif" />

In another embodiment, the conjugates of the present invention use the thiol containing N thiol² -deacetyl-TV² (4-methyl-4-mercapto-l-oxopentyl) maitansin (eg, DM4) as the cytotoxic agent. DM4 is represented by the following Structural Formula (IV):

<img file="AR080301A1_D0072.tif" />

Another maitansinoid comprising a side chain containing a sterically hindered thiol bond is the N² -deacetyl- / V-² (4-mercapto-l-oxopentyl) -maitansin (called DM3) represented by the following Structural Formula (V).

<img file="AR080301A1_D0073.tif" />

(V)

Each of the maitansinoids taught in US Pat. No. 5,208,020 and 7,276,497 can also be used in the conjugate of the present invention.

<img file="AR080301A1_D0074.tif" />

With respect to this, the full description of 5,208,020 and 7,276,697 is incorporated into this document as a reference.

Many positions in the maitansinoids can serve as the position to chemically bind to the link region. For example, the C-3 position having a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with hydroxy and the C-20 position having a hydroxy group, which are all expected to be useful. In certain modalities, the C-3 position is used. In certain embodiments, the C-3 position of maitansinol is used.

The structural representations of certain conjugates are shown below:

<img file="AR080301A1_D0075.tif" />

^ _n = 1-24 J 2-8

Ab-PEG-Mal-DM 1 / DM4

<img file="AR080301A1_D0076.tif" />

T °&gt;

Ab = Antibody&gt; 2-5

Ab-PEG4-Mal-DM1 (VII)

<img file="AR080301A1_D0077.tif" />

<img file="AR080301A1_D0078.tif" />

DM4: R = CH₃, q = 2, n = 1-24 J 2-8

Ab-PEG-SIA-DM1 / DM4 (VIII)

<img file="AR080301A1_D0079.tif" />

<img file="AR080301A1_D0080.tif" />

L

S '·&gt;

Ab = Antibody

Z.-J

Ab-SIA-DM1 (X)

<img file="AR080301A1_D0081.tif" />

<img file="AR080301A1_D0082.tif" />

<img file="AR080301A1_D0083.tif" />

Λ., (Xi)

<img file="AR080301A1_D0084.tif" />

(ΧΠ)

<img file="AR080301A1_D0085.tif" />

(xiii)

In a certain mode, the antibody is Movl9hu. In another embodiment, the antibody is FRI-21.

<img file="AR080301A1_D0086.tif" />

Several descriptions for producing such a maitansinoid-antibody conjugate are provided in US Pat. no. 6,333,410, 6,441,163, 6,716,821, and 7,368,565 each of which is incorporated in this document in its entirety.

In general, a solution of an antibody in aqueous buffer can be incubated with a molar excess of maitansinoids having a disulfide region carrying a reactive group. The reaction mixture can be quenched by the addition of an excess of amine (such as ethanolamine, taurine, etc.). The maitansinoid-antibody conjugate can then be purified by gel filtration. The number of bound maitansinoid molecules per antibody molecule can be determined by spectrophotometric measurement of the absorbance ratio at 252 nm and 280 nm. An average of 1-10 maitansinoid molecules / antibody molecule is used and an average of 2-5 is also used in the determined modalities. The average number of maitansinoid / antibody molecules can be, for example, about 1-10, 2-5, 3-4, 3.54 or 3.5. In one aspect, the average number of maitansinoid / antibody molecules is approximately 3.5 ± 0.5. In one aspect, the average number of maitansinoid / antibody molecules is approximately 3.5-4.

Antibody conjugates with maitansinoid drugs can be evaluated for their ability to suppress the proliferation, in vitro, of several unwanted cell lines. For example, cell lines such as the human KB cell line can easily be used for the evaluation of the cytotoxicity of these compounds. The cells to be evaluated can be exposed to the compounds for 4 to 5 days and the surviving cell fractions are measured in direct assays by known methods. The IC50 values ​​can then be calculated from the test results.

The benzodiazepine compounds described, for example, in the publication of US Pat. no. 2010/0203007 (for example, indolinobenzodiazepines or oxazolidinobenzodiazepines), derivatives thereof, intermediates thereof, could also be used to prepare fragments or conjugates of the anti-FOLR 1 antibody.

Useful benzodiazepines include the compounds of Formulas (XIV), (XV) and (XVI), in which the dimer compounds optionally carry a linking group that allows the binding agents to bind to the cell.

<img file="AR080301A1_D0087.tif" />

where the double line ~ between N and C represents a single bond or a double bond, providing that when it is a double bond X is absent and Y is H, and when it is a single bond, X is H or an amine protection group which converts the compound into a pro drug;

And selected from -OR, an ester represented by -OCOR ', a carbonate represented by -COOR', a carbamate represented by -OCONR'R ", an amine or a hydroxyl amine represented by NR'R", amide represented by - NRCOR ', a peptide represented by NRCOP, where P is an amino acid or a polypeptide containing between 2 to 20 amino acid units, a thioether represented by SR', a sulfoxide represented by SOR ', a sulfone represented by -SO2R', a sulfite -SO3, a bisulfite -OSO3, a halogen, a cyano, an azido or a thiol, where R, R 'and R "are the same or different and are selected from H, alkyls, alkenyls or substituted and unsubstituted, branched or cyclic linear alkyls having 1 at 10 carbon atoms, a unit of polyethylene glycol (-OCH₂CH₂) n, where n is an integer from 1 to 2000, arii having 6 to 10 carbon atoms, heterocyclic ring having 3 to 10 carbon atoms where the substituent is selected from a halogen, OR7, NRgRg, NO₂,

NRCOR ', SR10, a sulfoxide represented by SOR', a sulfone represented by SO₂R ', a sulfite -SO3, a bisulfite -OSO3, a sulfonamide represented by SO₂NRR ',

<img file="AR080301A1_D0088.tif" />

cyano, an azido, -CORn, OCORn or OCONR11R12, where the definitions for R₇, Rg, R9, Rio, Ru and R12 are given as above, optionally R "is OH;

W is C = O, C = S, CH₂, BH, SO or SO₂;

Ri, R2, R3, R4, Ri ', R2', R3 'and R4' are each independently selected from H, alkyl, alkenyl or substituted and unsubstituted, branched or cyclic linear alkynyls having 1 to 10 carbon atoms , a unit of polyethylene glycol (-OCH₂CH₂)_n, where n is an integer from 1 to 2000, 0 a substituent selected from a halogen, guanidinium [-NH (C = NH) NH₂], OR₇, NR₈R₉, DO NOT₂, NRCOR ', SR_W, a sulfoxide represented by SOR ', a sulfone represented by -SO₂R ', a sulfite -SO3, a bisulfite OSO3, a sulfonamide represented by SO₂NRR ', cyano, an azido, -CORn, OCORn θ OCONRnRi2 where R₇, Rg, R9, Rio, Rn and R12 are each independently selected from H, alkyls, alkenyl or substituted and unsubstituted, branched or cyclic linear alkyls having 1 to 10 carbon atoms, a polyethylene glycol unit (OCH₂CH₂)_n, where n is an integer number from 1 to 2000, arii having 6 to 10 carbon atoms, heterocyclic ring having 3 to 10 carbon atoms, optionally Rio is SR13 0 COR13, where Rj₃ is selected from an alkyl, alkenyl or alkynyl lineai, branched or cyclic having 1 to 10 carbon atoms, a polyethylene glycol unit (-OCH₂CH₂)_n, where n is an integer from 1 to 2000, arii having 6 to 10 carbon atoms, heterocyclic ring having 3 to 10 carbon atoms, optionally Rn is OR14, where R14 has the same definition as R, of optionally, any one of Ri, R₂, R₃, R4, Rf, R₂', R₃', or R4' is a linking group that is capable of binding a binding agent to the cell through a covalent bond or is selected from a polypyrrolo, poly-indolyl, polyimidazolyl, polypyroloimidazolyl, poly unit -pyrol-indolyl or polyimidazole-indolyl optionally bearing a linking group that is capable of binding to a cell binding agent;

Z is selected from (CH₂)_n, where n is 1, 2 or 3, CRi₅Ri6, NR17, O or S, where R15, Ri6 and Ri₇each independently of H, from a linear, branched or cyclic alkyl having 1 to 10 carbon atoms, a polyethylene glycol unit (OCH)₂CH₂)_n, where n is an integer from 1 to 2000;

R is OR, SR or NRR ', where R and R' have the same definition as given above;

<img file="AR080301A1_D0089.tif" />

X 'is selected from CH₂, NR, CO, BH, SO or SO₂ where R has the same definition as given above;

Y 'is O, CH₂, NR or S, where R has the same definition as given above;

Z 'is CH₂ or (CH₂)_n, where n is 2, 3 or 4, providing that X ', Y' and Z 'are not all CH₂ at the same time;

A and A 'are the same or different and are selected from O, -CRR'O, S, -CRR'S, -NR15 0 CRR'NHR]₅, where R and R 'have the same definition as above; and where Ri 5 has the same definition as given above for R;

D and D 'are the same or different and independently selected from linear, branched or cyclic alkyls, alkenyls or alkyls having 1 to 10 carbon atoms, optionally substituted γοοη any halogen, OR7, NRgRg, NO₂, NRCOR ', SR10, a sulfoxide represented by SOR', a sultana by -SO₂R ', a sulfite -SO3, a bisulfite -OSO3, a sulfonamide represented by SO₂NRR ', cyano, an azido, -CORn, OCORn or OCONR11R12, where the definitions of R7, Re, R9, Rio, Rii and R12 are given above, a polyethylene glycol unit (-OCH2CH₂) n, where n is an integer from 1 to 2000;

L is an optional phenyl group or a heterocyclic ring having 3 to 10 carbon atoms that are optionally substituted, where the substituent is a linking group that allows binding to a cell binding agent through a covalent bond , 0 is selected from linear, branched or cyclic alkyls, alkenyls or alkyls having 1 to 10 carbon atoms, optionally substituted with any halogen, OR7, NRsRç, NO₂, NRCOR ', SRæ, a sulfoxide represented by SOR', a sultana represented by -SO₂R ', a sulfite -SO3, a bisulfite -OSO3, a sulfonamide represented by SO₂NRR ', cyano, an azido,, -CORn, OCORn θ OCONRnRi2, where the definitions of R7, Rg, R9, Rio, Rii and R12 are given above, a polyethylene glycol unit (-OCH₂CH₂) n, where n is an integer from 1 to 2000; optionally, L itself is a linkage group capable of binding a binding agent to the cell through a covalent bond; or its solvates, salts, hydrates or hydrated salts, their optical isomers, racemates, diastereomers, enantiomers, or the crystalline polymorphic structures of these pharmaceutically acceptable compounds, providing that the compound has no more than a linking group capable of binding an agent of cell binding through a covalent bond.

<img file="AR080301A1_D0090.tif" />

In one aspect the double line ~ between N and C represents a single link or a double link, providing that when it is a double link X it is absent and Y is H, and when it is a single link, X is H or a protection group amine, which converts the compound into a prodrug;

And is selected from -OR, NR'R ", a sulphite -SO3, or a bisulfite -OSO3, where R is selected from linear, branched or cyclic alkyls, alkenyls or alkyls, having 1 to 10 carbon atoms , a unit of polyethylene glycol (-OCH₂CH₂)_n, where n is an integer from 1 to 2000, aryl having 6 to 10 carbon atoms, heterocyclic ring having 3 to 10 carbon atoms;

W is C = O, CH₂ bear₂;

Ri, R₂, R3, R4, R] '. R₂'. R3 'and R4' are each independently selected from H, NO₂ Or a linking group capable of binding a binding agent to the cell through a covalent bond;

R₆ is ORig, where Rig has the same definition as R;

Z is selected from (CH₂)_n, where n is 1, 2 or 3, CR15R16, NRp, O or S, where R15, Ru and R17 are each independently selected from H, from a linear, branched or cyclic alkyl having 1 to 10 carbon atoms, a unit of polyethylene glycol (OCH₂CH₂)_n, where n is an integer from 1 to 2000;

X 'is selected from CH₂, or C = O;

Y 'is O, NR, or S, where R is defined as above;

Z 'is CH₂ or (CH₂)₂;

A and A 'are each Ο;

D and D 'are the same or different and are independently selected from linear, branched or aromatic alkyls, alkenyls or alkyls, of 1 to 10 carbon atoms;

L is an optional phenyl group or a heterocyclic ring having 3 to 10 carbon atoms, which is optionally substituted, where the substituent is a linking group that allows binding to the cell binding agent through a covalent bond , or is selected from linear, branched or cyclic alkyls, alkenyl or alkyls having 1 to 10 carbon atoms, is optionally substituted with any halogen, OR₇, NRgRg, NO₂, NRCOR ', SR10, a sulfoxide represented by SOR', a sulfone represented by -SO₂R ', a sulfite -SO3, a bisulfite -OSO3, ima sulfonamide represented by SO₂NRR ', cyano, an azido, -CORn, OCORn or OCONRnRi₂, a

<img file="AR080301A1_D0091.tif" />

<img file="AR080301A1_D0092.tif" />

polyethylene glycol unit (-OCH₂CH2) n, where n is an integer from 1 to 2000; optionally, L itself is a linking group capable of binding a binding agent to the cell through a covalent bond, or its solvates, salts, hydrates, hydrated salts, its optical isomers, racemates, diastereomers, enantiomers or crystalline polymorphic structures of these pharmaceutically acceptable compounds.

In another aspect the compound is represented by Formula (XVII): L '(XVII) where the double line ~ between N and C represents a single link, or a double link, providing that when it is a double link X is absent and Y is H, and when it is a simple link, X is H or an amine protection group that converts the compound into a prodrug, and Y is selected from an OH, an ether represented by -OR, a sulphite -SO3, or a bisulfite -OSO3, where R is selected from alkyls, alkenyls, alkyls linear, branched or cyclic, that carry 1 to 10 carbon atoms,

One of R2, R3 is a linking group capable of binding a binding agent to the cell through a covalent bond and the other is H,

One of L ', L "ό L'" is a linking group capable of binding to a cell binding agent while the others are H; L 'which may be the linking group and G is CH or N. Other examples are described in the US patent application: no. 61 / 150,201, the total content of which is incorporated herein by reference. Thus in a certain embodiment, the Mol9hu antibody is conjugated to a benzodiazepene having a structure shown in XIX-XXII above. In another embodiment, the FR-1-21 antibody is conjugated to a benzodiazepene having a structure shown in XIX-XXII above.

IV. Polynucleotides

In certain embodiments, the invention includes polynucleotides comprising polynucleotides encoding a polypeptide that specifically binds to the receptor.

<img file="AR080301A1_D0093.tif" />

human FOLR1 or a fragment of this polypeptide. For example, the invention provides a polynucleotide comprising a nucleic acid sequence that encodes an antibody for a human FOLR1 or encodes a fragment of this antibody. The polynucleotides of the invention may be in the form of RNA or in the form of DNA.

The DNA includes cDNA, genomic DNA and synthetic DNA and can be double stranded or single stranded and if it is single stranded they can be a coding chain or a non-coding (anti-sense) chain.

In certain embodiments, polynucleotides are isolated. In certain embodiments the polynucleotides are substantially pure.

The invention provides a polynucleotide comprising a polynucleotide encoding a polypeptide comprising a sequence selected from a group consisting of sec. with no. ID: 4, 10, 11, 41, 42, and 88-103. A polynucleotide encoding a polypeptide having at least about 95%, at least about 96% at least about 97%, at least about 98%, at least about 99% of a sequence identity with sec. with no. Ident .: 4, 10, 11.41, 42, and 88-103.

The sec polynucleotides. with no. of ident .: 5, 14, and 15 comprise the sequence coding for the variable domain of a Movl9hu heavy chain, the variable domain of a light chain version 1.00, and the variable domain of a light chain version 1.60, respectively.

The invention further provides a polynucleotide comprising a sequence that is selected from a group consisting of sec. with no. Ident .: 5, 14, 15, 37, 38, 43, 44, 47, 48, and 120-127. Also included is a polynucleotide having at least about 95%, at least about 96%, at least about 97% at least about 98%, at least about 99% sequence identity with sec. with no. Ident .: 5, 14, 15, 37, 38, 43, 44, 47, 48, and 120-127. Thus, in certain embodiments, the polynucleotide comprises (a) a polynucleotide that has at least 95% sequence identity with sec. with no. of ident: 5, and / or (b) a polynucleotide having at least about 95% sequence identity with sec. with no. ID: 14 ​​or 15. In certain embodiments, the polynucleotide comprises (a) a polynucleotide having the amino acid sequence of sec. with no. of ident: 5, and / or (b)

<img file="AR080301A1_D0094.tif" />

a polynucleotide having the amino acid sequence of sec. with no. Ident .: 14 or sec. with no. of ident. : fifteen

In certain embodiments, the polynucleotides comprise the sequence encoding the mature polypeptide that is fused in the same reading frame to a polynucleotide that aids, for example, in the expression and secretion of a polypeptide from a host cell (for example a sequence leader that works like a secretory sequence to control the transport of the polypeptide from the cell). The polypeptide having a leader sequence is a pre-protein and may have a leader sequence cleaved by the host cell to form the mature form of the polypeptide. The polynucleotides can also code for a proprotein that is the mature protein in addition to amino acid residues added to the 5 'end. A mature protein that has a pro-sequence is a pro-protein and is an inactive form of the protein. Once the pro-sequence is cleaved, the active mature protein remains.

In certain embodiments the polynucleotides comprise the sequence that codes for the mature polypeptide that is fused in the same reading frame to a marker sequence that allows, for example, purification of the encoded polypeptide. For example, the marker sequence may be a hexa-histidine label supplied by the pQE-9 vector to provide purification of the mature polypeptide fused with the label in the case of a bacterial host, or the marker sequence may be a hemagglutinin label. (HA) derived from the hemagglutinin influenza protein, when a mammalian host (for example COS-7 cells) is used.

The present invention also relates to the variants of the coding polynucleotides described hereinbefore, for example, fragments, analogs and derivatives.

The polynucleotide variants may contain alterations in the coding regions, in the non-coding regions, or in both. In some embodiments, polynucleotide variants contain alterations that produce silent substitutions, additions or deletions, but do not alter the properties or activities of the encoded polypeptide. In some embodiments, nucleotide variants are produced by silent substitutions due to the degeneracy of the genetic code. Polynucleotide variants can be produced for a variety of reasons, for example for

<img file="AR080301A1_D0095.tif" />

optimize codon expression for a particular host (codon changes in human mRNA to those preferably by the bacterial host such as E.coli).

Vectors and cells comprising the polynucleotides described herein are also provided.

V. Methods of use and pharmaceutical compositions.

The FOLR-1 binding agents (which include antibodies, immunoconjugates and polypeptides) of the invention are useful in a variety of applications that include, but are not limited to, methods of therapeutic treatment, such as cancer treatment. In certain embodiments, agents are useful for inhibiting tumor growth, inducing differentiation, reducing tumor volume, and / or reducing the malignancy of a tumor. The methods of use could be in vitro, ex vivo, or in vivo methods. In certain embodiments, the FOLR1 binding agent, or the antibody or the immunoconjugate, or the polypeptide is a human FOLR1 antagonist, to which it binds.

In one aspect, the anti-FOLR1 antibodies and immunoconjugates of the invention are useful for detecting the presence of FOLR1 in a biological sample. The term "detect" as used herein includes quantitative or qualitative detection. In certain embodiments, a biological sample comprises a cell or tissue. In certain embodiments, such tissues include normal or cancerous tissues that express FOLR1 at higher levels relative to other tissues. In certain modalities, overexpression of FOLR1 detects the presence of ovarian cancer, lung cancer, brain cancer, marna cancer, uterine cancer, renai cancer and pancreatic cancer.

In one aspect, the invention provides a method for detecting the presence of FOLR1 in a biological sample. In certain embodiments, the method comprises contacting the biological sample with an anti-FOLRl antibody under permissive conditions for the binding of the anti-FOLRl antibody to FOLR1, and detecting if a complex is formed between the anti-FOLRl antibody and FOLR1.

In one aspect, the invention provides a method for diagnosing a disorder associated with an increased expression of FOLR1. In certain embodiments, the method comprises contacting a test cell with an anti-FOLRl antibody, determining the level of expression (either quantitatively or qualitatively) of FOLR1 by the cell of

<img file="AR080301A1_D0096.tif" />

test to detect the binding of the anti-FOLRl antibody to FOLR1; and comparing the level of FOLR1 expression in the test cell with the level of FOLR1 expression by a control cell (for example, a normal cell of tissue origin similar to the test cell, or a cell expressing FOLR1 at levels comparable to that of a normal cell) where a higher expression level of FOLR1 by the test cell when compared to the control cell indicates the presence of a disorder associated with the increased expression of FOLR1. In certain embodiments, the test cell is obtained from an individual suspected of having a disorder associated with the increased expression of FOLR1. In certain embodiments, the disorder is a cell proliferative disorder, such as a cancer or a tumor.

In certain embodiments, a method for diagnosis or detection, such as those described above, comprises detecting the binding of the anti-FOLR1 antibody to FOLR1 that is expressed on the surface of a cell or in a preparation of a membrane that is obtained from a cell expressing FOLR1 on its surface. In certain embodiments, the method comprises contacting a cell with an anti-FOLRl antibody under permissive conditions for the binding of the anti-FOLRl antibody with FOLR1 on the cell surface. An example assay to detect the binding of an anti-FOLR1 antibody to FOLR1 that is expressed on the surface of a cell is a "FACS" assay.

Other determined methods can be used to detect the binding of anti-FOLR1 antibodies to FOLR1. These methods include, but are not limited to, antigen binding assays that are well known in the art such as immunoblotting, radioimmunoassays, ELISA (enzyme-linked immunosorbent assay), sandwich sandwich immunoassays, immunoprecipitation assays, immunoassays. fluorescence, protein A immunoassays and immuno histochemistry (IHC).

In certain embodiments, anti-FOLRl antibodies are labeled. The labels include, but are not limited to, the labels or portions that are directly detected (such as fluorescent, chromophoric, electro-dense, chemiluminescent, and radioactive labels), as well as the portions, such as enzymes or ligands, which are detected indirectly, for example, through an enzymatic reaction or a molecular interaction.

In certain embodiments, anti-FOLRl antibodies are immobilized on an insoluble matrix. Immobilization involves separating anti-FOLRl antibodies from

<img file="AR080301A1_D0097.tif" />

any FOLR1 that remains free in the solution. This is conventionally achieved either by the insolubilization of the anti-FOLRl antibody before the test procedure, as by absorption to a water-insoluble matrix or surface ((Bennich et al., U.S. Patent No. 3,720,760), or by covalent coupling (for example by cross-linking with glutaraldehyde), or by insolubilization of the anti-FOLR1 antibody after the formation of a complex between the anti-FOLR1 antibody and FOLR1, for example, by immunoprecipitation.

Any of the above diagnostic or detection modalities could be carried out using an immunoconjugate of the invention instead of or in addition with an anti-FOLRl antibody.

In certain embodiments, the disease that is treated with the FOLR1 binding agent or antagonist (for example a Movl9hu antibody or immunoconjugate) is a cancer. In certain embodiments, the cancer is characterized by tumors expressing the folate receptor 1 that binds to the binding agent FOLR1 (eg, antibody).

The present invention provides methods for relying on cancer which comprises administering a therapeutically effective amount of a FOLR1 binding agent to a subject (for example a subject in need of treatment). In certain embodiments, the cancer is a cancer selected from a group consisting of colorectal cancer, pancreatic cancer, lung cancer, ovarian cancer, liver cancer, marna cancer, brain cancer, kidney cancer, prostate cancer, cancer gastrointestinal, melanoma, cervical cancer, bladder cancer, glioblastoma, head and neck cancer. In certain modalities, cancer is an ovarian cancer. In certain modalities the cancer is a lung cancer. In certain modalities the subject is a human.

The present invention further provides methods for inhibiting tumor growth using the antibodies or other agents described herein. In certain embodiments, the method for inhibiting tumor growth comprises contacting the cell in vitro with a FOLR1 binding agent (for example the antibody). For example, an immortalized cell line or a cancer cell line that expresses FOLR1 is cultured in the medium to which the antibody or other agent is added to inhibit tumor growth. In some embodiments, the tumor cells are isolated from a patient sample such as, for example, a tissue biopsy, pleural effusion or blood sample,

<img file="AR080301A1_D0098.tif" />

and grown in a medium to which a binding agent is added to FOLR1 to inhibit tumor growth.

In some embodiments, the method for inhibiting tumor growth comprises contacting the tumor or tumor cells in vivo with the binding agent FOLR1 (for example the antibody). In certain embodiments, contacting a tumor or a tumor cell line with the FOLR1 binding agent is addressed in an animal model. For example, FOLR1 binding agents can be administered to xenografts that express one or more of the FOLR1s that are grown in immuno-compromised mice (eg, NOD / SCID mice) to inhibit tumor growth. In some embodiments, mature cancer cells are isolated from a patient sample such as a tissue biopsy, a pleural effusion, or blood sample and are injected into immunocompromised mice that are then administered a binding agent to FOLR1 to inhibit tumor growth. In certain embodiments, the FOLR1 binding agent is administered at the same time or shortly after the introduction of tumorigenic cells into the animai to prevent tumor growth. In some embodiments, the FOLR1 binding agent is administered as a therapeutic, after the tumor cells have grown to a specific size.

In certain embodiments, the method of inhibiting tumor growth comprises administering to a subject a therapeutically effective amount of the FOLR1 binding agent. In certain modalities the subject is a human. In certain modalities, the subject has a tumor or had a tumor removed.

In certain embodiments, the tumor expresses the folate receptor to which the FOLR1 binding agent or antibody binds. In certain modalities, the tumor overexpresses human FOLR1.

In certain embodiments, the tumor is a tumor selected from the group consisting of brain tumor, colorectal tumor, pancreatic tumor, lung tumor, ovarian tumor, liver tumor, marna tumor, kidney tumor, prostate tumor, gastrointestinal tumor, melanoma, cervical tumor, bladder tumor, glioblastoma and head and neck tumor. In certain modalities, the tumor is an ovarian tumor.

In addition, the invention provides a method for reducing the malignancy of a tumor in a subject, which comprises administering to the subject a therapeutically effective amount of the binding agent to FOLR1. In certain modalities, the tumor comprises

<img file="AR080301A1_D0099.tif" />

mother cancer cells. In certain modalities, the frequency of cancer stem cells in the tumor is reduced by the administration of the agent.

Thus, in certain embodiments the inventions provide methods for treating cancer using the Movl9hu antibody and immunoconjugates. In certain embodiments, the Movl9hu immunoconjugate is Movl9hu-SPDB-DM4; Movl9hu-sulfoSPP-DM1; Movl9hu-SPP-DMl; or Movl9hu-PEG4-Mal-DM4.

The invention further provides methods of differentiation of tumor cells into non-tumor cells that comprise contacting the tumor cells with a FOLR1 binding agent (for example, administering the FOLR1 binding agent to a subject having a tumor comprising the cells tumor or had a tumor removed). In certain embodiments, tumor cells are ovarian tumor cells.

The present invention further provides methods for reducing the activation of myofibroblasts in the stroma of a solid tumor, which comprises contacting the stroma with an effective amount of the FOLR1 binding agent, polypeptide or antibody.

The present invention further provides pharmaceutical compositions comprising one or more of the FOLR1 binding agents described herein. In certain embodiments, the pharmaceutical compositions further comprise a pharmaceutically acceptable carrier. These pharmaceutical compositions find use in inhibiting tumor growth and treating cancer in human patients.

In certain embodiments, the formulations are prepared for storage and use by combining a purified antibody or agent of the present invention, with a pharmaceutically acceptable carrier (eg, carrier, excipient) (Remington, The Science and Practice of Pharmacy 20ma Mack Publishing Edition, 2000). Pharmaceutically acceptable vehicles include, but are not limited to, non-toxic buffers such as phosphate, citrate and other organic acids; salts such as sodium chloride; antioxidants that include ascorbic acid and methionine; preservatives (for example, ammonium octadecyldimethylbenzyl chloride; hexamethonium chloride, benzalkonium chloride, benzetonium chloride, phenol, butyl or benzyl alcohol; alkyl parabens taies as methyl or propyl paraben, catechol, resorcinol; cyclohexanol; 3 pentanol and m-cresol); low molecular weight polypeptides (for example, less than about 10 amino acid residues); taies proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone; amino acids

<img file="AR080301A1_D0100.tif" />

taies such as gliein, glutamine, asparagine, histidine, arginine or lysine; carbohydrates such as monosaccharides, disaccharides, glucose, mannose, or dextrins; chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol; against ions forming salts such as sodium horn; metal complexes (for example Zn-protein complexes); and non-ionic surfactants such as TWEEN or polyethylene glycol (PEG).

The pharmaceutical compositions of the present invention can be administered in any number of ways by either local or systemic treatment. Administration may be typical (such as a mucosal membrane that includes vaginal and rectal delivery) as well as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, aerosols, liquids and powders; pulmonary (for example by inhalation or insufflation of powders or aerosols, including nebulizer, intratracheal, intranasal, epidermal and transdermal); oral; or parenteral, including intravenous, intra arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial administration (for example, intrathecal or intraventricular).

An antibody or immunoconjugate of the invention may be combined in a pharmaceutical combination formulation, or dosage regimen such as combination therapy, with a second compound having anti-cancer properties. The second compound of a pharmaceutical combination formulation or dosage regimen preferably has complementary activities to the ADC of the combination such that they do not adversely affect each other. Pharmaceutical compositions comprising the binding agent FOLR1 and the second anti-cancer agent are also provided.

For the treatment of the disease, the appropriate dose of an antibody or an agent of the present invention depends on the type of disease to be treated, the severity and the course of the disease, the sensitivity of the disease, whether the antibody or the agent It is administered for therapeutic or preventive purposes, prior to therapy, the patient's medical history, and so on in the opinion of the doctor who guarantees the disease. The antibody or agent can be administered once or in a series of treatments that last from several days to several months, or until cure occurs or a decrease in disease status is achieved (for example the reduction in tumor size). Optimum dosing schedules can be calculated from measurements of drug accumulation in the patient's body and will vary depending on the relative potency of a drug.

<img file="AR080301A1_D0101.tif" />

antibody or individual agent. The doctor who administers it can easily determine the optimal doses, dosage methodologies and repetition rates. In certain modalities the dose is from 0.01 pg to 100 mg per kg of body weight and can be given once or more daily, weekly, monthly or annually. In certain embodiments an antibody or other FOLR1 binding agent is administered once every two weeks or once every three weeks. In certain embodiments, the dose of the antibody or other FOLR1 binding agent is from about 0.1 mg to about 20 mg per kg of body weight. The doctor who confides the subject can estimate the repetition rates per dose based on the measurement of residence times and drug concentrations in body fluids or tissues.

Combination therapy can provide "synergy" and prove "synergism," that is, the effect achieved when the active ingredients are used together is greater than the sum of the effects that result from using the compounds separately. A synergistic effect can be obtained when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined unit dose formulation, (2) delivered alternately or in parallel with the separate formulations; or (3) by any other regime. When delivered in an alternative therapy, a synergistic effect can be obtained when the compounds are administered or delivered sequentially, for example by different injections into separate syringes. In general, during the alternative therapy, an effective dose of each active ingredient is administered sequentially, that is, consecutively, while in a combination therapy, the effective doses of two or more active ingredients are administered together.

SAW. Kits comprising FOLR1 binding agents

The present invention provides kits comprising the antibodies, immunoconjugates, or other agents described herein and can be used to perform the methods described herein. In certain embodiments, a kit comprises at least one antibody purified against human folate receptor 1 in one or more containers. In some modalities, the kits comprise all the necessary and / or sufficient components to carry out a detection test, which include all the controls, instructions for carrying out the measurements, and some program necessary for the analysis and presentation of the results. A person with knowledge in the technique,

<img file="AR080301A1_D0102.tif" />

It will readily recognize that the disclosed antibodies, immunoconjugates and other agents of the present invention can easily be incorporated into one of the established kit formats that are well known in the art.

In addition, kits comprising the FOLR1 binding agent (for example, a FOLR1 binding antibody) are thus provided as well as a second agent against cancer. In certain embodiments, the second agent against cancer is a chemotherapeutic agent (for example, gemeitabine or irinotecan).

The modalities of the present disclosure can be defined further by reference for the following non-limiting examples, which describe in detail the preparation of antibodies determined from the present disclosure and the methods for using the antibodies of the present disclosure. It will be evident to those with knowledge in the art that many modifications, both for materials and methods, can be practiced without diverting the scope of this disclosure.

EXAMPLES

It is understood that the examples and modalities described herein are for illustrative purposes only and that various modifications or changes in light thereof are suggested to persons with experience in the subject and will be included in the spirit and scope of this application.

Example 1

Chimerization of the Movi9 murine monoclonal antibody

The amino acid sequences of the Movi9 variable region were obtained from the NCBI database (accesses CAA68253 for a light chain (section with ID no .: 24) and CAA68252 for a heavy chain (section with no. ident .: 23)) and then codons were optimized and synthesized by Blue Heron Biotechnology. The light chain variable region was cloned into the EcoRI and BsiWI sites of the plasmid pAbKZeo and the heavy chain variable region was cloned into the HindlII and Apal sites of the plasmid pAbGINeo.

<img file="AR080301A1_D0103.tif" />

<img file="AR080301A1_D0104.tif" />

Example 2

Humanization of the murine monoclonal antibodies Movi 9 and FRI-21

The Movl9 antibody was humanized following the surface rediseno methods of the framework regions described above (Roguska M. et al., Proc.

Nati Acad. Sci. United States, February 1994; 91: 969-973) and (Roguska et al., Protein Eng. 9 (10): 895-904 (1996)). In summary, the average solvent accessibility of each residue of the variable region framework region was calculated by using the closely related antibody-resolved structures of the PDB database, and positions with more than 30% of Average accessibility was marked as surface debris (Pedersen JT et al., J. Mol. Biol. 1994; 235: 959-973). The surface of the human replacement sequence was selected by alignment of the surface positions of the murine antibody sequences with the corresponding positions of the human antibody germline sequences in the Kabat database (Johnson, G. and Wu, TT (2001) Nucleic Acids Research, 29: 205-206). The surface of the human light chain variable region with the maximum homology (don DPK19, IMGT locus IGKV2D-30 * 01 for Movl9 and IMGT locus IGKV1 / OR2-0 * 01 for FR1-21) and surface of the variable chain region human weighing with the maximum homology (clone 8M27, IMGT locus IGHV1-69 * O8 for Movl9 and IMGT locus IGHV5-51 * 02 for FR1-21) was selected to replace the surface positions of the murine frame region of Movl9, 6 CDRs (Table 1) are left unchanged. The surface positions and residues of Movl9 and FRI-21, murine and human are presented in Figures 1A-D.

CDR Movi 9

Light chain

CDR1: KASQSVSFAGTSLMH (sec. With ID no .: 7)

CDR2: RASNLEA (sec. With ID no .: 8)

CDR3: QQSREYPYT (sec. With ID no .: 9)

Heavy chain

CDR1: GYFMN (sec. With ID no .: 1)

CDR2 (AbM): RIHPYDGDTF (sec. With ID no .: 2)

CDR3: YDGSRAMDY (sec. With ID No.:3)

CDR2 Movl9 HC defined from Kabat

Murine

HC CDR2: RIHPYDGDTFYNQNFKD (sec, with ID no .: 128)

Human

HC CDR2: RIHPYDGDTFYNQKFQG (sec. With ID no .: 129)

<img file="AR080301A1_D0105.tif" />

Light chain

CDR1: KASDHINNWLA (sec. With ID no .: 27)

CDR2: GATSLET (sec. With ID No. 28)

CDR3: QQYWSTPFT (sec. With ID no .: 29)

Heavy chain

CDR1: SSYGMS (sec. With ID No. 30)

CDR2 (AbM): TISSGGSYTY (sec. With ID number: 31)

CDR3: DGEGGLYAMDY (sec. With ID no .: 32)

CDR2 FR1-21 HC defined from Kabat

Murine

HC CDR2: TISSGGSYTYYPDGVKG (sec. With ID No.:33)

Human

HC CDR2: TISSGGSYTYYSPGFQG (sec. With ID No.:34)

Table IA: The CDR heavy and light chains of Movl9 and FRI-21 as defined for surface redesign are provided. Kabat's definition for heavy chain CDR2 is also given for murine and human antibodies.

None of the residue changes caused problems due to the affectation of the interactions of any of the Movl9 or FRI-21 CDRs with their objective epitopes at folate receptor 1, so they were not considered surface reverse mutations for humanized sequences of both antibodies The redesigned Movl9 sequence however introduced a consensus N-N74 glycosylation site of a light chain (version of a light chain 1.00), so a second humanized version of a light chain was made to remove this site. A review of the Kabat database of human light chain sequence revealed that threonine is the most common residue

<img file="AR080301A1_D0106.tif" />

it is located at position 74 of a light chain, so version 1.60 of a humanized light chain from Movl9 was built with a threonine at position 74. Position 74 is not a surface residue so this replacement of residues It has no impact on humanization through surface redesign. The alignments of the murine and humanized Movl9 and FR 1-21 variable region sequences are presented in Figure 2.

For coding sequences of the humanized Movl9 and FRI-21 region the codons were optimized and synthesized by Blue Héron Biotechnology. The sequences are flanked by sites for restriction enzymes to facilitate cloning in frame with the respective sequences in single-chain plasmids for mammalian expression. The light chain variable region was cloned into the EcoRI and BsiWI sites of the plasmid pAbKZeo. The DNAs of the resulting plasmids encoding a light chain of huMovl9 were deposited in the ATCC as no. deposit of the ATCC PTA10773 and PTA-10774 and the DNA of the resulting plasmid encoding a light chain of the huFRl-21 was deposited as no. deposit of the ATCC PTA-10776. The heavy chain variable region was cloned at the HindlII and Apal sites of the plasmid pAbGINeo. The DNA of the resulting plasmid encoding a heavy chain of huMovl9 was deposited in the ATCC as no. deposit of the ATCC PTA-10772 and the DNA of the resulting plasmid encoding a heavy chain of huFRl-21 was deposited in the ATCC as no. deposit of the ATCC PTA-10775. These plasmids were transfected as described in Example 3 to produce huMovl9. The plasmid encoding any of the huMovl9 light chains (ie, those that were deposited as no. deposit of the ATCC PTA-10773 or PTA-10774) can be combined with the plasmid encoding a huMovl9 heavy chain to create a huMovl9 antibody according to the methods set forth herein since they are well known to someone with experience in the subject.

Example 3

Expression of recombinant antibodies

Chimeric and humanized antibody constructs occurred transiently, either in the HEK-293T adherent cells with a standard calcium phosphate procedure (BD Biosciences, CalPhos Mammalian Transfection Kit, Cat # 631 312) or in HEK- cells 293T adapted to suspension with a PEI procedure

<img file="AR080301A1_D0107.tif" />

modified [Durocher Y, Perret S, Kamen A High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res. January 15, 2002; 30 (2): E9] in shaking flasks. Transient PEI transfections were performed as previously described (Durocher, Y. et al., Nucleic Acids Res. 30 (2): E9 (2002)), except that HEK-293T cells were grown in Freestyle 293 (Invitrogen) and the volume of the culture was not diluted after the addition of PEI-DNA complexes. Both transient suspending adherent transfections were incubated for one week and then the clarified supernatant was purified by a protein A column followed by chromatography on a single ion exchange CM column as described below. As shown in Figure 3, the expression of huMovl9 was at least 10 times higher than the expression of chimeric Movi9 in transfected cells.

Example 4

Antibody purification

The antibodies were purified from the clarified cell culture supernatant, using standard methods, such as chromatography with Protein A or G (HiTrap Protein A or G HP, 1 ml, Amersham Biosciences). In summary, the supernatant was prepared for chromatography by adding 1/10 of the volume of 1 M Tris / HCl, pH 8.0. The supernatant with adjusted pH was filtered through a 0.22 μτη filtration membrane and loaded onto the equilibrated column with binding buffer (PBS, pH 7.3). The column was washed with binding buffer until a stable baseline was obtained without absorbance at 280 nm. The antibody was eluted with 0.1 M acetic acid buffer containing 0.15 M NaCl, pH 2.8, with a flow of 0.5 ml / min. Fractions of approximately 0.25 ml were collected and neutralized by the addition of 1/10 of the volume of 1 M Tris-HCl, pH 8.0. The maximum fraction (s) was dialyzed overnight against PBS 1x and sterilized by filtration through a 0.2 pm filtration membrane. The purified antibody was quantified by absorbance at A₂8th

The fractions purified with protein A were further purified by using the single-exchange chromatography (IEX) with the carboxymethyl cellulose (CM) chromatography. In summary, samples of the purification with protein A were changed to the buffer buffer (10 mM potassium phosphate, sodium chloride 10

<img file="AR080301A1_D0108.tif" />

mM, pH 7.5) and filtered through 0.22 pm filing cabinet. The prepared sample was then loaded onto a fast flow CM resin (GE Lifesciences) that was equilibrated with the starting buffer, at a flow of 120 cm / hr. The size of the column was chosen to have sufficient capacity to bind all the antibodies in the sample. The column was washed with binding buffer until a stable baseline was obtained without absorbance at 280 nm. The antibody was eluted by starting a gradient of 10 mM to 500 mM sodium chloride in 20 column volumes (CV). Fractions were collected with the UV reading above 50 mAu of the main peak. Purity (the percentage of monomer and soluble aggregates of high molecular weight) was evaluated by size exclusion chromatography (SEC) on a TSK G3000SWXL gel, 7.8 x 300 mm, with a SWXL safety column, 6.0 x 40 mm (Tosoh Bioscience, Montgomeryville, PA) through the use of an Agilent HPLC 1100 system (Agilent, Santa Clara, CA). Fractions with the desired purity (&gt; 95%) were pooled, the buffer was exchanged for PBS (pH 7.4) using the TFF system, and sterilized by filtration through a 0.2 pm filtration membrane. The purified antibody was further examined for purity by the SEC and the IgG concentration was determined by measuring absorbance at 280 nm by using an extinction coefficient of 1.47. Dilution was made when necessary. On the other hand, ceramic hydroxyapatite (CHT) can be used to polish both murine and humanized antibodies with greater selectivity. Type II CHT resin with 40 pm particle size (Bio-Rad Laboratories) was applied to polished antibodies with a protocol similar to IEX chromatography. The CHT starting buffer was 20 mM sodium phosphate, pH 7.0 and the antibody was eluted with a gradient of 20 to 160 mM sodium phosphate above 20 CV.

Example 5

Development of murine anti-FOLRl antibodies

There were two different series of immunization detection. The first series has resulted in the generation of the FRI-21 gift, the second series has resulted in the generation of clones FR1-48, FR1-49, FR1-57 and FR1-65. In the first series the mice were immunized subcutaneously with approximately 5x10⁶ KB cells expressing FOLR1 (American Tissue Culture Collection, ATCC CCL-17). In the second series, 300-19 Nature cells, 317: 353-355 (1985)), were transfected with the pSRa-FolRl expression plasmid to stably express high levels of FOLR1 on the cell surface

<img file="AR080301A1_D0109.tif" />

human. The standard immunization protocols known to those with experience, for example, as used in ImmunoGen, Inc. were applied to both series. Mice immunized with antigen were challenged three days before being sacrificed for hybridoma generation. The spleens of the mice were collected according to the standard protocols of the animals, such as, for example, grinding the tissue between two sterile frozen microscopic slides, to obtain a suspension of individual cells in RPMI-1640 medium. Spleen cells were centrifuged, sedimented, washed, and fused with a murine myeloma, such as, for example, P3X63Ag8.653 cells (Keamey et al., J. Immunol., 123: 1548-1550 (1979)) by polyethylene glycol-1500 (Roche 783 641). The molten cells were resuspended in RPMI-1640 selection medium containing hypoxanthine-aminopterin-thymidine (HAT) (Sigma, H0262) and selected for growth in flat-bottom 96-well culture plates (Coming-Costar 3596, 0.2 ml of cell suspension per well) at 37 ° C with CO₂ at 5% After 5 days of incubation, 0.1 ml of culture supernatant was removed from each well and replaced with 0.1 ml of RPMI-1640 containing the hypoxanthine-thymidine (HT) supplement (Sigma, H-0137). Incubation at 37 ° C with CO₂ 5% was continued until the clusters of hydridomas were ready for antibody detection. Other hybridization immunization and production techniques can also be used, including those described in Langone et al. (Eds., "Immunochemical Techniques, Part I", Methods in Enzymology, Academie Press, Volume 121, Florida) and Harlow and others ( "Antibodies: A Laboratory Manual"; Cold Spring Harbor Laboratory Press, New York (1988)).

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<img file="AR080301A1_D0121.tif" />

Example 6

Hybridoma detection and selection

FOLR1-300-19 cells transfected with human FOLR1 and KB cells are used in the first and second series of detections accordingly. The hybridoma culture supernatants were selected by flow cytometry for secretion of monoclonal antibodies that bind to FOLR1 positive cells, such as 300-19 cells expressing FOLR1 or KB cells, but not FOLR1 negative cells, taies as 300-19 non-transfected cells. 0.1 ml of hybridoma supernatant was incubated for 3 hours, either with FOLR1-positive cells or 300-19 non-transfected cells (1 xlO⁵ cells per sample) in 0.1 ml of FACS buffer (RPMI-1640 medium supplemented with 2% normal goat serum). The cells were then centrifuged, sedimented, washed and incubated for 1 hour with 0.1 ml of a goat anti-mouse IgG antibody conjugated to PE (such as those obtained from, for example, the Jackson Laboratory, 6 pg / ml in FACS buffer). The cells were centrifuged, sedimented again, washed with FACS buffer and resuspended in 0.2 ml of PBS with 1% formaldehyde. The cell-associated fluorescence was measured using a FACSCalibur flowmeter with the HTS multi-well sampler or with a FACSArray flowmeter and analyzed using CellQuest Pro (all from BD Biosciences, San Diego, United States). Positive hybridoma clones were subcloned by limiting dilution. A subclone of each hybridoma, which showed the same reactivity against FOLR1 as the parental cells by flow cytometry, was chosen for further analysis. Stable subclones were cultured and the isotype of each secreted anti-FOLRl antibody was identified using commercial isotyping reagents (Roche 1493027). The murine antibodies were purified with protein A from the cleared hybridoma culture media as described above. These antibodies were designated as FR-1 antibodies.

<img file="AR080301A1_D0122.tif" />

Example 7

Purification of the murine monoclonal antibody Antibodies were purified from hybridoma subclone supernatants using standard methods, such as, for example, protein chromatography A or G (HiTrap Protein A or G HP, 1 ml, Amersham Biosciences) . In summary, the supernatant was prepared for chromatography by adding 1/10 of the volume of 1 M Tris / HCl, pH 8.0. The supernatant with adjusted pH was filtered through a 0.22 pm filtration membrane and charged to the equilibrated column with binding buffer (PBS, pH 7.3). The column was washed with binding buffer until a stable baseline was obtained without absorbance at 280 nm. The antibody was eluted with 0.1 M acetic acid buffer containing 0.15 M NaCl, pH 2.8, with a flow of 0.5 ml / min. Fractions of approximately 0.25 ml were collected and neutralized by the addition of 1/10 of the volume of 1 M Tris-HCl, pH 8.0. The maximum fraction (s) was dialyzed overnight against PBS lx and sterilized by filtration through a 0.2 pm filtration membrane. The purified antibody was quantified by absorbance at A₂8th Example 8

Characterization of the binding by flow cytometry The specificity of binding was checked by flow cytometry through the use of purified antibodies. FACS histograms demonstrating the binding of anti-FOLRl antibodies to 300-19 cells expressing FOLR1 and the absence of binding to parental 300-19 cells are shown in Figure 4. Each antibody was incubated for 3 hours with the 300-19 cells expressing FOLR1 or with non-transfected 300-19 cells (lxlO⁵cells per sample) in 0.1 ml of FACS buffer (RPMI-1640 medium supplemented with 2% normal goat serum). The cells were then pelleted, washed and incubated for 1 hour with 0.1 ml of a goat anti-mouse IgG antibody conjugated to FITC (such as those obtainable from, for example, the Jackson Laboratory, 6 pg / ml in FACS buffer). The cells were pelleted again, washed with FACS buffer and resuspended in 200 pi of PBS with 1% formaldehyde. The samples were obtained

<img file="AR080301A1_D0123.tif" />

100 by using a FACSCalibur flow cytometer with the HTS multi-well sampler or a FACSArray flow cytometer and analyzed using CellQuest Pro (all from BD Biosciences, San Diego, United States). FACS histograms of antiFOLRl antibodies showed a change in fluorescence, while parental 300-19 cells did not. In addition, no significant fluorescence change was detected when any of the cell lines was incubated only with a goat anti-human IgG antibody conjugated to FITC alone.

Example 9

Cloning and sequencing of the VL and VH regions of muFRl-21

Total cellular RNA was prepared from 5x 10⁶ hybridoma cells by using an RNeasy kit (QIAGEN) according to the manufacturer's protocol. The cDNA was subsequently synthesized from total RNA using the cDNA synthesis kit, SuperScript II (Invitrogen). The procedure for the first round of degenerate PCR reaction on cDNA derived from hybridoma cells was based on the methods described in Wang et al. ((2000) J Immunol Methods. January 13; 233 (1-2): 167-77 ) and Co et al. ((1992) J Immunol. fifteen February; 148 (4): 1149-54). The VH sequences were amplified by PCR using the following degenerated primers: EcoMHl

CTTCCGGAATTCSARGTNMAGCTGSAGSAGTC (sec. With ID no .: 50) £ coMH2 CTTCCGGAATTCSARGTNMAGCTGSAGSAGTCWGG (sec. The VL sequences were amplified by PCR using the following degenerate primers: SacIMK

GGAGCTCGAYATTGTGMTSACMCARWCTMCA (sec. With identification number: 53) and HindKL TATAGAGCTCAAGCTTGGATGGTGGGAAGATGGATACAGTTGGTGC (sec. With identification number: 54). (Mixed bases are defined as follows: N = G + A + T + C, S = G + C, Y = C + T, M = A + C, R = A + G, W = A + T).

The PCR reaction mixtures were run on a 1% low fusion agarose gel, the 300-400 bp bands were cut, purified by using Zymo DNA mini columns, and sent to Agencourt Biosciences for sequencing The respective 5'and 3 'PCR primers were used as sequencing primers to generate the cDNA of

<img file="AR080301A1_D0124.tif" />

101 the variable region of both directions. The amino acid sequences of the VH and VL regions were obtained by translating the DNA resulting from sequencing with the VectorNTI software.

To identify sequencing artifacts with the 5 'end primer in the preliminary cDNA sequences of the variable region, the NCBI IgBlast site (www.ncbi.nlm.nih.gov/igblast/) was used to search the sequences from the mouse germ line from which the antibody sequences were derived. The clean sequences of the variable region were combined with the NCBI reference sequences for the constant regions of specific antibodies to assemble full length sequences of the murine antibody. The molecular weight of the expected light and heavy murine Fri-21 chains was calculated and compared with the mass measured by liquid chromatography / mass spectrophotometric analysis (LC / MS). The heavy FRI-21 murine chain coincided with the measured mass, but for the light chain an sequencing effort of sequencing was required to determine the sequence of the 5 'end. The CD37lLCleadl PCR primer (ttttgaattcgccaccatgaagtttccttctcaacttct) was designed to align with the germline sequence linked to the murine antibody leader so that this new PCR reaction gave a complete variable region cDNA sequence, unaltered by the primers. PCR reactions, band purifications, and sequencing were performed as described above and the new complete sequence encoded a light chain that coincided with the mass of a Fr 1-21 light chain measured by LC / MS.

Example 10

Expression of reference antibodies

The amino acid sequence of the Morphotech anti-FOLRl antibody, MorAb-003 (Farletuzumab), was obtained from the list of Common International Denominations for Pharmaceutical Substances (INN) of the World Health Organization (WHO), and codons were optimized and were synthesized by Blue Héron Biotechnology. The sequence of the light chain variable region is flanked by the sites for the restriction enzymes EcoRI and BsiWI and the sequence of the variable region of a heavy chain flanked by the sites for the restriction enzyme HindlII and Apal for cloning in the

<img file="AR080301A1_D0125.tif" />

102

<img file="AR080301A1_D0126.tif" />

framework of the respective constant sequences in single chain plasmids for expression in mammals. Cloning, expression and purification were carried out as described for Movl9 and Fr 1-21 humanized cited above.

Example 11

ADCC activity of huMovl9

A lactate dehydrogenase (LDH) release assay was used to measure antibody-dependent cell-mediated cytotoxicity (ADCC) of tumor cell lines by using freshly isolated human natural killer cells (NK) as effector cells (e.g. , Shields, J. Biol. Chem., 276 (9): 6591-6604 (2001)). NK cells were first isolated from human blood from a normal donor (Research Blood Components, Inc., Brighton, MA), using a modified protocol for the NK Isolation Kit II (Miltenyi Biotech, 130-091 -152). The blood was diluted twice with PBS lx. 25 ml of diluted blood were carefully placed over 25 ml of

Ficoll Paque in a 50 ml conical tube and centrifuged at 400 g for 45 min at RT. Peripheral blood mononuclear cells (CMSP) were obtained from the interface, transferred to a new 50 ml conical tube, and washed once with PBS lx. The cells were resuspended in 2 ml of NK isolation buffer (1 x PBS, 0.5% BSA, 2 mM EDTA), and 500 pi of the antibody-biotin cocktail was added to the cell suspension. The antibody-biotin cocktail contains biotinylated antibodies that bind to lymphocytes, with the exception of NK cells, resulting in a negative selection of NK cells. The mixture was incubated at 4 ° C for 10 minutes and then 1.5 ml of NK isolation buffer and 1 ml of anti-biotin micro beads were added. The mixture of antibodies and cells was incubated for another 15 minutes at 4 ° C. The cells were then washed once with 50 ml of NK isolation buffer and resuspended in 3 ml of NK isolation buffer. Then, a MACS LS column was mounted in the autoMACS separator (Miltenyi Biotech) and pre-washed with 3 ml of NK isolation buffer. The cell suspension was automatically applied to the spine, washed and the effluent fraction with the NK cells without a label was collected in a new 50 ml conical tube. The resulting NK cells are

<img file="AR080301A1_D0127.tif" />

103 seeded in 30 ml of complete RPMI medium (RPMI-1640 supplemented with 5% fetal bovine serum, 1% penicillin-streptomycin, 1 mM HEPES, 1 raM sodium pyruvate, 1% 100X MEM non-essential amino acid solution) during the night. Subsequent analysis and all dilutions were performed in RHBP medium (RPMI 1640 medium supplemented with

20 mM HEPES, pH 7.4, 0.1% BSA and 1% streptomycin penicillin). Several concentrations of antibodies in the RHBP medium were distributed in duplicate in 50 μΐ aliquots / well in a round bottom 96-well plate. Target cells were resuspended to IO⁶ cells / ml in RHBP medium and 100 μΐ / well were added in each well containing antibody dilutions. The plate containing the target cells and the antibody dilutions was incubated for 30 minutes at 37 ° C. The NK cells were added to the wells containing the target cells at 50 µΐ / well. The typical ratio was approximately 1 target cell to 3-4 NK cells. At least the following controls were established for each experiment: NK cells alone, target cells alone (spontaneous LDH release), target cells with NK cells (antibody independent LDH release), target cells with 10% Triton X-100 (maximum LDH release). The mixtures were incubated at 37 ° C for 4 hours to allow cell lysis. The plates were centrifuged for 10 minutes at 1200 rpm, and 100 μΐ of the supernatant was carefully transferred to a new flat-bottom 96-well plate. The LDH reaction mixture (100 μΐ / well) of the Cytotoxicity Detection Kit (Roche 1 644 793) was added to each well and incubated at room temperature for 5 to 30 min. The optical density of the samples was measured at 490 nm (OD490). The specific lysis percentage of each sample was determined by using the following formula: specific lysis percentage = (sample value - spontaneous release) / (maximum release - spontaneous release) * 100.

Incubation with huMovl9 led to good ADCC activity against cells

IGROV-1 in the presence of NK effector cells. ADCC activity in IGROV-1 cells was compared for huMovl9, huFR-1-21, Mor003 and chTKl (isotype control) (Figure 6). Treatment with 0.9 ng / ml of huMovl9 resulted in approximately 30% lysis of IGROV-1 cells, similar to the activity observed with the other anti-FOLR1 antibodies. ADCC activity for huMovl9 had an EC50 of 0.20 ng / ml, huFr-1-21 had

<img file="AR080301A1_D0128.tif" />

an EC₅or of 0.11 ng / ml, Mor003 of 0.16 ng / ml and chTKl showed no activity against IGROV-1 cells.

Example 12

Preparation of anti-FOLRl immunoconjugates

Preparation of huMOV19vl.6-sulfo-SPDB-DM4

The exemplary ligand 2-sulfo-SPDB was dissolved in DMA. The huMOV19vl.6 antibody was incubated at 8 mg / ml with a 12-fold molar excess of the 2-sulfo-SPDB ligand for approximately 2 hours at 25 ° C at pH 7.5. The reaction mixture was purified by using a Sephadex ™ G25F column equilibrated with 50 mM potassium phosphate buffer containing 50 mM NaCl, 2 mM EDTA, pH 6.5. DM4 maitansinoid was dissolved in dimethylacetamide (DMA, the final concentration is 5%) and a 1.7-fold molar excess compared to the ligand was added dropwise to the sulfo-SPDB modified antibody. The reaction mixture was adjusted to pH 7.5 with 1 m of HEPES buffer. After overnight incubation at room temperature, the conjugated antibody was purified by Sephadex ™ G25F chromatography equilibrated with 10 mM histidine, 250 mM gliein, 1% sucrose, pH 5.5, the number of DM4 molecules bound per molecule of antibody was determined using the previously reported antibody and maitansinoid extinction coefficients (Widdison, WC, et al., J Med Chem, 49: 4392-4408 (2006)). The percentage of total free maitansinoid species was determined as described above. Conjugates with 3.5-4 DM4 molecules per huMovl9vl.6 antibody were obtained with &lt;1% present as unconjugated maitansinoid.

Preparation of huMOV19vl.6-SPP-DMl

The exemplary N-succinimidyl 4- (2-pyridyldithio) pentanoate (SPP) ligand was dissolved in ethanol. The huMOV19vl.6 antibody was incubated at 8 mg / ml with a 6.5 to 6 fold molar excess of the SPP ligand for approximately 2 hours at room temperature in 50 mM potassium phosphate buffer (pH 6.5) containing 50 mM NaCl, EDTA 2 mM, and 5% ethanol. The modified SPP antibody was diluted twice in PBS, pH 6.5, and modified, with a

<img file="AR080301A1_D0129.tif" />

105 1.5-fold molar excess of DM1 maitansinoid by the addition of a concentrated solution (15-30 mM) of DM1 in dimethylacetamide (DMA). The DMA concentration was adjusted to 5% and after incubation overnight at room temperature, the conjugated antibody was purified by chromatography on 10 mM equilibrated Sephadex ™ G25F, 250 mM glycine, 1% sucrose pH 5.5. The number of linked DM1 molecules per antibody molecule was determined by using the antibody extinction coefficients and DM1 reported above (Liu et al., Proc. Nati. Acad. Sci. United States, 93, 8618-8623 ( nineteen ninety six)). The percentage of free maitansinoid present after the conjugation reaction was determined by injecting 20 to 50 pg of the conjugate into a HiSep ™ column equilibrated in 25% acetonitrile in 100 mM ammonium acetate buffer, pH 7.0 and eluted in acetonitrile. The peak area of ​​the total free maitansinoid species (eluted in the gradient and identified by comparison of elution time with known standards) was measured with an absorbance detector set at a wavelength of 252 nm and compared with the area of peaks related to bound maitansinoid (eluted at the conjugate peak in the flow column through fractions) to calculate the percentage of total free maitansinoid species. Conjugates with 3.5-4 molecules of DM1 by huMOV19vl.6 were obtained with &lt;1% present as unconjugated maitansinoid.

Preparation of huMOV19vl.6 SPDB-DM4

The exemplary ligand N-succinimidyl 4- (2-pyridyldithio) butanoate (SPDB) was dissolved in ethanol. The huMOV19vl.6 antibody was incubated at 8 mg / ml with a 5.5-5 fold molar excess of the SPDB ligand for approximately 2 hours at room temperature in 50 mM potassium phosphate buffer (pH 6.5) containing 50 mM NaCl, EDTA 2 mM, and 3% ethanol. The SPDB-modified antibody was diluted twice in PBS, pH 6.5, and modified, with a 1.5-fold molar excess of DM4 maitansinoid by the addition of a concentrated solution (15-30 mM) of DM4 in dimethylacetamide (DMA). After overnight incubation at room temperature, the conjugated antibody was purified by Sephadex ™ G25F chromatography equilibrated with 10 mM histidine, 250 mM glycine, 1% sucrose pH 5.5. The number of DM4 molecules coupled per antibody molecule

<img file="AR080301A1_D0130.tif" />

105 was determined by using the extinction coefficients for the antibody and maitansinoid previously reported (Widdison, WC, et al., J Med Chem, 49: 4392-4408 (2006)). The percentage of total free maitansinoid species was determined as described above. Conjugates with 3.5-4 DM4 molecules per huMOV19vl.6 antibody were obtained with &lt;1% present as unconjugated maitansinoid.

Preparation of huMOV19vl.O-3-sulfo-mal-DM4

The NHS-3-sulfo-mal ligand and DM4 were dissolved separately in DMA. The ligand and thiol DM4 were mixed in a DMA solution containing 40% 200 mM succinate buffer, 2 mM EDTA, pH5.0 to give a molar ratio of DM4 to ligand of 1.6: 1 and a final DM4 concentration equal to 10 mm The mixture was reacted for 2 hours at 25C. Without purification, the reaction mixture was added so that an equivalent of 9.6 molar excess of the ligand to the antibody was added to a solution of the huMOV19vl.O antibody in phosphate buffer (pH 7.5) under conditions of final conjugation of the 4mg / ml antibody, 90% phosphate buffer / 10% DMA pH 7.5 (v / v). After an overnight incubation at room temperature, the conjugation mixture was purified by Sephadex G25 chromatography equilibrated in PBS pH 7.5. The huMOV19vl.O-3-sulfo-mal-DM4 was then dialyzed in a buffer containing 9.55 mM phosphate, 139.6 mM NaCl, pH6.5. The number of DM4 molecules coupled per antibody molecule was determined using the extinction coefficients of the antibody and maitansinoid previously reported (Widdison, WC, et al., J Med Chem, 49: 4392-4408 (2006)). The percentage of the total free maitansinoid species was determined as described above. Conjugates with 3.5-4 DM4 molecules per huMOV19vl.O antibody were obtained with &lt;1% present as unconjugated maitansinoid.

Preparation of huMOV19vl.0-SMCC-DMl

The NHS-sulfo-SMCC and DM1 ligand were dissolved separately in the DMA. The thiol ligand and DM1 were mixed in a DMA solution containing 40% 200 mM succinate buffer, 2 mM EDTA, pH5.0 to give a DM1 to ligand molar ratio of 1.2: 1 and an equal DM1 final concentration at 3.75 mM. The mixture was reacted

<img file="AR080301A1_D0131.tif" />

107 for 75 minutes at 20 ° C. Without purification, the reaction mixture was added so that an equivalent of 6.4 molar excess of the ligand to antibody was added to a solution of the huMOV19vl.O antibody in phosphate buffer (pH 7.5) under final conjugation conditions of 4mg / ml of antibody , 88% 50 mM potassium phosphate, 50 mM NaCl, 2 mM EDTA, pH

7.5 / 12% DMA pH 7.5 (v / v). After 2 hours of incubation at 20 ° C, the conjugation mixture was purified by Sephadex G25 chromatography equilibrated in PBS pH 7.5. The huMOV19vl.O-SMCC-DMl was then dialyzed in a buffer containing 250 mM glycine, 10 mM histidine pH 5.5. The number of DM1 molecules coupled per antibody molecule was determined using the previously reported antibody and maitansinoid extinction coefficients (Widdison, WC, et al., J Med Chem, 49: 4392-4408 (2006)). The percentage of total free maitansinoid species was determined as described above. Conjugates with 3.5-4 DM1 molecules per huMOV19vl.O antibody were obtained with &lt;2.8% present as unconjugated maitansinoid.

Preparation of huMOV19vl.O-PEG4-mal-DMl

The reagent of step 1 NHS-PEG4-mal-DMl was dissolved in the DMA. The huMovl9vl.O antibody was incubated at 5mg / ml with a 5.7-fold molar excess of NHS-PEG4-mal-DMl overnight at 25 ° C in 50 mM KPi, 50 mM NaCl, 2 mM EDTA, pH 7.5 and 10 % of DMA by volume. The reaction mixture was purified by a Sephadex G25 column equilibrated in

PBS pH 7.5. The huMOV19vl.O-PEG4-mal-DMl was dialyzed in buffer containing 250 mM glycine, 10 mM histidine pH 5.5. The number of DM1 molecules coupled per antibody molecule was determined by using the antibody and maitansinoid extinction coefficients reported previously (Widdison, WC, et al., J Med Chem, 49: 4392-4408 (2006)). The percentage of total free maitansinoid species was determined as described above. Conjugates with 3.5-4 DM1 molecules per huMOV19vl.O antibody were obtained with &lt;1.1% present as unconjugated maitansinoid.

<img file="AR080301A1_D0132.tif" />

108

Example 13

Affinity binding of antibodies and conjugates

The binding affinity of the anti-FOLRl antibodies and their conjugates SPDB-DM4, PEG4Mal-DM4, SMCC-DM1, or anti-FOLRl-sulfo-SPDB-DM4 were analyzed by flow cytometry. SKOV3 cells expressing FOLR1 were incubated with different concentrations of anti-FOLRl antibodies or their conjugates and processed as described above for flow cytometry analysis. Data analysis was performed using CellQuest Pro (BD Biosciences, San Diego, United States) and for each sample the average fluorescence intensity for FL1 (IFM) is exported and plotted based on the concentration of antibodies in a graph semi-logarithmic A dose-response curve was generated by nonlinear regression and the apparent dissociation equilibrium constant (Kd) value of the samples examined for binding to SKOV3 cells was calculated using GraphPad Prism v4 (GraphPad software, San Diego, CA) and was presented in Figure 5. The results show that the conjugation of either DM1 as

DM4 through any of the ligands that were used, does not significantly alter the affinity of any of the antibodies (for example, huMovl9).

Example 14

In vitro cytotoxicity assays

The ability of the exemplary conjugates muFRl-9, muFRl-13, muFRl-22, muFRl23, huFRl-23, muFRl-21, and huFRl-21 to inhibit cell growth was measured by the use of in vitro cytotoxicity assays by the method described in Kovtun YV et al. (Cancer Res 66: 3214-3221 (2006)). A PEG4-mal-DM4 conjugate in various concentrations was added to KB cells expressing FOLR1 in a 96-well plate at

one 000 cells per well in 100 μΐ of complete RPMI medium (RPMI-1640, 10% fetal bovine serum, 2 mM glutamine, 1% gentamicin, all Invitrogen reagents). The antibodies and conjugates were diluted in RPMI complete medium with a 3-fold dilution series and 100 µΐ were added per well. The final concentration was typically in the 3xl0 'range⁸ M at 4.6xl0 '¹² M. The control wells that contain the cells and the

<img file="AR080301A1_D0133.tif" />

109 medium, but lacking conjugates, and wells with medium alone, were included in each test plate. The plates were incubated for four to six days at 37 ° C in a humidified atmosphere with 5% CO2. The WST-8 reagent, 10% v / v (Dojindo Molecular Technologies, Gaithersburg, MD, United States), was added to the wells and the plates were incubated at 37 ° C for 2-6 h. WST-8 was reduced by dehydrogenases in living cells to an orange (maximum product formation that is soluble in tissue culture medium. The amount of formation produced is directly proportional to the number of living cells. The plates were analyzed by absorbance measurement at 450 nm (A450) and at 650 nm (A ^ o) in a multi-well plate reader. First, the background of the opalescence of the cells (A550) was subtracted from A050 · ​​The result of A * 45o was used to determine the fraction of the surviving cells. The absorbance background A * 4so was that of the wells with WST-8 medium only. The fraction of survivors was calculated as follows: Viability percentage = 100 x (A * 45o treated sample - fund A *₄₅o) / (A * 45o untreated sample - fund A *₄₅or). The values ​​of the surviving fraction were plotted against the concentration of the antibody or conjugate in a semi-log plot for each treatment. From these data, IC50 values ​​were determined by using GraphPad Prism v4 (GraphPad software, San Diego, CA) and presented in Figure 5. The results shown in Figure 5 demonstrate that all conjugations are equally active in their cytotoxic potency against KB cells expressing FOLR1. In order to verify the specificity of the anti-FOLRl-maitansinoid conjugates towards FOLR1, their activities were evaluated in the presence of an excess of unconjugated antibodies against KB cells. The addition of excess unconjugated competition antibodies to the conjugates suppressed their cytotoxicity, as seen in Figure 7. These data indicate that the conjugates kill KB cells in an antigen-dependent manner. Additional data demonstrated that huMovl9SPDB-DM4 induced cell cycle arrest in the G2 / M phase in KB cells in in vitro assays.

<img file="AR080301A1_D0134.tif" />

110

Example 15

Efficacy, in vivo, of the huMovl9-PEG4Mal-DM4 and huMovl9-SPDB-DM4 conjugates compared to similar conjugates that are not targeted in a xenograft KB model

The cleavable conjugate huMovl9-SPDB-DM4 that is directed to FOLR1 compared to huC242-SPDB-DM4 that is not targeted, and the non-cleavable conjugate huMovl9-PEG4-Mal-DM4 compared to huC242-PEG4Mal-DM4 that is not targeted they were tested by using an established model of KB cell xenotransplantation implanted subcutaneously in SCID mice. Mice were randomized by body weight in the treatment groups and treated individually (SPDB conjugates) on day 3 after cell inoculation, or three times per week on days 3, 10 and 17 after inoculation of cells with 5 and 10 mg / kg of a conjugate, respectively. The mean tumor volume of the different treatment groups is represented in Figure 8. The treatments, either with huMovl9-SPDB-DM4, or huMovl9-PEG4Mal-DM4 resulted in a decrease in the average tumor volume compared to the PBS control, while treatments with any of the respective conjugates that are not targeted, they produced no significant effect.

Example 16

Efficacy, in vivo, of anti-FOLRl-PEG4Mal-DM4 conjugates in a KB xenograft model

PEG4Mal-DM4 conjugates of the exemplary anti-FOLRl antibodies huMovl9, muFR-1-9, muFR-1-13, muFR-1-22, muFR-1-23, and huFR-1-21 were tested by using an established model of KB cell xenografts implanted subcutaneously in SCID mice. Mice were randomized by body weight in the treatment groups and were treated once on day 3 after inoculation of cells with 10 mg / kg of one of the conjugates listed above or only with PBS. It was previously shown that HuMovl9-PEG4Mal-DM4 is similar to the PEG4Mal-DM4 conjugate of muFR-1-9, muFR-1-13, muFR-1-22, muFR-1-23, and huFR-1-21 in its potency Cytotoxic in vitro. The HuMovl9-PEG4Mal-DM4 and the huFR-l-21-PEG4Mal-DM4 were

<img file="AR080301A1_D0135.tif" />

significantly more potent in vivo than any of the other conjugates, resulting in a more pronounced decrease in average tumor volume (Figures 9 and 10). It was also shown that the potency is dose dependent (Figure 11) and the choice of ligand also plays a role (Figures 12 and 13).

Example 17

Efficacy, in vivo, of anti-FOLRl-sulfo-SPDB-DM4 conjugates in a xenograft models

The anti-FOLRl huMovl9-sulfo-SPDB-DM4 conjugates were tested on three xenografts of ovarian serous adenocarcinoma: OVCAR-3, IGROV-1, and OV-90. Each of these tumor xenografts showed FOLR1 expression levels comparable to the patients' tumors when measured by a calibrated method of immunohistochemical staining (IHC) on sections included in paraffin and fixed in formalin. Mice carrying xenograft of established subcutaneous tumors (approximately 100 mm³) were treated with a single intravenous injection of the huMovl9-sulfo-SPDBDM4 conjugate at 1.2, 2.5 and 5.0 mg / kg (based on antibody concentration; Figures 14-16 show the concentration of conjugated maitansinoid in pg / kg). The conjugate is activated in the three models evaluated. In the OVCAR-3 xenograft, the minimum effective dose (SMD) was 1.2 mg / kg (Figure 14). The highest dose levels were highly active, resulting in complete regression (CR) in 4/6 and 2/6 mice in the 2.5 and 5.0 mg / kg treatment groups, respectively. Treatment with the conjugate resulted in a strong antitumor activity in both models of IGROV-1 and OV-90 xenograft, with a DME of 2.5 mg / kg, with a single injection (Figures 15 and 16). These data demonstrate the strong anti-tumor activity of the huMovl9-sulfo-SPDB-DM4 conjugates against ovarian tumor xenografts with FOLR1 expression levels comparable to patient tumors.

<img file="AR080301A1_D0136.tif" />

112

Example 18

Effect of couplers on the efficacy of immunoconjugates

The anti-FOLRl huMovl9 antibody was coupled to DM1 or DM4 through couplers containing SPP, SPDB, or sulfo SPDB cleavable disulfides, or by the non-cleavable SMCC ligand. The in vitro cytotoxic activity of these conjugates on the KB, IGROV-1 and JEG-3 cell lines was examined. FACS analysis indicated that KB cells (cervix) had&gt; 2,000,000 antibody binding sites per cell. IGROV-1 (ovary) cells had 260,000 binding sites per cell, and JEG-3 (choriocarcinoma) cells had 40,000 binding sites per cell. The results of cytotoxicity in vitro are summarized in Table 2 below. The cleavable conjugates exhibited markedly greater in vitro activities compared to the SMCC conjugates.

Table 2: Effect of immunoconjugate ligands on cytoxicity in vitro.

IC50, nM (n = 3), based- Ab

<td>Cells</td><td>SPP-DM1</td><td>SPDB-DM4</td><td>Sulfo-SPDM-DM4</td><td>SMCC-DM1</td>

<td>KB</td><td> 0.1</td><td> 0.1</td><td> 0.1</td><td> 0.1</td>

<td>Igrov 1</td><td> 0.1</td><td> 0.1</td><td> 0.3</td><td> 1.0</td>

<td>Jeg3</td><td> 0.2</td><td> 0.2</td><td> 3.0</td><td> 20</td>

The in vivo activities of the conjugates in the tumor models of KB and OVCAR-3positives for FOLR1 were also tested. The results shown in Figure 17 demonstrate that the SPDB-DM4 and sulfo-SPDB-DM4 cleavable conjugates are more potent than the non-cleavable SMCC-DM1 conjugate in vivo. In addition, among the cleavable conjugates, the SPP-DM1 conjugate was less active than either SPDB-DM4 or sulfo-SPDB-DM4 conjugate, in both xenograft models (Figure 18). The last two conjugates were equally active against KB tumors, while the sulfo-SPDB-DM4 conjugate was more active against the OVCAR-3 model. The data obtained through the OVCAR-3 model are summarized in Table 3 below.

<img file="AR080301A1_D0137.tif" />

Table 3: Effect of immunoconjugate ligands on tumor size in OVCAR-3 xenograft model.

<td>Conjugate</td><td>Tumor about control (%)</td><td>Answer partial</td><td>Answer complete</td><td>Answer</td>

<td>SPP-DM1</td><td> 54</td><td> 0/6</td><td> 0/6</td><td>Inactive</td>

<td>SPDB-DM4</td><td> 9</td><td> 6/6</td><td> 1/6</td><td>Very active</td>

<td>Sulfo-DPDB-</td><td> 0</td><td> 6/6</td><td> 4/6</td><td>Very active</td>

DM4

These data demonstrate that immunoconjugates containing a cleavable ligand 5 show greater efficacy both in vitro as in vivo, and anti-FOLRl immunoconjugates containing sulfo-SPDB are very active in tumor models.

I Example 19!

ί

Efficacy in vitro and in vivo of the SMCC-DM1 conjugates of the huFRl 10 antibody The Anti-FOLRl huFRl-48, huFRl-49, huFRl-57, and huFRl-65 were conjugated with the SMCC and DM1 ligand and, as described above , the effects on KB cells were analyzed, in vivo, using the xenograft models described above. Although each of the antibodies showed similar efficacy in the KB cell model, the immunocojugates huFRl-48, huFRl-49, huFRl-57, and huFRl-65 showed variable, but significant efficacy in vivo at a dose of 200 pg / kg in a xenograft model system (Table 4 and Figure 19).

Table 4: In vitro and in vivo efficacy of the SMCC-DM1 conjugates of the huFRl antibody

<td>Clone#</td><td>Apparent Affinity (nM)</td><td>HuAbsmcc-DMl activity in KB in vitro (nM)</td><td>HuAbsmcc-DMl activity in vivo</td>

<td>huFRl-48</td><td> 0.13</td><td> 0.05</td><td> +</td>

<td>huFRl-49</td><td> 0.08</td><td> 0.10</td><td> +</td>

<td>huFRl-57</td><td> 0.14</td><td> 0.10</td><td> +</td>

<td>huFRl-65</td><td> 0.15</td><td> 0.10</td><td> +</td>

114

<img file="AR080301A1_D0138.tif" />

huMovl9 0.06

<img file="AR080301A1_D0139.tif" />

All publications, patents, patent applications, Internet sites, and access numbers / sequence databases (including polynucleotide and polypeptide sequences) cited herein are incorporated by reference in their entirety for all purposes, in the same extent as each individual publication, patent, patent application, website, or access number / sequence databases indicated specifically and individually to be incorporated as reference.

<img file="AR080301A1_D0140.tif" />

115

<img file="AR080301A1_D0141.tif" />

SEQUENCES sec. with no. Ident .: 1 - huMovl9 vHC CDR1 GYFMN sec. with no. ID: 2 - huMovl9 vHC CDR2

RIHPYDGDTFYNQKFQG sec. with no. ID: 3 - huMovl9 vHC CDR3

YDGSRAMDY sec. with no. of ident. : 4 - huMovl9 vHC

QVQLVQSGAEWKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDGD

TFYNQKFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQGTT

VTVSS sec. with no. of ident.:5 - nucleic acid sequence huMovl9 HCV aagcttgccaccatgggttggtcatgcatcatcctcttcttggttgcaactgctaccggagtgcacagtcaggtacagctcgtgcagtccg gcgccgaggtggtgaagcctggtgccagcgtgaagatctcctgtaaagccagtggatacacattcaccggttattttatgaattgggtga aacagagcccaggccaatccctcgaatggatagggcgaatccacccatatgacggggacaccttttacaaccagaaattccagggga aagccactctgacagtggacaagagttccaacactgcacacatggagcttctctccctgaccagcgaagacttcgctgtttattactgtac ccgttatgatggttcccgtgcaatggactactggggccaagggaccactgtcaccgtaagttccgccagcaccaagggccc sec. with no. of ident.:6 - amino acid sequence huMovl9 HC

QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDGD 25 TFYNQKFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQGTT

VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF

PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP

CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH

NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ

<img file="AR080301A1_D0142.tif" />

PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS

DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK sec. with num. ID: 7 - huMovl9 vLC CDR1 5 KASQSVSFAGTSLMH sec. with no. of ident.:8 - huMovl9 vLC CDR2 RASNLEA sec. with no. from ident.:9 - huMovl9 vLC CDR3 QQSREYPYT sec. with no. Ident .: 10 - huMovl9 vLCvl.OO

DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLE 15 AGVPDRFSGSGSKTDFTLNISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKR sec. with no. ID: 11 - huMovl9 vLCvl.60

DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLE

AGVPDRFSGSGSKTDFTLTISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKR sec. with no. ID: 12 - huMovl9 LCvl.OO

DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLE

AGVPDRFSGSGSKTDFTLNISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKRTVAAP

SVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD

STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC sec. with no. ID: 13 - huMovl9 LCvl.60

DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLE

AGVPDRFSGSGSKTDFTLTISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKRTVAAPS

<img file="AR080301A1_D0143.tif" />

117

VFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS

TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC sec. with no. ident .: 14 - nucleic acid 00 5 huMovl9 LCvl gaattcgccaccatgggctggagctgcattatcctttttctggtagccacagctacaggcgtgcatagcgatatcgtgctgacacaatccc ccctctctctggccgtgtcactcggacagcccgctatcatcagctgcaaagccagccagtctgtcagcttcgctggaacaagtcttatgc attggtatcatcagaagcctggccagcaacccaggctgctgatctatcgagcctcaaacttggaagcaggagtgccagaccggttttctg ggtccgggagtaaaaccgattttacacttaatatctcacctgtcgaggccgaggacgccgccacctactactgtcagcagagccgagag tacccttacacttttggcggtgggactaaactggaaataaaacgtacg sec. with no. of ident.:15 - nucleic acid huMovl9 LCvl.60 gaattcgccaccatgggctggtcttgtatcatcctgtttctggtggccaccgcaaccggtgttcactccgacattgtgctgacacagtcccc cctttcactggctgtatccctcggccagcccgctatcatcagctgcaaggctagccagagcgtgagttttgccggcacttcacttatgcatt ggtaccatcagaaaccaggccagcaacctaggctgctgatttatcgggctagcaacctggaggccggcgtgcccgaccgctttagcg ggagcggctccaagactgacttcactctgaccatctcccccgtagaagcagaagatgctgcaacctactactgtcagcagtctcgcgag tatccttatacattcggaggcggaactaaactggagattaaacgtacg sec. with no. Ident .: 16 - muMovl9 vHC CDR2 RIHPYDGDTFYNQNFKD sec. with no. of ident..T7 - muMovl9 vHC_CAA68252

QVQLQQSGAELVKPGASVKISCKASGYSFTGYFMNWVKQSHGKSLEWIGRIHPYDGD

TFYNQNFKDKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQGTT

VTVS sec. with no. ID: 18 - muMovl9 vLC_CAA68253

DIELTQSPASLAVSLGQRAIISCKASQSVSFAGTSLMHWYHQKPGQQPKLLIYRASNLE

AGVPTRFSGSGSKTDFTLNIHPVEEEDAATYYCQQSREYPYTFGGGTKL sec. with no. ID: 19 - chMovl9 HC

<img file="AR080301A1_D0144.tif" />

QVQLQQSGAELVKPGASVKISCKASGYSFTGYFMNWVKQSHGKSLEWIGRIHPYDGD

TFYNQNFKDKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQGTT

VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF

PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP

CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK sec. with no. ID: 20 - chMovl9 LC

DIELTQSPASLAVSLGQRAIISCKASQSVSFAGTSLMHWYHQKPGQQPKLLIYRASNLE

AGVPTRFSGSGSKTDFTLNIHPVEEEDAATYYCQQSREYPYTFGGGTKLEIKRTVAAP

SVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD

STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC sec. with no. of ident.:21 - nucleic acid chMovl9 HC aagcttgccaccatgggttggtcttgtattatcctctttctcgtcgcaaccgcaacaggcgtccattcacaagtccaactgcagcaatccgg cgccgaactcgttaaacctggagcatctgttaaaatctcatgtaaagcatcaggatactcatttactggctattttatgaactgggtcaaaca atcacacggaaaatcacttgaatggatcggacgtattcacccctatgatggcgatactttttacaaccagaacttcaaagacaaagctaca gatcaagagctatggattattggggacaaggaacaacagtcacagtctcatctgcatcaactaagggccca ctcaccgttgacaaatcatctaacaccgctcacatggaactcctttcactcacatctgaagacttcgctgtttattactgtactagatacgatg sec. with no. of ident.:22 - nucleic acid LC chMovl9 gaattcgccaccatgggttggtcttgtattatcctctttctcgtcgcaaccgcaacaggcgtccattcagatatcgaactcacacaatcacc agcttccctcgcagtctctctcggtcaacgcgcaatcatctcttgtaaagcctcccaatcagtctcattcgccggcacgtccctcatgcatt ggtaccatcaaaaacccggtcagcaacccaaactccttatctatagagcaagcaacctcgaagcaggcgttcccaccagatttagcgga tcaggaagtaaaaccgatttcacactcaacattcatccagtcgaagaagaagatgcagctacttattattgccaacagtctagagaatatc catacacattcggagggggtaccaaacttgaaattaaacgtacg sec. with no. ID: 23 - muMovl9 vHC_CAA68252

<img file="AR080301A1_D0145.tif" />

119

<img file="AR080301A1_D0146.tif" />

QVQLQQSGAELVKPGASVKISCKASGYSFTGYFMNWVKQSHGKSLEWIGRIHPYDGD

TFYNQNFKDKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQGTT

VTVS sec. with no. from ident.:24 - muMovl9 vLC_CAA68253

DIELTQSPASLAVSLGQRAIISCKASQSVSFAGTSLMHWYHQKPGQQPKLLIYRASNLE

AGVPTRFSGSGSKTDFTLNIHPVEEEDAATYYCQQSREYPYTFGGGTKL sec. with no. ID: 25 - Human folate receptor 1 MAQRMTTQLLLLLVWVAVVGEAQTRIAWARTELLNVCMNAKHHKEKPGPEDKLHE

QCRPWRKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSP

NLGPWIQQVDQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSG

FNKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPN

EEVARFYAAAMSGAGPWAAWPFLLSLALMLLWLLS

SEC. WITH NUMBER DE IDENT.:26 - human folate receptor 1 nucleic acid sequence

Atggctcagcggatgacaacacagctgctgctccttctagtgtgggtggctgtagtaggggaggctcagacaaggattgcatgggcca ggactgagcttctcaatgtctgcatgaacgccaagcaccacaaggaaaagccaggccccgaggacaagttgcatgagcagtgtcgac cctggaggaagaatgcctgctgttctaccaacaccagccaggaagcccataaggatgtttcctacctatatagattcaactggaaccact gtggagagatggcacctgcctgcaaacggcatttcatccaggacacctgcctctacgagtgctcccccaacttggggccctggatcca gcaggtggatcagagctggcgcaaagagcgggtactgaacgtgcccctgtgcaaagaggactgtgagcaatggtgggaagattgtc gcacctcctacacctgcaagagcaactggcacaagggctggaactggacttcagggtttaacaagtgcgcagtgggagctgcctgcc aacctttccatttctacttccccacacccactgttctgtgcaatgaaatctggactcactcctacaaggtcagcaactacagccgagggagt ggccgctgcatccagatgtggttcgacccagcccagggcaaccccaatgaggaggtggcgaggttctatgctgcagccatgagtggg gctgggccctgggcagcctggcctttcctgcttagcctggccctaatgctgctgtggctgctcagc sec. with no. ID: 27 - FR1-21 vLC CDR1 KASDHINNWLA sec. with no. ID: 28 - FRI-21 vLC CDR2

GATSLET

<img file="AR080301A1_D0147.tif" />

sec. with no. ID: 29 - FR1-21 vLC CDR3 QQYWSTPFT sec. with no. ID 30: FRI-21 vHC CDR1 SSYGMS sec. with no. ID: 31 - FR1-21 vHC CDR2 TISSGGSYTY sec. with no. ID: 32 - FR1-21 vHC CDR3 DGEGGLYAMDY sec. with no. ID: 33 - FR1-21 Murine Kabat CDR-H2

TISSGGSYTYYPDGVKG sec. with no. ID: 34 - FR1-21 Human Kabat CDR-H2 TISSGGSYTYYSPGFQG sec. with no. from ident.:35 - muFRl-21 vLC

DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISGATSLETGV

PSRFSGSGSGKDYTLSISSLQTEDVATYYCQQYWSTPFTFGSGTKLEIKR sec. with no. from ident.:36 - muFRl-21 vHC

EVKLVESGGDLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLECVATISSGGSY TYYPDGVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARDGEGGLYAMDYWGQ GTSVTVSS sec. with no. of ident.:37 - muFRl-21 vLC DNA sequence

<img file="AR080301A1_D0148.tif" />

gacatccagatgacacaatcttcatcctacttgtctgtatctctaggaggcagagtcaccattacttgcaaggcaagtgaccacataaataa ttggttagcctggtatcagcagaaaccaggaaatgctcctaggctcttaatatctggtgcaaccagtttggaaactggggttccttcaagat tcagtggcagtggatctggaaaggattacactctcagcatttccagtcttcagactgaagatgttgctacttattactgtcaacagtattgga gtactccattcacgttcggctcggggacaaagttggaaataaaacg sec. with no. of ident.:38 - muFRl-21HCvarPat gaagtgaagctggtggagtctgggggagacttagtgaagcctggagggtccctgaaactctcctgtgcagcctctggattcactttcagt agctatggcatgtcttgggttcgccagactccagacaagaggttggagtgtgtcgcaaccattagtagtggtggtagttacacctactatc cagacggtgtgaaggggcgattcaccatctccagagacaatgccaagaacaccctgtacctgcaaatgagcagtctgaagtctgagga cacagccatgtattactgtgcaagggacggcgaggggggcctctatgctatggactactggggtcaaggaacctcagtcaccgtctcct ca sec. with no. of ident.:39 - muFRl-21 LC

DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISGATSLETGV

PSRFSGSGSGKDYTLSISSLQTEDVATYYCQQYWSTPFTFGSGTKLEIKRADAAPTVSÏ FPPSSEQLTSGGASWCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYS MSSTLTLTKCEYKKRKNYNKRKNYK. with no. ID: 40 - muFRl-21 HC

EVKLVESGGDLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLECVATISSGGSY TYYPDGVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARDGEGGLYAMDYWGQ GTSVTVSSAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSV HTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCP PCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVWDVSEDDPDVQISWFVN

NVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKI KGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPV LDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK sec. with no. from ident.:41 - huFRl-21 vLC

122

<img file="AR080301A1_D0149.tif" />

DIQMTQSSSSLSVSVGGRVTITCKASDHINNWLAWYQQKPGKAPKLLISGATSLETGV

PSRFSGSGSGKDYTLSISSLQPEDVATYYCQQYWSTPFTFGQGTKLEIKR sec. with no. from ident.:42 - huFRl-21 vHC

EVQLVESGGDVVKPGGSLKLSCAASGFTFSSYGMSWVRQTPGKGLECVATISSGGSY TYYSPGFQGRFTISRDKSKNTLYLQMSSLKAEDTAMYYCARDGEGGLYAMDYWGQ GTSVTVSS sec. with no. of ident.:43 - huFRl-21VH_co aagcttgccaccatgggatggtcatgcatcattctttttctcgtcgccactgccacaggtgtgcattccgaggtgcaacttgtagaatctgg cggggatgttgtgaagcctggaggtagtctcaagttgtcctgtgctgcatctgggtttaccttctcttcctacggaatgagctgggtgagac agactcctggcaaggggctggagtgcgttgccaccattagtagtggaggttcttacacctactattcacctggttttcagggacgctttaca atctcccgcgataagtctaagaacaccctttacctccagatgagtagccttaaggctgaggacacagccatgtattattgcgctcgcgatg gggagggagggctttacgctatggactactggggccagggtaccagcgtgaccgtttcctctgctagtaccaagggccc sec. with no. of ident.:44 - huFR21 VLco gaattcgccaccatgggatggtcatgtatcattctgttcttggtagcaacagcaactggcgtccattctgacatccagatgacccaatcctc cagcagcttgtcagtatccgttgggggccgcgttactattacctgtaaggcctccgaccatataaataactggcttgcatggtatcaacag aagcctgggaaggcacctaaactgcttatctctggggccacaagcctggagaccggcgtgccttccaggttctctggaagtggatctgg caaggactataccttgagcattagtagccttcaacctgaggacgtcgccacctactattgtcagcagtattggtctacaccctttacctttgg acagggcactaaattggagataaaacgtacg sec. with no. ID: 45 - huFRl-21 LC

DIQMTQSSSSLSVSVGGRVTITCKASDHINNWLAWYQQKPGKAPKLLISGATSLETGV

PSRFSGSGSGKDYTLSISSLQPEDVATYYCQQYWSTPFTFGQGTKLEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKGKSNRKVKSKRNGVKSRVGSKRVGSKRKVNGVKRKDKRGSKRGSKRKGNGKRGKDGSKRGKSKRGGNGKRGGNGKRGGNGGGNGDGNGRKKRKKRGNGKRKKRGNGNKRGNGKRGNGKRGNGKRGNGKRGNGNGKRKKRKVNGVKRNGVGNGKRKKRKVNGVKRNGVGNGKRKKRKVNGVKRNGVGNGKGG with no. from ident.:46 - huFRl-21 HC

<img file="AR080301A1_D0150.tif" />

EVQLVESGGDVVKPGGSLKLSCAASGFTFSSYGMSWVRQTPGKGLECVATISSGGSY

TYYSPGFQGRFTISRDKSKNTLYLQMSSLKAEDTAMYYCARDGEGGLYAMDYWGQ

GTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV

HTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT

CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG sec. with no. of ident.:47 - DNA sequence huFRl-21LC gacatccagatgacccaatcctccagcagcttgtcagtatccgttgggggccgcgttactattacctgtaaggcctccgaccatataaata actggcttgcatggtatcaacagaagcctgggaaggcacctaaactgcttatctctggggccacaagcctggagaccggcgtgccttcc aggttctctggaagtggatctggcaaggactataccttgagcattagtagccttcaacctgaggacgtcgccacctactattgtcagcagt attggtctacaccctttacctttggacagggcactaaattggagataaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctg atgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataa tgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttca cgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgc acaggggagagtgt sec. with no. of ident.:48 - DNA sequence huFRl-21HC gaggtgcaacttgtagaatctggcggggatgttgtgaagcctggaggtagtctcaagttgtcctgtgctgcatctgggtttaccttctcttcc tacggaatgagctgggtgagacagactcctggcaaggggctggagtgcgttgccaccattagtagtggaggttcttacacctactattca cctggttttcagggacgctttacaatctcccgcgataagtctaagaacaccctttacctccagatgagtagccttaaggctgaggacacag ccatgtattattgcgctcgcgatggggagggagggctttacgctatggactactggggccagggtaccagcgtgaccgtttcctctgcta gtaccaagggcccatcagttttccccttggctccaagttctaaatccacaagcggtggaacagctgcactgggatgcctcgttaaagatta tttccctgagcctgtgacagtgagctggaatagcggagcattgacttcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgt actcactgtccagtgtcgtaaccgtcccttctagcagcttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaa ggtggataagaaggttgaaccaaagagctgtgataagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgt gttcctgtttccccccaaacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatc ccgaggttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattctacatat

<img file="AR080301A1_D0151.tif" />

cgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaacaaggctcttcccgc tcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgcccccatctagagacgagctgacc aagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtagagtgggagtctaacggacagccagaaaac aactacaagacaactcccccagtgctggacagcgacgggagcttcttcctctactccaagttgactgtagacaagtctagatggcagca aggaaacgttttctcctgctcagtaatgcatgaggctctgcacaatcactatacccagaaatcactgtcccttagcccaggg sec. with no. of EcoRI DNA ident.:secuencia to Xbal49 - huFolRl gaattcgccaccatggcacagcgcatgaccactcagctcctgcttctgttggtttgggtggcagtcgtgggagaggcccagaccaggat tgcttgggcacgcacagagctgcttaatgtttgcatgaacgcaaagcaccataaagagaaacccggtcccgaggataagttgcacgaa cagtgccgcccttggagaaagaatgcatgctgtagcacgaacacctctcaggaggcgcataaagacgtaagctatttgtatagatttaac tggaaccattgcggtgaaatggcacctgcctgtaaacggcactttatccaggatacttgcttgtacgagtgtagcccgaatctcgggccct ggattcagcaagttgatcagagttggcgcaaagagagggtgctgaacgttccgctttgcaaggaggactgcgagcaatggtgggaag actgtagaaccagctacacctgtaagtctaactggcacaaaggatggaactggacatccgggtttaacaaatgcgctgtcggcgctgcc tgccagccatttcatttctactttccaactcccactgtcctgtgtaacgagatttggacgcattcatataaagtcagcaactacagccggggc tccggccgctgcattcagatgtggttcgaccctgcacagggcaaccctaacgaggaggtcgcacgcttctacgctgcagcaatgtctgg agccggtccttgggctgcttggccatttctccttagcctcgccctcatgcttctctggctgttgtcataatctaga sec. with no. Ident .: 50 - EcoMHl Primer CTTCCGGAATTCSARGTNMAGCTGSAGSAGTC sec. with no. ID: 51 - Primer EcoMH2 CTTCCGGAATTCSARGTNMAGCTGSAGSAGTCWGG sec. with no. from ident.:52 - BamlgGl primer

GGAGGATCCATAGACAGATGGGGGTGTCGTTTTGGC sec. with no. ID: 53 - SacIMK GGAGCTCGAYATTGTGMTSACMCARWCTMCA sec. with no. from ident.:54 - HindKL

TATAGAGCTCAAGCTTGGATGGTGGGAAGATGGATACAGTTGGTGC

<img file="AR080301A1_D0152.tif" />

The mixed bases are defined as follows: N = G + A + T + C, S = G + C, Y = C + T, M = A + C, R = A + G, W = A + T sec. with no. from ident.:55 - cd37-lLClead ttttgaattcgccaccatgaagtttccttctcaacttct sec. with no. of ident.:56 - CDR2 of Movl9 human and chimeric HCV compound RIHPYDGDTFYNQXaaiFXaa₂Xaa₃

Xaai = Q, H, K, or R Xaa₂ = R, Q, Η, ο N Xaa₃ = E, T, S, G, A, ο V sec. with no. ID: 57 - FRl-48vL CDR1

RASENIYSNLA sec. with no. ID: 58 - FRl-48vL CDR2 AATNLAD sec. with no. ID: 59 - FRl-48vL CDR3 QHFWASPYT sec. with no. ID: 60 - FRl-48vH CDR1 TNYWMQ sec. with no. ID: 61 - FRl-48vH CDR2 AIYPGNGDSR sec. with no. ID: 62 - FRl-48vH CDR3

RDGNYAAY

<img file="AR080301A1_D0153.tif" />

126

<img file="AR080301A1_D0154.tif" />

sec. with no. of ident.:63 - FRl-49vL CDR1 RASENIYTNLA sec. with no. ID: 64 - FRl-49vL CDR2 TASNLAD sec. with no. ID: 65 - FRl-49vL CDR3 QHFWVSPYT sec. with no. from ident.:66 - FRl-49vH CDR1 TNYWMY sec. with no. ID: 67 - FRl-49vH CDR2 15 AIYPGNSDTT sec. with no. of ident.:68 - FRl-49vH CDR3 RHDYGAMDY sec. with no. from ident.:69 - FRl-57vL CDR1 RASQNINNNLH sec. with no. ID: 70 - FRl-57vL CDR2 YVSQSVS sec. with no. ID: 71 - FRl-57vL CDR3 QQSNSWPHYT sec. with no. ID: 72 - FRl-57vH CDR1

SSFGMH

<img file="AR080301A1_D0155.tif" />

127

<img file="AR080301A1_D0156.tif" />

sec. with no. ID: 73 - FRl-57vH CDR2 YISSGSSTIS sec. with no. from ident.:74 - FRl-57vH CDR3 EAYGSSMEY sec. with no. ID: 75 - FRl-65vL CDR1 KASQNVGPNVA sec. with no. of ident.:76 - FRl-65vL CDR2 SASYRYS sec. with no. ID: 77 - FRl-65vL CDR3 15 QQYNSYPYT sec. with no. ID: 78 - FRl-65vH CDR1 TSYTMH sec. with no. from ident.:79 - FRl-65vH CDR2 YINPISGYTN sec. with no. ID: 80 - FRl-65vH CDR3 GGAYGRKPMDY sec. with no. ID: 81 - CDR2 muFRl-48 defined from Kabat HC AIYPGNGDSRYTQKFKG sec. with no. ID: 82 - CDR2 huFRl-48 defined by Kabat HC

AIYPGNGDSRYTQKFQG

<img file="AR080301A1_D0157.tif" />

128

<img file="AR080301A1_D0158.tif" />

sec. with no. ID: 80 - muFRl-49 defined from Kabat HC AIYPGNSDTT YNLKFKG sec. with no. ID: 83 - CDR2 huFRl-49 defined from Kabat HC AIYPGNSDTTYNQKFQG sec. with no. ID: 84 - CDR2 muFRl-57 defined from Kabat HC YISSGSSTISYADTVKG sec. with no. ID: 85 - CDR2 huFRl-57 defined from Kabat HC YISSGSSTISYADSVKG sec. with no. ID: 86 - CDR2 muFRl-65 defined by Kabat HC

YINPISGYTNYNQKFKD sec. with no. ID: 87 - CDR2 huFRl-65 defined from Kabat HC YINPISGYTNYNQKFQG sec. with no. ID: 88 - muFRl-48vL

DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYAATNLADG

VPSRFSGSESGTQYSLKINSLQSEDFGSYYCQHFWASPYTFGGGTKLEIKR sec. with no. from ident.:89 - muFRl-48vH

QVQLQQSGAELARPGASVKLSCRASGYTFTNYWMQWIKQRPGQGLEWIGAIYPGNG DSRYTQKFKGKATLTADKSSSTAYMQVSSLTSEDSAVYYCARRDGNYAAYWGQGTL VTVSA sec. with no. ID: 90 - muFRl-49vL

<img file="AR080301A1_D0159.tif" />

129

<img file="AR080301A1_D0160.tif" />

DIQMTQSPASLSVSVGETVTITCRASENIYTNLAWYQQKQGKSPQLLVYTASNLADG

VPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWVSPYTFGGGTKLEIKR sec. with no. from ident.:91 - muFRl-49vH

EVQLQQSGTVLARPGASVKMSCKASGYKFTNYWMYWIKQRPGQGLELIGAIYPGNS DTTYNLKFKGKAKLTAVTSANTVYMEVSSLTNEDSAVYYCTKRHDYGAMDYWGQG TSVTVSS sec. with no. ID: 92 - muFRl-57vL

DIVLTQSPATLSVTPGDSVSLSCRASQNINNNLHWYQQKSHESPRLLIKYVSQSVSGIP SRFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPHYTFGGGTKLEIKR sec. with no. ID: 93 - muFRl-57vH

DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISSGSST 15 ISYADTVKGRFTISRDNSKKTLLLQMTSLRSEDTAMYYCAREAYGSSMEYWGQGTSV

TVSS sec. with no. ID: 94 - muFRl-65vL

DIVMTQSQKFMSTSVGDRVSVTCKASQNVGPNVAWYQQKPGQSPKALIYSASYRYS 20 EVPDRFTGSGSGTDFTLTISNMQSADLAEYFCQQYNSYPYTFGGGTKLEIKR sec. with no. ID: 95 - muFRl-65vH

QVQLQQSGAELARPGASVKMSCKASGYTFTSYTMHWVKQRPGQGLAWIGYINPISG

YTNYNQKFKDKATLTADKSSSTAYMQLNSLTSEDSAVYYCASGGAYGRKPMDYWG

QGTSVTVSS sec. with no. from ident.:96 - huFRl-48vL

DIQMTQSPSSLSVSVGERVTITCRASENIYSNLAWYQQKPGKSPKLLVYAATNLADGV

PSRFSGSESGTDYSLKINSLQPEDFGSYYCQHFWASPYTFGQGTKLEIKR

<img file="AR080301A1_D0161.tif" />

130

<img file="AR080301A1_D0162.tif" />

sec. with no. ID: 97 - huFRl-48vH

QVQLVQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQRPGQGLEWIGAIYPGNG

DSRYTQKFQGKATLTADKSSSTAYMQVSSLTSEDSAVYYCARRDGNYAAYWGQGTL

VTVSA sec. with no. ID: 98 - huFRl-49vL

DIQMTQSPSSLSVSVGERVTITCRASENIYTNLAWYQQKPGKSPKLLVYTASNLADGV

PSRFSGSGSGTDYSLKINSLQPEDFGTYYCQHFWVSPYTFGQGTKLEIKR sec. with no. of ident.:99 - huFRl-49vH

QVQLQQSGAVVAKPGASVKMSCKASGYTFTNYWMYWIKQRPGQGLELIGAIYPGNS

DTTYNQKFQGKATLTAVTSANTVYMEVSSLTSEDSAVYYCTKRHDYGAMDYWGQG

TSVTVSS sec. with no. Ident .: 100 - huFRl-57vL

EIVLTQSPATLSVTPGDRVSLSCRASQNINNNLHWYQQKPGQSPRLLIKYVSQSVSGIP

DRFSGSGSGTDFTLSISSVEPEDFGMYFCQQSNSWPHYTFGQGTKLEIKR sec. with no. ID: 101 - huFRl-57vH

EVQLVESGGGLVQPGGSRRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSGSST

ISYADSVKGRFTISRDNSKKTLLLQMTSLRAEDTAMYYCAREAYGSSMEYWGQGTL

VTVSS sec. with no. ID: 102 - huFRl-65vL

EIVMTQSPATMSTSPGDRVSVTCKASQNVGPNVAWYQQKPGQSPRALIYSASYRYSG

VPARFTGSGSGTDFTLTISNMQSEDLAEYFCQQYNSYPYTFGQGTKLEIKR sec. with no. ID: 103 - huFRl-65vH

<img file="AR080301A1_D0163.tif" />

131

QVQLVQSGAEVAKPGASVKMSCKASGYTFTSYTMHWVKQRPGQGLAWIGYINPISG

YTNYNQKFQGKATLTADKSSSTAYMQLNSLTSEDSAVYYCASGGAYGRKPMDYWG

QGTSVTVSS sec. with no. ID: 104 - muFRl-48LC

DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYAATNLADG

VPSRFSGSESGTQYSLKINSLQSEDFGSYYCQHFWASPYTFGGGTKLEIKRADAAPTVS

IFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYS

MSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC sec. with no. ID: 105 - muFRl-48HC

QVQLQQSGAELARPGASVKLSCRASGYTFTNYWMQWIKQRPGQGLEWIGAIYPGNG

DSRYTQKFKGKATLTADKSSSTAYMQVSSLTSEDSAVYYCARRDGNYAAYWGQGTL

VTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHT

FPAVLESDLYTLSSSVTVPSSMRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTV PEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPRE EQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIP PPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSK LNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK sec. with mim. ID: 106 - muFRl-49LC

DIQMTQSPASLSVSVGETVTITCRASENIYTNLAWYQQKQGKSPQLLVYTASNLADG

VPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWVSPYTFGGGTKLEIKRADAAPTV

SIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTY SMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFN RNEC sec. with no. ID: 107 - muFRl-49HC

<img file="AR080301A1_D0164.tif" />

EVQLQQSGTVLARPGASVKMSCKASGYKFTNYWMYWIKQRPGQGLELIGAIYPGNS

DTTYNLKFKGKAKLTAVTSANTVYMEVSSLTNEDSAVYYCTKRHDYGAMDYWGQG

TSVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVH

TFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCK

CPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTA QTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVR APQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDS DGSYFMYSKLRVEKKNWVERNSYSCSWHEGLHNHHTTKSFSRTPGK sec. with no. ID: 108 - muFRl-57LC

DIVLTQSPATLSVTPGDSVSLSCRASQNINNNLHWYQQKSHESPRLLIKYVSQSVSGIP

SRFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPHYTFGGGTKLEIKRADAAPTVSI

FPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYS

MSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSF

NRNEC sec. with no. ID: 109 - muFRl-57HC

DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISSGSST

ISYADTVKGRFTISRDNSKKTLLLQMTSLRSEDTAMYYCAREAYGSSMEYWGQGTSV

TVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFP AVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPA PNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQ THREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQV YVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYF

MYSKLRVEKKNWVERNSYSCSWHEGLHNHHTTKSFSRTPGK sec. with no. ID: 110 - muFRl-65LC

DIVMTQSQKFMSTSVGDRVSVTCKASQNVGPNVAWYQQKPGQSPKALIYSASYRYS

EVPDRFTGSGSGTDFTLTISNMQSADLAEYFCQQYNSYPYTFGGGTKLEIKRADAAPT

133

<img file="AR080301A1_D0165.tif" />

VSIFPPSSEQLTSGGASWCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDST

YSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC sec. with no. ID: 111 - muFRl-65HC

QVQLQQSGAELARPGASVKMSCKASGYTFTSYTMHWVKQRPGQGLAWIGYINPISG YTNYNQKFKDKATLTADKSSSTAYMQLNSLTSEDSAVYYCASGGAYGRKPMDYWG QGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSG VHTFPAVLESDLYTLSSSVTVPSSMRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCI CTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQ

PREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVY TIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFV YSKLNVQKSLTCSGHNHLS. with no. ID: 112 - huFRl-48LC

DIQMTQSPSSLSVSVGERVTITCRASENIYSNLAWYQQKPGKSPKLLVYAATNLADGV PSRFSGSESGTDYSLKINSLQPEDFGSYYCQHFWASPYTFGQGTKLEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC sec. with no. ID: 113 - huFRl-48HC

QVQLVQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQRPGQGLEWIGAIYPGNG

DSRYTQKFQGKATLTADKSSSTAYMQVSSLTSEDSAVYYCARRDGNYAAYWGQGTL

VTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF

PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP

CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG sec. with no. ID: 114 - huFRl-49LC

<img file="AR080301A1_D0166.tif" />

DIQMTQSPSSLSVSVGERVTITCRASENIYTNLAWYQQKPGKSPKLLVYTASNLADGV

PSRFSGSGSGTDYSLKINSLQPEDFGTYYCQHFWVSPYTFGQGTKLEIKRTVAAPSVFI

FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS

LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC sec. with no. ID: 115 - huFRl-49HC

QVQLQQSGAVVAKPGASVKMSCKASGYTFTNYWMYWIKQRPGQGLELIGAIYPGNS

DTTYNQKFQGKATLTAVTSANTVYMEVSSLTSEDSAVYYCTKRHDYGAMDYWGQG

TSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH

TFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG sec. with no. ID: 116 - huFRl-57LC

EIVLTQSPATLSVTPGDRVSLSCRASQNINNNLHWYQQKPGQSPRLLIKYVSQSVSGIP

DRFSGSGSGTDFTLSISSVEPEDFGMYFCQQSNSWPHYTFGQGTKLEIKRTVAAPSVFI

FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS

LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC sec. with no. ID: 117 - huFRl-57HC

EVQLVESGGGLVQPGGSRRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSGSST

ISYADSVKGRFTISRDNSKKTLLLQMTSLRAEDTAMYYCAREAYGSSMEYWGQGTL

VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV

LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

135

<img file="AR080301A1_D0167.tif" />

sec. with no. ID: 118 - huFRl-65LC

EIVM TQSPATMSTSPGDRVSVTCKASQNVGPNVAWYQQKPGQSPRALIYSASYRYSG

VPARFTGSGSGTDFTLTISNMQSEDLAEYFCQQYNSYPYTFGQGTKLEIKRTVAAPSV

FIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC sec. with no. ID: 19 - huFRl-65HC

QVQLVQSGAEVAKPGASVKMSCKASGYTFTSYTMHWVKQRPGQGLAWIGYINPISG

YTNYNQKFQGKATLTADKSSSTAYMQLNSLTSEDSAVYYCASGGAYGRKPMDYWG QGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA

KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDLAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG sec. with no. ident .: 120 - huFRl-48_VL gaattcgccaccatgggatggagttgtatcatcctgtttcttgtggctacagccacaggggtacactccgatattcaaatgacacagtccc cttcatccctgtccgtcagtgtgggggaaagggttaccatcacctgccgtgcatcagagaacatctattccaacctcgcctggtaccaac agaaacctggcaagtcccctaagctgttggtctacgccgctacaaacctcgccgatggggtgccttcccgtttcagtgggtcagagtca ggcaccgactattctctgaagatcaactccctccagcctgaggatttcggctcctattactgtcagcacttctgggctagtccatatactttc ggccagggaaccaaacttgaaattaaacgtacg sec. with no. ident .: 121 - huFRl-48_VH aagcttgccaccatggggtggagctgcatcatcctttttctggtggccactgccaccggcgtgcactctcaggtccaacttgtgcagagc ggagccgaggtggccaaacccggagctagtgttaagctctcatgtaaagcatctggctacacctttactaactactggatgcagtggatc aagcaacggccaggccagggcctggagtggattggtgctatttatcccggaaacggggatagcaggtacactcagaaatttcaggga aaggctacccttaccgccgataagagttcttccacagcatatatgcaagtctcctctctgacctcagaggatagtgctgtctattactgcgct cgccgggatggcaactatgcagcctattggggtcaaggcacccttgtgactgtatccgcagcaagcaccaagggccc

136

<img file="AR080301A1_D0168.tif" />

sec. with no. of ident.:122 - huFRl-49_VL gaattcgccaccatgggttggtcatgcattatcctgtttctggtcgcaacagcaacaggtgtgcacagtgacattcagatgacccaaagcc cctccagtctgagcgtttccgtgggggaacgtgtcactatcacatgcagagcttccgagaatatttacactaacctcgcatggtaccagca gcaccgactattctttgaaaattaattccctgcagcctgaggattttggtacctactattgccagcatttttgggtatcaccatacacttttgga gaaacccgggaagtctccaaaacttctcgtatatacagccagcaacttggcagatggggtgcccagccggtttagcggatctggttcag cagggaacaaagctggagatcaagcgtacg sec. with no. ident .: 123 - huFRl-49_VH aagcttgccaccatgggctggtcttgtattattctttttcttgtggccacagccacaggagtccattcacaggtacagctccaacagtctggc gcagttgtcgccaagcccggcgcctctgtgaagatgagttgcaaggcctctggctacaccttcactaattattggatgtactggatcaaac aacgccccggccagggtctggaactcattggagccatctacccaggcaactccgacacaacatacaatcagaagtttcagggcaaag caaccctgaccgctgtaacctcagctaataccgtgtacatggaggtaagtagcttgactagtgaagattccgcagtatactattgcaccaa gcgccatgattacggcgccatggattactggggccaaggtaccagtgtgaccgtgtcttccgcttccaccaagggccc sec. with no. ident .: 124 - huFRl-57_VL gaattcgccaccatgggctggtcatgcattattttgttcctggtcgccaccgcaaccggcgttcattccgaaattgttcttactcagagccct gcaaccttgagtgtgacacccggcgatcgggtctcactgagttgcagagcttcccagaatatcaacaataatctgcactggtatcagcag aagcctggccagtctcctcgcttgctgattaagtatgtctcacagagcgtgtcaggtatccctgaccgtttctccgggtcaggttcaggca ccgacttcacactgtccatttctagcgtggagcctgaggatttcggaatgtacttttgccagcagagcaatagctggcctcactacaccttt ggccaagggaccaagctggagatcaagcgtacg sec. with no. ident .: 125 - huFRl-57_VH aagcttgccaccatgggctggagctgtatcatcttgttccttgtggccacagctactggcgtgcactccgaggtgcagctggtcgaatcc ggcggaggcctggtgcagcctggggggagtagacggctgtcctgcgctgcctctgggtttactttctcaagtttcggtatgcactgggtg cgtcaggcccccgggaagggcctggaatgggttgcttatatatcatctggcagctccaccatttcttatgctgattccgttaagggacgctt caccatttccagagacaacagtaagaaaacccttctgctgcagatgacctctctccgcgccgaagacaccgcaatgtattattgtgctag agaggcctacggcagtagtatggaatactgggggcaggggaccctggtgaccgtgtcttccgcatctactaagggccc sec. with no. ID: 126 - huFRl-65_VL l

Ì!

i î

J i

I

Ì

I '&lt;

ij

j

<img file="AR080301A1_D0169.tif" />

gaattcgccaccatgggctggtcttgcattattctgttcctggttgcaacagccactggcgtccattccgaaatcgtgatgacccaatctcc cgccaccatgtctacctctcccggggaccgggtgtctgtgacctgcaaggcctctcagaatgttggcccaaacgtggcatggtatcaac agaaaccagggcagtcacccagagccctgatttactccgcttcttacagatattcaggagttcccgcccggttcacaggtagtgggtccg gcactgactttaccttgaccatttccaacatgcaatccgaggacctggccgaatacttctgtcagcagtacaattcatatccctatacattcg gccaggggaccaagctggaaataaagcgtacg sec. with no. ID: 127 - huFRl-65_VH

Aagcttgccaccatgggctggtcatgcataatcctgttcctggtcgcaaccgctacaggtgtacactcccaggtgcagttggtgcagag cggggccgaagttgctaagcccggtgcaagtgtaaaaatgtcctgcaaagctagcgggtacacattcacatcctatactatgcattgggt aaaacagcgcccaggacaggggctcgcctggataggctatattaacccaatatcaggatacacaaactacaatcagaaatttcaggga aaggcaaccctgaccgccgacaagtcctcttctaccgcatatatgcagctcaactccctgaccagtgaagatagcgcagtgtattactgt gcctccggcggtgcttatggccggaaacccatggattactggggacaaggcacctccgtcacagtgagtagcgcctcaaccaagggc ce sec. with no. ID: 128 - CDR2 defined by Kabat Movl9 HC Murino RIHPYDGDTFYNQNFKD sec. with no. ID: 129 - CDR2 defined by Kabat Movl9 HC Human RIHPYDGDTFYNQKFQG}

hee

!

<img file="AR080301A1_D0170.tif" />

138

<img file="AR080301A1_D0171.tif" />

SEQUENCE LIST &lt;110&gt; Immunogen, Inc.

AB, Olga

TAVARES, Daniel

RUI, Lingyun

PAYNE, Gillian

GOLDMAKHER, Viktor S.

&lt;120&gt; Antibodies and Immunoconjugates of Folate Receptor 1 and its uses &lt;130&gt;

&lt;140&gt;

&lt;141&gt;

&lt;150&gt;

&lt;151&gt;

&lt;150&gt;

&lt;151&gt;

&lt;150&gt;

&lt;151&gt;

&lt;160&gt;

2921.002GC03

To be assigned

attached

US 61 / 413,172

2010-11-12

US 61 / 346,595

2010-05-20

US 61 / 307,797

2010-02-24

130

<img file="AR080301A1_D0172.tif" />

139 &lt;170&gt; Patentln version 3.5 &lt;210&gt; 1 &lt;211&gt; 5 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; huMovl9 vHC CDR1 &lt;400&gt; 1

Gly Tyr Phe Met Asn

one &lt;210&gt; 2 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; huMovl9 vHC CDR2 &lt;400&gt; 2!

Ì

I i

!

i

Item'

<img file="AR080301A1_D0173.tif" />

140

Arg Ile His Pro Tyr Asp Gly Asp Thr Phe Tyr Asn Gin Lys Phe Gin

Gly &lt;210&gt; 3 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;2 23&gt; huMovl9 vHC CDR3 &lt;400&gt; 3

Tyr Asp Gly Ser Arg Wing Met Asp Tyr

one 5 &lt;210&gt; 4 &lt;211&gt; 118 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence

<img file="AR080301A1_D0174.tif" />

&lt;220&gt;

&lt;2 2 3&gt; huMovl9 vHC j

?

ä j

j &lt;400&gt; 4!

I 5 ï

jj Gin Val Gin Leu Val Gin Ser Gly Ala Glu Val Val Lys Pro Gly Ala!

j 15 10 15 ï

I j

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

25 30

Phe Met Asn Trp Val Lys Gin Ser Pro Gly Gin Ser Leu Glu Trp Ile

35 40 45

<td>j</td><td> 20</td><td>Gly</td><td>Arg fifty</td><td colspan="2">Ile His</td><td colspan="3">Pro Tyr Asp 55</td><td>Gly</td><td colspan="2">Asp Thr</td><td>Phe</td><td colspan="2">Tyr asn 60</td><td>Gin</td><td>Lys</td><td>Phe</td>

<td>j i</td><td></td><td>Gin</td><td>Gly</td><td>Lys</td><td>To</td><td>Thr</td><td>Leu</td><td>Thr</td><td>Val</td><td>Asp</td><td>Lys</td><td>Be</td><td>Be</td><td>Asn</td><td>Thr</td><td>To</td><td>His</td>

<td> 1 1</td><td></td><td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>j j one F</td><td> 25</td><td>Met</td><td>Glu</td><td>Leu</td><td>Leu</td><td>Be</td><td>Leu</td><td>Thr</td><td>Be</td><td>Glu</td><td>Asp</td><td>Phe</td><td>To</td><td>Val</td><td>Tyr</td><td>Tyr</td><td>Cys</td>

<td>j</td><td></td><td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<img file="AR080301A1_D0175.tif" />

142

Thr Arg Tyr Asp Gly Ser Arg Wing Met Asp Tyr Trp Gly Gin Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115 &lt;210&gt; 5 &lt;211&gt; 440 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;2 2 3&gt; huMovl9 HCV &lt;400&gt; 5 aagcttgcca ccatgggttg gtcatgcatc atcctcttct tggttgcaac tgctaccgga gtgcacagtc aggtacagct cgtgcagtcc ggcgccgagg tggtgaagcc tggtgccagc gtgaagatct cctgtaaagc cagtggatac acattcaccg gttattttat gaattgggtg aaacagagcc caggccaatc cctcgaatgg atagggcgaa tccacccata tgacggggac

120

180

240

<img file="AR080301A1_D0176.tif" />

143 accttttaca ccaggggaaa accagaaatt gccactctga cagtggacaa actgcacaca gagttccaac tggagcttct ctccctgacc agcgaagact tcgctgttta ttactgtacc cgttatgatg gttcccgtgc aatggactac tggggccaag ggaccactgt caccgtaagt tccgccagca ccaagggccc &lt;210&gt; 6 &lt;211&gt; 448 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;2 2 3&gt; huMovl9 HC &lt;400&gt; 6

Gin Val Gin Leu Val Gin Ser Gly Ala Glu Val Val Lys Pro Gly Ala

10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

<img file="AR080301A1_D0177.tif" />

j Phe Met Asn Trp Val Lys Gin Ser Pro Gly Gin Ser Leu Glu Trp Ile l 35 40 45 î

i 5 Gly Arg Ile His Pro Tyr Asp Gly Asp Thr Phe Tyr Asn Gin Lys Phe

I j 50 55 60 ί Gin Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala His j 10 65 70 75 80 j

I î Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Phe Ala Val Tyr Tyr Cys j

i 85 90 95 j

j 15 j Thr Arg Tyr Asp Gly Ser Arg Wing Met Asp Tyr Trp Gly Gin Gly Thr i

«? 100 105 110 î

ii

i? ²⁰ j Thr Val Thr Val Ser Be Ala Ser Thr Lys Gly Pro Ser Val Phe Pro î

j 115 120 125

I j

i) 25 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly ï 130 135 140

<img file="AR080301A1_D0178.tif" />

145

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Be Gly Ala Leu Thr Be Gly Val His Thr Phe Pro Ala Val Leu Gin

165 170 175

Be Be Gly Leu Tyr Be Leu Be Be Val Val Thr Val Pro Be Be

180 185 190

Ser Leu Gly Thr Gin Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Giu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Giu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

<img file="AR080301A1_D0179.tif" />

146

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Be His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys

355 360 365

<img file="AR080301A1_D0180.tif" />

£

147

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Wing Val Glu Trp Glu Ser

370 375 380

Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Be Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met Hìs Giu Ala

420 425 430

Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445 &lt;210&gt; 7 &lt;211&gt; 15 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence

<img file="AR080301A1_D0181.tif" />

148 &lt;220&gt;

&lt;223&gt; huMovl9 vLC CDR1 &lt;400&gt; 7

Lys Ala Ser Gin Ser Val Ser Phe Ala Gly Thr Ser Leu Met His

10 15 &lt;210&gt; 8 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; huMovl9 vLC CDR2 &lt;400&gt; 8

Arg Ala Being Asn Leu Glu Ala

5

<td></td><td> &lt;210&gt;</td><td> 9</td>

<td> 25</td><td> &lt;211&gt;</td><td> 9</td>

<td></td><td> &lt;212&gt;</td><td>PRT</td>

<td></td><td> &lt;213&gt;</td><td>Artificial Sequence</td>

<img file="AR080301A1_D0182.tif" />

&lt;220&gt;

&lt;2 23&gt; huMovl9 vLC CDR3 &lt;400&gt; 9

Gin Gin Ser Arg Glu Tyr Pro Tyr Thr

5 &lt;210&gt; 10 &lt;211&gt; 112 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; huMovl9 vLCvl.OO &lt;400&gt; 10

Asp Ile Val Leu Thr Gin Ser Pro Leu Ser Leu Ala Val Ser Leu Gly

10 15

Gin Pro Ala Ile Ile Ser Cys Lys Ala Ser Gin Ser Val Ser Phe Ala

<img file="AR080301A1_D0183.tif" />

150

Gly Thr Ser Leu Met His Trp Tyr His Gin Lys Pro Gly Gin Gin Pro

40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ala Gly Val Pro Asp

55 60

Arg Phe Ser Gly Ser Gly Ser Lys Thr Asp Phe Thr Leu Asn Ile Ser

70 75 80

Pro Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Ser Arg

90 95

Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110 &lt;210&gt; 11 &lt;211&gt; 112 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

<img file="AR080301A1_D0184.tif" />

151

<img file="AR080301A1_D0185.tif" />

&lt;223&gt; huMovl9 vLCvl.60 &lt;400&gt; 11

Asp Ile Val Leu Thr Gin Ser Pro Leu Ser Leu Ala Val Ser Leu Gly

10 15

Gin Pro Ala Ile Ile Ser Cys Lys Ala Ser Gin Ser Val Ser Phe Ala

Gly Thr Ser Leu Met His Trp Tyr His Gin Lys Pro Gly Gin Gin Pro

40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ala Gly Val Pro Asp

55 60

Arg Phe Ser Gly Ser Gly Ser Lys Thr Asp Phe Thr Leu Thr Ile Ser

70 75 80

Pro Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Ser Arg

<img file="AR080301A1_D0186.tif" />

Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110 &lt;210&gt; 12 &lt;211&gt; 218 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; huMovl9 LCvl.OO &lt;400&gt; 12

Asp Ile Val Leu Thr Gin Ser Pro Leu Ser Leu Ala Val Ser Leu Gly

10 15

Gin Pro Ala Ile Ile Ser Cys Lys Ala Ser Gin Ser Val Ser Phe Ala

25 30

Gly Thr Ser Leu Met His Trp Tyr His Gin Lys Pro Gly Gin Gin Pro

<img file="AR080301A1_D0187.tif" />

153

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ala Gly Val Pro Asp

55 60

Arg Phe Ser Gly Ser Gly Ser Lys Thr Asp Phe Thr Leu Asn Ile Ser

70 75 80

Pro Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Ser Arg

85 90 95

Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg i

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gin

115 120 125 i !

I ²⁰ 1

I

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr | ξ

130 135 140 j

I

Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Î

Î

145

150

155

160

<img file="AR080301A1_D0188.tif" />

Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 &lt;210&gt; 13 &lt;211&gt; 218 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; huMovl9 LCvl.60 &lt;400&gt; 13 ï

<img file="AR080301A1_D0189.tif" />

<img file="AR080301A1_D0190.tif" />

Leu Ala Val

Ser Leu Gly ì

:ï

Asp Ile Val

Leu Thr Gin

Ser

Pro

Leu

Ser

Gin Pro Ala Ile Ile Ser Cys Lys Ala Ser Gin Ser Val Ser Phe Ala

25 30

Gly Thr Ser Leu Met His Trp Tyr His Gin Lys Pro Gly Gin Gin Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ala Gly Val Pro Asp

55 60

Arg Phe Ser Gly Ser Gly Ser Lys Thr Asp Phe Thr Leu Thr Ile Ser

70 75 80

Pro Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Ser Arg

90 95

Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110 f

j i

i

<img file="AR080301A1_D0191.tif" />

156

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Giu Gin

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser

145 150 155 160

Gly Asn Ser Gin Giu Ser Val Thr Giu Gin Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Giu Lys

180 185 190

His Lys Val Tyr Ala Cys Giu Val Thr His Gin Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Giu Cys

210 215

<img file="AR080301A1_D0192.tif" />

157 &lt;210&gt; 14 &lt;211&gt; 408 &lt;212&gt; ADN &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; huMovl9 LCvl.OO acido nucleico &lt;400&gt; 14 gaattcgcca ccatgggctg gagctgcatt atcctttttc tggtagccac agctacaggc gtgcatagcg atatcgtgct gacacaatcc cccctctctc tggccgtgtc actcggacag 120 cccgctatca tcagctgcaa agccagccag tctgtcagct tcgctggaac aagtcttatg 180 cattggtatc atcagaagcc tggccagcaa cccaggctgc tgatctatcg agcctcaaac 240 ttggaagcag gagtgccaga ccggttttct gggtccggga gtaaaaccga ttttacactt 300 aatatctcac ctgtcgaggc cgaggacgcc gccacctact actgtcagca gagccgagag 360 tacccttaca cttttggcgg tgggactaaa ctggaaataa aacgtacg

408 &lt;210&gt; 15

158

<img file="AR080301A1_D0193.tif" />

&lt;211&gt; 408 &lt;212&gt; ADN &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; huMovl9 LCvl.60 &lt;400&gt; 15 gaattcgcca ccatgggctg gtcttgtatc atcctgtttc tggtggccac cgcaaccggt gttcactccg acattgtgct gacacagtcc cccctttcac tggctgtatc cctcggccag 120 cccgctatca tcagctgcaa ggctagccag agcgtgagtt ttgccggcac ttcacttatg 180 cattggtacc atcagaaacc aggccagcaa cctaggctgc tgatttatcg ggctagcaac 240 ctggaggccg gcgtgcccga ccgctttagc gggagcggct ccaagactga cttcactctg 300 accatctccc ccgtagaagc agaagatgct gcaacctact actgtcagca gtctcgcgag 360 tatccttata cattcggagg cggaactaaa ctggagatta aacgtacg

408

<td></td><td> &lt;210&gt;</td><td> 16</td>

<td> 25</td><td> &lt;211&gt;</td><td> 17</td>

<td></td><td> &lt;212&gt;</td><td>PRT</td>

&lt;213&gt; Secuencia Artificial

<img file="AR080301A1_D0194.tif" />

159 &lt;220&gt;

&lt;223&gt; muMovl9 vHC CDR2 &lt;400&gt; 16

Arg Ile His Pro Tyr Asp Gly Asp Thr Phe Tyr Asn Gin Asn Phe Lys

10 15

Asp &lt;210&gt; 17 &lt;211&gt; 117 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; muMovl9 vHC_CAA68252 &lt;400&gt; 17

Gin Val Gin Leu Gin Gin Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

<img file="AR080301A1_D0195.tif" />

160

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

25 30

Phe Met Asn Trp Val Lys Gin Ser His Gly Lys Ser Leu Glu Trp Ile

40 45

Gly Arg Ile His Pro Tyr Asp Gly Asp Thr Phe Tyr Asn Gin Asn Phe

55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala His

65 70 75 80

Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Phe Ala Val Tyr Tyr Cys

90 95

Thr Arg Tyr Asp Gly Ser Arg Ala Met Asp Tyr Trp Gly Gin Gly Thr

100 105 110

Thr Val Thr Val Ser

115

<img file="AR080301A1_D0196.tif" />

&lt;210&gt; 18 &lt;211&gt; 108 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; muMovl9 vLC_CAA68253 &lt;400&gt; 18

Asp Ile Glu Leu Thr Gin Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

10 15

Gin Arg Ala Ile Ile Ser Cys Lys Ala Ser Gin Ser Val Ser Phe Ala

25 30

Gly Thr Ser Leu Met His Trp Tyr His Gin Lys Pro Gly Gin Gin Pro

40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ala Gly Val Pro Thr

<img file="AR080301A1_D0197.tif" />

162

Arg Phe Ser Gly Ser Gly Ser Lys Thr Asp Phe Thr Leu Asn Ile His

70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Ser Arg

90 95

Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu

100 105 &lt;210&gt; 19 &lt;211&gt; 448 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; chMovl9 HC &lt;400&gt; 19

Gin Val Gin Leu Gin Gin Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

1

<img file="AR080301A1_D0198.tif" />

163

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

25 30

Phe Met Asn Trp Val Lys Gin Ser His Gly Lys Ser Leu Glu Trp Ile

40 45

Gly Arg Ile His Pro Tyr Asp Gly Asp Thr Phe Tyr Asn Gin Asn Phe

55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala His

70 75 80 i Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Phe Ala Val Tyr Tyr Cys j 85 90 95

Thr Arg Tyr Asp Gly Ser Arg Ala Met Asp Tyr Trp Gly Gin Gly Thr

100 105 110 ί 25 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

Ì 115 120 125 j

j j

j {' i

<img file="AR080301A1_D0199.tif" />

164 ì

s j

j j

<img file="AR080301A1_D0200.tif" />

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130

135

140 j

j i

$ ì

j s

i

I

Cys Leu Val Lys

145

Ser Gly Ala Leu

Ser Ser Gly Leu

180

Ser Leu Gly Thr

195

Asn Thr Lys Val

210

Asp Tyr Phe Pro Glu Pro

150

Thr Ser Gly Val His Thr

165 170

Tyr Ser Leu Ser Ser Val

185

Gin Thr Tyr Ile Cys Asn

200

Asp Lys Lys Val Glu Pro

215

Val Thr Val Ser Trp Asn

155 160

Phe Pro Ala Val Leu Gin

175

Val Thr Val Pro Ser Ser

190

Val Asn His Lys Pro Ser

205

Lys Ser Cys Asp Lys Thr

220

I

Î

J

I ?

i.

His Thr Cys Pro Pro Cys Pro Ala Pro Giu Leu Leu Gly Gly Pro Ser

225 230 235 240

I i

i

<img file="AR080301A1_D0201.tif" />

165

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Giu Val Thr Cys Val Val Val Asp Val Ser His Giu Asp Pro

260 265 270

Giu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Giu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu

340 345 350

<img file="AR080301A1_D0202.tif" />

166

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445 &lt;210&gt; 20

<img file="AR080301A1_D0203.tif" />

167 &lt;211&gt; 218 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; chMovl9 LC &lt;400&gt; 20

Asp Ile Glu Leu Thr Gin Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

10 15

Gin Arg Ala Ile Ile Ser Cys Lys Ala Ser Gin Ser Val Ser Phe Ala

25 30

Gly Thr Ser Leu Met His Trp Tyr His Gin Lys Pro Gly Gin Gin Pro

40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ala Gly Val Pro Thr

55 60

Arg Phe Ser Gly Ser Gly Ser Lys Thr Asp Phe Thr Leu Asn Ile His

70 75 80 i

<img file="AR080301A1_D0204.tif" />

168

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Ser Arg

90 95

Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gin

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser

145 150 155 160

Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

169

<img file="AR080301A1_D0205.tif" />

His Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 &lt;210&gt; 21 &lt;211&gt; 441 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; chMovl9 HC acido nucleico &lt;400&gt; 21

Ala Ala Gly Cys Thr Thr Gly Cys Cys Ala Cys Cys Ala Thr Gly Gly

10 15

Gly Thr Thr Gly Gly Thr Cys Thr Thr Gly Thr Ala Thr Thr Ala Thr

<img file="AR080301A1_D0206.tif" />

170

Cys Cys Thr Cys Thr Thr Thr Cys Thr Cys Gly Thr Cys Gly Cys Ala

40 45

Ala Cys Cys Gly Cys Ala Ala Cys Ala Gly Gly Cys Gly Thr Cys Cys

55 60

Ala Thr Thr Cys Ala Cys Ala Ala Gly Thr Cys Cys Ala Ala Cys Thr

70 75 80

Gly Cys Ala Gly Cys Ala Ala Thr Cys Cys Gly Gly Cys Gly Cys Cys

85 90 95

Gly Ala Ala Cys Thr Cys Gly Thr Thr Ala Ala Ala Cys Cys Thr Gly

100 105 110

Gly Ala Gly Cys Ala Thr Cys Thr Gly Thr Thr Ala Ala Ala Ala Thr

115 120 125

130

135

140

Cys Thr Cys Ala Thr Gly Thr Ala Ala Ala Gly Cys Ala Thr Cys Ala

<img file="AR080301A1_D0207.tif" />

Gly Gly Ala Thr Ala Cys Thr Cys Ala Thr Thr Thr Ala Cys Thr Gly

145 150 155 160

Gly Cys Thr Ala Thr Thr Thr Thr Ala Thr Gly Ala Ala Cys Thr Gly

165 170 175

Gly Gly Thr Cys Ala Ala Ala Cys Ala Ala Thr Cys Ala Cys Ala Cys

180 185 190

Gly Gly Ala Ala Ala Ala Thr Cys Ala Cys Thr Thr Gly Ala Ala Thr

195 200 205

Gly Gly Ala Thr Cys Gly Gly Ala Cys Gly Thr Ala Thr Thr Cys Ala

210 215 220

Cys Cys Cys Cys Thr Ala Thr Gly Ala Thr Gly Gly Cys Gly Ala Thr

225 230 235 240

Ala Cys Thr Thr Thr Thr Thr Ala Cys Ala Ala Cys Cys Ala Gly Ala

245

250

255

<img file="AR080301A1_D0208.tif" />

172

<img file="AR080301A1_D0209.tif" />

Ala Cys Thr Thr Cys Ala Ala Ala Gly Ala Cys Ala Ala Ala Gly Cys

260 265 270

Thr Ala Cys Ala Cys Thr Cys Ala Cys Cys Gly Thr Thr Gly Ala Cys

275 280 285

Ala Ala Ala Thr Cys Ala Thr Cys Thr Ala Ala Cys Ala Cys Cys Gly

290 295 300

Cys Thr Cys Ala Cys Ala Thr Gly Gly Ala Ala Cys Thr Cys Cys Thr

305 310 315 320

Thr Thr Cys Ala Cys Thr Cys Ala Cys Ala Thr Cys Thr Gly Ala Ala

325 330 335

Gly Ala Cys Thr Thr Cys Gly Cys Thr Gly Thr Thr Thr Ala Thr Thr

340 345 350

Ala Cys Thr Gly Thr Ala Cys Thr Ala Gly Ala Thr Ala Cys Gly Ala

355

360

365

<img file="AR080301A1_D0210.tif" />

Thr Gly Gly Ala Thr Cys Ala Ala Gly Ala Gly Cys Thr Ala Thr Gly

370 375 380

Gly Ala Thr Thr Ala Thr Thr Gly Gly Gly Gly Ala Cys Ala Ala Gly

385 390 395 400

Gly Ala Ala Cys Ala Ala Cys Ala Gly Thr Cys Ala Cys Ala Gly Thr

405 410 415

Cys Thr Cys Ala Thr Cys Thr Gly Cys Ala Thr Cys Ala Ala Cys Thr

420 425 430

Ala Ala Gly Gly Gly Cys Cys Cys Ala

435 440

<td> &lt;210&gt;</td><td> 22</td>

<td> &lt;211&gt;</td><td> 408</td>

<td> &lt;212&gt;</td><td>ADN</td>

<td> &lt;213&gt;</td><td>Secuencia Artificial</td>

<img file="AR080301A1_D0211.tif" />

174

<img file="AR080301A1_D0212.tif" />

&lt;220&gt;

&lt;223&gt; chMovl9 LC àcido nucleico &lt;400&gt; 22 gaattcgcca ccatgggttg gtcttgtatt atcctctttc tcgtcgcaac cgcaacaggc gtccattcag atatcgaact cacacaatca ccagcttccc tcgcagtctc tctcggtcaa

120 cgcgcaatca tctcttgtaa agcctcccaa tcagtctcat tcgccggcac gtccctcatg cattggtacc atcaaaaacc cggtcagcaa cccaaactcc ttatctatag agcaagcaac

180

240 ctcgaagcag gcgttcccac cagatttagc ggatcaggaa gtaaaaccga tttcacactc

300 aacattcatc cagtcgaaga agaagatgca gctacttatt attgccaaca gtctagagaa

360 tatccataca cattcggagg gggtaccaaa cttgaaatta aacgtacg

408

<td> 20</td><td> &lt;210&gt; &lt;211&gt;</td><td> 23 117</td>

<td></td><td> &lt;212&gt;</td><td>PRT</td>

<td></td><td> &lt;213&gt;</td><td>Secuencia Artificial</td>

<td> 25</td><td> &lt;220&gt;</td><td></td>

&lt;223&gt; muMovl9 vHC_CAA68252

<img file="AR080301A1_D0213.tif" />

&lt;400&gt; 23

Gin Val Gin Leu Gin Gin Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

25 30

Phe Met Asn Trp Val Lys Gin Ser His Gly Lys Ser Leu Glu Trp Ile

40 45

Gly Arg Ile His Pro Tyr Asp Gly Asp Thr Phe Tyr Asn Gin Asn Phe

55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala His

65 70 75 80

Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Phe Ala Val Tyr Tyr Cys

90 95

Thr Arg Tyr Asp Gly Ser Arg Ala Met Asp Tyr Trp Gly Gin Gly Thr

100 105 110

<img file="AR080301A1_D0214.tif" />

176

Thr Val Thr Val Ser

115 &lt;210&gt; 24 &lt;211&gt; 108 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; muMovl9 vLC CAA68253 &lt;400&gt; 24

Asp Ile Glu Leu Thr Gin Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

10 15

Gin Arg Ala Ile Ile Ser Cys Lys Ala Ser Gin Ser Val Ser Phe Ala

25 30

Gly Thr Ser Leu Met His Trp Tyr His Gin Lys Pro Gly Gin Gin Pro

40 45

<img file="AR080301A1_D0215.tif" />

177

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ala Gly Val Pro Thr

55 60

Arg Phe Ser Gly Ser Gly Ser Lys Thr Asp Phe Thr Leu Asn Ile His

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Ser Arg

Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu

100 105 &lt;210&gt; 25 &lt;211&gt; 257 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; receptor 1 de folato humano &lt;400&gt; 25

<img file="AR080301A1_D0216.tif" />

Met Ala Gin Arg Met Thr Thr Gin Leu Leu Leu Leu Leu Val Trp Val

10 15

Ala Val Val Gly Glu Ala Gin Thr Arg Ile Ala Trp Ala Arg Thr Glu

25 30

Leu Leu Asn Val Cys Met Asn Ala Lys His His Lys Glu Lys Pro Gly

35 40 45

Pro Glu Asp Lys Leu His Glu Gin Cys Arg Pro Trp Arg Lys Asn Ala

55 60

Cys Cys Ser Thr Asn Thr Ser Gin Glu Ala His Lys Asp Val Ser Tyr

70 75 80

Leu Tyr Arg Phe Asn Trp Asn His Cys Gly Glu Met Ala Pro Ala Cys

90 95

Lys Arg His Phe Ile Gin Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn

100 105 110

<img file="AR080301A1_D0217.tif" />

Leu Gly Pro Trp Ile Gin Gin Val Asp Gin Ser Trp Arg Lys Glu Arg

115 120 125

Val Leu Asn Val Pro Leu Cys Lys Glu Asp Cys Glu Gin Trp Trp Glu

130 135 140

Asp Cys Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp

145 150 155 160

Asn Trp Thr Ser Gly Phe Asn Lys Cys Ala Val Gly Ala Ala Cys Gin

165 170 175

Pro Phe His Phe Tyr Phe Pro Thr Pro Thr Val Leu Cys Asn Glu Ile

180 185 190

Trp Thr His Ser Tyr Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg

195 200 205

Cys Ile Gin Met Trp Phe Asp Pro Ala Gin Gly Asn Pro Asn Glu Glu

210

215

220

<img file="AR080301A1_D0218.tif" />

180

Val Ala Arg Phe Tyr Ala Ala Ala Met Ser Gly Ala Gly Pro Trp Ala

225 230 235 240

Ala Trp Pro Phe Leu Leu Ser Leu Ala Leu Met Leu Leu Trp Leu Leu

245 250 255

Ser &lt;210&gt; 26 &lt;211&gt; 771 &lt;212&gt; ADN &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; secuencia de acido nucleico del receptor 1 de folato humano &lt;400&gt; 26 atggctcagc ggatgacaac acagctgctg ctccttctag tgtgggtggc tgtagtaggg gaggctcaga caaggattgc atgggccagg actgagcttc tcaatgtctg catgaacgcc

120 aagcaccaca aggaaaagcc aggccccgag gacaagttgc atgagcagtg tcgaccctgg

180

<img file="AR080301A1_D0219.tif" />

181 aggaagaatg cctgctgttc taccaacacc agccaggaag cccataagga tgtttcctac

240 ctatatagat tcaactggaa ccactgtgga gagatggcac ctgcctgcaa acggcatttc

300 atccaggaca cctgcctcta cgagtgctcc cccaacttgg ggccctggat ccagcaggtg

360 gatcagagct ggcgcaaaga gcgggtactg aacgtgcccc tgtgcaaaga ggactgtgag

420 caatggtggg aagattgtcg cacctcctac acctgcaaga gcaactggca caagggctgg

480 aactggactt cagggtttaa caagtgcgca gtgggagctg cctgccaacc tttccatttc

540 tacttcccca cacccactgt tctgtgcaat gaaatctgga ctcactccta caaggtcagc aactacagcc gagggagtgg ccgctgcatc cagatgtggt tcgacccagc ccagggcaac

600

660 cccaatgagg aggtggcgag gttctatgct gcagccatga gtggggctgg gccctgggca

720 gcctggcctt tcctgcttag cctggcccta atgctgctgt ggctgctcag c

771

<td></td><td> &lt;210&gt;</td><td> 27</td>

<td></td><td> &lt;211&gt;</td><td> 11</td>

<td> 25</td><td> &lt;212&gt;</td><td>PRT</td>

&lt;213&gt; Secuencia Artificial

<img file="AR080301A1_D0220.tif" />

182 &lt;220&gt;

&lt;223&gt; FR1-21 vLC CDR1 &lt;400&gt; 27

Lys Ala Ser Asp His Ile Asn Asn Trp Leu Ala &lt;210&gt; 28 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; FR1-21 vLC CDR2 &lt;400&gt; 28

Gly Ala Thr Ser Leu Glu Thr

5

<td></td><td> &lt;210&gt;</td><td> 29</td>

<td> 25</td><td> &lt;211&gt;</td><td> 9</td>

<td></td><td> &lt;212&gt;</td><td>PRT</td>

<td></td><td> &lt;213&gt;</td><td>Secuencia Artificial</td>

183

<img file="AR080301A1_D0221.tif" />

&lt;220&gt;

&lt;223&gt; FR1-21 vLC CDR3 &lt;400&gt; 29

Gin Gin Tyr Trp Ser Thr Pro Phe Thr

5 &lt;210&gt; 30 &lt;211&gt; 6 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; FR1-21 vHC CDRl &lt;400&gt; 30

Ser Ser Tyr Gly Met Ser

5 &lt;210&gt; 31 &lt;211&gt; 10 &lt;212&gt; PRT

<img file="AR080301A1_D0222.tif" />

184

<img file="AR080301A1_D0223.tif" />

&lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; FRl-21 vHC CDR2 &lt;400&gt; 31

Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr

10 &lt;210&gt; 32 &lt;211&gt; 11 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; FRl-21 vHC CDR3 &lt;400&gt; 32

Asp Gly Glu Gly Gly Leu Tyr Ala

Met Asp Tyr &lt;210&gt; 33 &lt;211&gt; 17

185

<img file="AR080301A1_D0224.tif" />

&lt;212&gt;

&lt;213&gt;

PRT

Secuencia Artificial &lt;220&gt;

&lt;223&gt; CDR-H2 FR1-21 murino de Kabat &lt;400&gt; 33

Thr Ile Ser Ser Gly Gly Ser

Tyr Thr Tyr Tyr Pro Asp Gly Val Lys

1

Gly &lt;210&gt; 34 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; CDR-H2 FR1-21 humano de Kabat &lt;400&gt; 34

Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Ser Pro Gly Phe Gin

<img file="AR080301A1_D0225.tif" />

186

Gly &lt;210&gt; 35 &lt;211&gt; 108 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; muFRl-21 vLC &lt;400&gt; 35

Asp Ile Gin Met Thr Gin Ser Ser Ser Tyr Leu Ser Val Ser Leu Gly

10 15

Gly Arg Val Thr Ile Thr Cys Lys Ala Ser Asp His Ile Asn Asn Trp

25 30

Leu Ala Trp Tyr Gin Gin Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile

<img file="AR080301A1_D0226.tif" />

Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

55 60

Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile Ser Ser Leu Gin Thr

70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gin Gin Tyr Trp Ser Thr Pro Phe

90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg

100 105 &lt;210&gt; 36 &lt;211&gt; 120 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; muFRl-21 vHC &lt;400&gt; 36

<img file="AR080301A1_D0227.tif" />

e

188

Glu Val Lys Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

25 30

Gly Met Ser Trp Val Arg Gin Thr Pro Asp Lys Arg Leu Glu Cys Val

40 45

Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Gly Val

55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

70 75 80

Leu Gin Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys

90 95

Ala Arg Asp

Gly

Glu Gly Gly

Leu Tyr

Ala Met Asp

Tyr

Trp Gly Gin

100 105 110

<img file="AR080301A1_D0228.tif" />

189

Gly Thr Ser Val Thr Val Ser Ser

115 120 &lt;210&gt; 37 &lt;211&gt; 323 &lt;212&gt; ADN &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; Secuencia de ADN muFRl-21 vLC &lt;400&gt; 37 gacatccaga tgacacaatc ttcatcctac ttgtctgtat ctctaggagg cagagtcacc attacttgca aggcaagtga ccacataaat aattggttag cctggtatca gcagaaacca

120 ggaaatgctc ctaggctctt aatatctggt gcaaccagtt tggaaactgg ggttccttca agattcagtg gcagtggatc tggaaaggat tacactctca gcatttccag tcttcagact

180

240 gaagatgttg ctacttatta ctgtcaacag tattggagta ctccattcac gttcggctcg

300 gggacaaagt tggaaataaa acg

323

<img file="AR080301A1_D0229.tif" />

190 &lt;210&gt;

&lt;211&gt;

&lt;212&gt;

&lt;213&gt;

360

ADN

Secuencia Artificial &lt;220&gt;

&lt;223&gt; muFRl-2lHCvarPat &lt;400&gt; 38 gaagtgaagc tggtggagtc tgggggagac ttagtgaagc ctggagggtc cctgaaactc tcctgtgcag cctctggatt cactttcagt agctatggca tgtcttgggt tcgccagact

120 ccagacaaga ggttggagtg tgtcgcaacc attagtagtg gtggtagtta cacctactat ccagacggtg tgaaggggcg attcaccatc tccagagaca atgccaagaa caccctgtac

180

240 ctgcaaatga gcagtctgaa gtctgaggac acagccatgt attactgtgc aagggacggc

300 gaggggggcc tctatgctat ggactactgg ggtcaaggaa cctcagtcac cgtctcctca

360

<td></td><td> &lt;210&gt;</td><td> 39</td>

<td></td><td> &lt;211&gt;</td><td> 214</td>

<td> 25</td><td> &lt;212&gt;</td><td>PRT</td>

&lt;213&gt; Secuencia Artificial

<img file="AR080301A1_D0230.tif" />

191 &lt;220&gt;

&lt;223&gt; muFRl-21 LC &lt;400&gt; 39 ⁵

Asp Ile Gin Met Thr Gin Ser Ser Ser Tyr Leu Ser Val Ser Leu Gly t

5 10 15 ί

Ì

Gly Arg Val Thr Ile Thr Cys Lys Ala Ser Asp His Ile Asn Asn Trp I i

25 30 j î

Leu Ala Trp Tyr Gin Gin Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile

35 40 45 ;

Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

55 60

I

Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile Ser Ser Leu Gin Thr

70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gin Gin Tyr Trp Ser Thr Pro Phe

90 95

<img file="AR080301A1_D0231.tif" />

192

<img file="AR080301A1_D0232.tif" />

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala

100 105 110

Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Giu Gin Leu Thr Ser Gly

115 120 125

Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile

130 135 140

Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gin Asn Gly Val Leu

145 150 155 160

Asn Ser Trp Thr Asp Gin Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser

165 170 175

Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr

180 185 190

Thr Cys Giu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser

195 200 205

I i

<img file="AR080301A1_D0233.tif" />

193

Phe Asn Arg Asn Glu Cys

210 &lt;210&gt; 40 &lt;211&gt; 456 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; muFRl-21 HC &lt;400&gt; 40

Glu Val Lys Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

25 30

Gly Met Ser Trp Val Arg Gin Thr Pro Asp Lys Arg Leu Glu Cys Val

40 45

194

<img file="AR080301A1_D0234.tif" />

Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Gly Val

55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn. Ala Lys Asn Thr Leu Tyr

70 75 80

Leu Gin Met Ser Ser Leu Lys Ser Giu Asp Thr Ala Met Tyr Tyr Cys

90 95

Ala Arg Asp Gly Giu Gly Gly Leu Tyr Ala Met Asp Tyr Trp Gly Gin

100 105 110

Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val

115 120 125

Tyr Pro Leu Ala Pro Gly Cys Gly Asp Thr Thr Gly Ser Ser Val Thr

130 135 140

Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Giu Ser Val Thr Val Thr

145

150

155

160

<img file="AR080301A1_D0235.tif" />

195

Trp Asn Ser Gly Ser Leu Ser Ser Ser Val His Thr Phe Pro Ala Leu

165 170 175

Leu Gin Ser Gly Leu Tyr Thr Met Ser Ser Ser Val Thr Val Pro Ser

180 185 190

Ser Thr Trp Pro Ser Gin Thr Val Thr Cys Ser Val Ala His Pro Ala

195 200 205

Ser Ser Thr Thr Val Asp Lys Lys Leu Giu Pro Ser Gly Pro Ile Ser

210 215 220

Thr Ile Asn Pro Cys Pro Pro Cys Lys Giu Cys His Lys Cys Pro Ala

225 230 235 240

Pro Asn Leu Giu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Asn Ile

245 250 255

Lys Asp Val Leu Met Ile Ser Leu Thr Pro Lys Val Thr Cys Val Val

260 265 270

<img file="AR080301A1_D0236.tif" />

196

Val Asp Val Ser Giu Asp Asp Pro Asp Val Gin Ile Ser Trp Phe Val

275 280 285

Asn Asn Val Giu Val His Thr Ala Gin Thr Gin Thr His Arg Glu Asp

290 295 300

Tyr Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile Gin His Gin

305 310 315 320

Asp Trp Met Ser Gly Lys Giu Phe Lys Cys Lys Val Asn Asn Lys Asp

325 330 335

Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Ile Lys Gly Leu Val

340 345 350

Arg Ala Pro Gin Val Tyr Ile Leu Pro Pro Pro Ala Giu Gin Leu Ser

355 360 365

Arg Lys Asp Val Ser Leu Thr Cys Leu Val Val Gly Phe Asn Pro Gly

370 375 380 f

I r

I ί:

<img file="AR080301A1_D0237.tif" />

197

<img file="AR080301A1_D0238.tif" />

j Asp Ile Ser Val Glu Trp Thr Ser Asn Gly His Thr Glu Glu Asn Tyr { 385 390 395 400 j ⁵

Lys Asp Thr Ala Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Ile Tyr

405 410 415 j

Ser Lys Leu Asn Met Lys Thr Ser Lys Trp Glu Lys Thr Asp Ser Phe

Î 420 425 430 j

I 15 Ser Cys Asn Val Arg His Glu Gly Leu Lys Asn Tyr Tyr Leu Lys Lys

I j 435 440 445

I ί

Thr Ile Ser Arg Ser Pro Gly Lys

450 455

<td> &lt;210&gt;</td><td> 41</td>

<td> &lt;211&gt;</td><td> 108</td>

<td> &lt;212&gt;</td><td>PRT</td>

<td> &lt;213&gt;</td><td>Secuencia Artificial</td>

<img file="AR080301A1_D0239.tif" />

198 &lt;220&gt;

&lt;223&gt;

huFRl-21 vLC fs •\

I

I

I

Ì î

i s

.?

&lt;400&gt; 41

Asp Ile Gin Met Thr Gin Ser Ser Ser Ser Leu Ser Val Ser Val Gly

10 15

Gly Arg Val Thr

Ile Thr Cys

Lys Ala Ser Asp His

Ile Asn Asn Trp i

î Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile j 15 35 40 45 j

S j

ï j Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

55 60

Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile Ser Ser Leu Gin Pro j 65 70 75 80

I ²⁵

I Glu Asp Val Ala Thr Tyr Tyr Cys Gin Gin Tyr Trp Ser Thr Pro Phe

<img file="AR080301A1_D0240.tif" />

199

<img file="AR080301A1_D0241.tif" />

Thr Phe Gly Gin Gly Thr Lys Leu Glu Ile Lys Arg

100 105 &lt;210&gt; 42 &lt;211&gt; 120 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; huFRl-21 vHC &lt;400&gt; 42

Glu Val Gin Leu Val Glu Ser Gly Gly Asp Val Val Lys Pro Gly Gly

10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

25 30

Gly Met Ser Trp Val Arg Gin Thr Pro Gly Lys Gly Leu Glu Cys Val

<img file="AR080301A1_D0242.tif" />

200

Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Ser Pro Gly Phe

55 60

Gin Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr Leu Tyr

70 75 80

Leu Gin Met Ser Ser Leu Lys Ala Giu Asp Thr Ala Met Tyr Tyr Cys

90 95

Ala Arg Asp Gly Giu Gly Gly Leu Tyr Ala Met Asp Tyr Trp Gly Gin

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120 &lt;210&gt; 43 &lt;211&gt; 446 &lt;212&gt; ADN &lt;213&gt; Secuencia Artificial &lt;220&gt;

<img file="AR080301A1_D0243.tif" />

201 &lt;223&gt; huFRl-2lVH_co &lt;400&gt; 43 aagcttgcca ccatgggatg gtcatgcatc attctttttc tcgtcgccac tgccacaggt gtgcattccg aggtgcaact tgtagaatct ggcggggatg ttgtgaagcc tggaggtagt ctcaagttgt cctgtgctgc atctgggttt accttctctt cctacggaat gagctgggtg agacagactc ctggcaaggg gctggagtgc gttgccacca ttagtagtgg aggttcttac acctactatt cacctggttt tcagggacgc tttacaatct cccgcgataa gtctaagaac

120

180

240

300 accctttacc tccagatgag tagccttaag gctgaggaca cagccatgta ttattgcgct cgcgatgggg agggagggct ttacgctatg gactactggg gccagggtac cagcgtgacc gtttcctctg ctagtaccaa gggccc &lt;210&gt; 44 &lt;211&gt; 396 &lt;212&gt; ADN &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; huFR21VL co

360

420

446 i

ί ί

J j

<img file="AR080301A1_D0244.tif" />

î

J i

I

I &lt;400&gt; 44 gaattcgcca ccatgggatg gtcatgtatc attctgttct tggtagcaac agcaactggc gtccattctg acatccagat gacccaatcc tccagcagct tgtcagtatc cgttgggggc cgcgttacta ttacctgtaa ggcctccgac catataaata actggcttgc atggtatcaa cagaagcctg ggaaggcacc taaactgctt atctctgggg ccacaagcct ggagaccggc gtgccttcca ggttctctgg aagtggatct ggcaaggact ataccttgag cattagtagc cttcaacctg aggacgtcgc cacctactat tgtcagcagt attggtctac accctttacc tttggacagg gcactaaatt ggagataaaa cgtacg

120

180

240

300

360

396 &lt;210&gt; 45 &lt;211&gt; 214 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; huFRl-21 LC &lt;400&gt; 45

<img file="AR080301A1_D0245.tif" />

203

<img file="AR080301A1_D0246.tif" />

i

Asp Ile Gin Met Thr Gin Ser Ser Ser Ser Leu Ser Val Ser Val Gly

10 15

Gly Arg Val Thr Ile Thr Cys

Leu Ala Trp Tyr Gin Gin Lys

35

Ser Gly Ala Thr Ser Leu Glu

55

Ser Gly Ser Gly Lys Asp Tyr

70

Glu Asp Val Ala Thr Tyr Tyr

Thr Phe Gly Gin Gly Thr Lys

100

Lys Ala Ser Asp His Ile Asn Asn Trp

30

Pro Gly Lys Ala Pro Lys Leu Leu Ile

45

Thr Gly Val Pro Ser Arg Phe Ser Gly

Thr Leu Ser Ile Ser Ser Leu Gin Pro

80

Cys Gin Gin Tyr Trp Ser Thr Pro Phe

95

Leu Glu Ile Lys Arg Thr Val Ala Ala

105 110 i

i

I

J

<img file="AR080301A1_D0247.tif" />

204

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin

145 150 155 160

Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<img file="AR080301A1_D0248.tif" />

205 &lt;210&gt; 46 &lt;211&gt; 449 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; huFRl-21 HC &lt;400&gt; 46

Glu Val Gin Leu Val Glu Ser Gly Gly Asp Val Val Lys Pro Gly Gly

10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

25 30

Gly Met Ser Trp Val Arg Gin Thr Pro Gly Lys Gly Leu Glu Cys Val

40 45

Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Ser Pro Gly Phe

<img file="AR080301A1_D0249.tif" />

206

Gin Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr Leu Tyr

70 75 80

Leu Gin Met Ser Ser Leu Lys Ala Glu Asp Thr Ala Met Tyr Tyr Cys

90 95

Ala Arg Asp Gly Glu Gly Gly Leu Tyr Ala Met Asp Tyr Trp Gly Gin

100 105 110

Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165

170

175

<img file="AR080301A1_D0250.tif" />

207

Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gin Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

<img file="AR080301A1_D0251.tif" />

208

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

385

390

395

400

Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

<img file="AR080301A1_D0252.tif" />

209

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly &lt;210&gt; 47 &lt;211&gt; 642 &lt;212&gt; ADN &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; huFRl-21LC &lt;400&gt; 47 gacatccaga tgacccaatc ctccagcagc ttgtcagtat ccgttggggg ccgcgttact

<img file="AR080301A1_D0253.tif" />

210 attacctgta aggcctccga ccatataaat aactggcttg catggtatca acagaagcct gggaaggcac ctaaactgct tatctctggg gccacaagcc tggagaccgg cgtgccttcc aggttctctg gaagtggatc tggcaaggac tataccttga gcattagtag ccttcaacct gaggacgtcg ccacctacta ttgtcagcag tattggtcta caccctttac ctttggacag ggcactaaat tggagataaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt

<td> &lt;210&gt;</td><td> 48</td>

<td> &lt;211&gt;</td><td> 1347</td>

<td> &lt;212&gt;</td><td>ADN</td>

&lt;213&gt; Secuencia Artificial

<img file="AR080301A1_D0254.tif" />

&lt;220&gt;

&lt;223&gt; huFRl-21HC &lt;400&gt; 48

<td>gaggtgcaac</td><td>ttgtagaatc</td><td>tggcggggat</td><td>gttgtgaagc</td><td>ctggaggtag</td><td>tctcaagttg</td>

<td>tcctgtgctg</td><td>catctgggtt</td><td>taccttctct</td><td>tcctacggaa</td><td>tgagctgggt</td><td>gagacagact</td>

<td>cctggcaagg</td><td>ggctggagtg</td><td>cgttgccacc</td><td>attagtagtg</td><td>gaggttctta</td><td>cacctactat</td>

<td>tcacctggtt</td><td>ttcagggacg</td><td>ctttacaatc</td><td>tcccgcgata</td><td>agtctaagaa</td><td>caccctttac</td>

<td>ctccagatga</td><td>gtagccttaa</td><td>ggctgaggac</td><td>acagccatgt</td><td>attattgcgc</td><td>tcgcgatggg</td>

<td>gagggagggc</td><td>tttacgctat</td><td>ggactactgg</td><td>ggccagggta</td><td>ccagcgtgac</td><td>cgtttcctct</td>

<td>gctagtacca</td><td>agggcccatc</td><td>agttttcccc</td><td>ttggctccaa</td><td>gttctaaatc</td><td>cacaagcggt</td>

<td>ggaacagctg</td><td>cactgggatg</td><td>cctcgttaaa</td><td>gattatttcc</td><td>ctgagcctgt</td><td>gacagtgagc</td>

<td>tggaatagcg</td><td>gagcattgac</td><td>ttcaggtgtg</td><td>cacacttttc</td><td>ccgctgtgtt</td><td>gcagtcctcc</td>

<td>ggtctgtact</td><td>cactgtccag</td><td>tgtcgtaacc</td><td>gtcccttcta</td><td>gcagcttggg</td><td>aacccagacc</td>

<td>tacatctgta</td><td>acgtcaacca</td><td>taaaccatcc</td><td>aacacaaagg</td><td>tggataagaa</td><td>ggttgaacca</td>

aagagctgtg ataagacaca tacatgccct ccttgtcctg caccagagct cctcggaggt

<img file="AR080301A1_D0255.tif" />

ccatctgtgt tcctgtttcc ccccaaaccc aaggacactc ttatgatctc tcgtactcca

780 gaggtcacct gtgttgttgt cgacgtgagc catgaagatc ccgaggttaa attcaactgg tacgtggatg gagtcgaggt tcacaatgcc aagaccaagc ccagggagga gcaatataat

840

900 tctacatatc gggtagtgag cgttctgacc gtgctccacc aagattggct caatggaaaa

960 gagtacaagt gcaaggtgtc caacaaggct cttcccgctc ccattgagaa aactatctcc

1020 aaagccaagg ggcagccacg ggaaccccag gtgtatacat tgcccccatc tagagacgag

1080 ctgaccaaga accaggtgag tctcacttgt ctggtcaagg ggttttaccc ttctgacatt gctgtagagt gggagtctaa cggacagcca gaaaacaact acaagacaac tcccccagtg

1140

1200 ctggacagcg acgggagctt cttcctctac tccaagttga ctgtagacaa gtctagatgg

1260 cagcaaggaa acgttttctc ctgctcagta atgcatgagg ctctgcacaa tcactatacc

1320 cagaaatcac tgtcccttag cccaggg

1347

<td> 25</td><td> &lt;210&gt;</td><td> 49</td>

<td></td><td> &lt;211&gt;</td><td> 792</td>

&lt;212&gt; ADN

<img file="AR080301A1_D0256.tif" />

&lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; huFolRl Secuencia de ADN EcoRI a Xbal &lt;400&gt; 49 gaattcgcca ccatggcaca gcgcatgacc actcagctcc tgcttctgtt ggtttgggtg gcagtcgtgg gagaggccca gaccaggatt gcttgggcac gcacagagct gcttaatgtt tgcatgaacg caaagcacca taaagagaaa cccggtcccg aggataagtt gcacgaacag tgccgccctt ggagaaagaa tgcatgctgt agcacgaaca cctctcagga ggcgcataaa gacgtaagct atttgtatag atttaactgg aaccattgcg gtgaaatggc acctgcctgt aaacggcact ttatccagga tacttgcttg tacgagtgta gcccgaatct cgggccctgg attcagcaag ttgatcagag ttggcgcaaa gagagggtgc tgaacgttcc gctttgcaag gaggactgcg agcaatggtg ggaagactgt agaaccagct acacctgtaa gtctaactgg cacaaaggat ggaactggac atccgggttt aacaaatgcg ctgtcggcgc tgcctgccag ccatttcatt tctactttcc aactcccact gtcctgtgta acgagatttg gacgcattca tataaagtca gcaactacag ccggggctcc ggccgctgca ttcagatgtg gttcgaccct

120

180

240

300

360

420

480

540

600

660

214

<img file="AR080301A1_D0257.tif" />

<img file="AR080301A1_D0258.tif" />

gcacagggca accctaacga ggaggtcgca cgcttctacg ctgcagcaat gtctggagcc

720 ggtccttggg ctgcttggcc atttctcctt agcctcgccc tcatgcttct ctggctgttg tcataatcta ga

780

792 &lt;210&gt; 50 &lt;211&gt; 32 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; Cebador EcoMHl &lt;400&gt; 50

Cys Thr Thr Cys Cys Gly Gly Ala Ala Thr Thr Cys Ser Ala Arg Gly

1 5 10 15

Thr Asn Met Ala Gly Cys Thr Gly Ser Ala Gly Ser Ala Gly Thr Cys &lt;210&gt; 51

215

<img file="AR080301A1_D0259.tif" />

&lt;211&gt;

&lt;212&gt;

&lt;213&gt;

PRT

Secuencia Artificial &lt;220&gt;

&lt;223&gt; Cebador EcoMH2 &lt;400&gt; 51

Cys Thr Thr Cys Cys Gly Gly Ala Ala Thr Thr Cys Ser Ala Arg Gly

10 15

Thr Asn Met Ala Gly Cys Thr Gly Ser Ala Gly Ser Ala Gly Thr Cys

25 30

Trp Gly Gly &lt;210&gt; 52 &lt;211&gt; 36 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

216

<img file="AR080301A1_D0260.tif" />

&lt;400&gt; 52

Gly Gly Ala Gly Gly Ala Thr Cys Cys Ala Thr Ala Gly Ala Cys Ala

10 15

Gly Ala Thr Gly Gly Gly Gly Gly Thr Gly Thr Cys Gly Thr Thr Thr

25 30

Thr Gly Gly Cys &lt;210&gt; 53 &lt;211&gt; 31 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; SacIMK &lt;400&gt; 53

Gly Gly Ala Gly Cys Thr Cys Gly Ala Tyr Ala Thr Thr Gly Thr Gly

<img file="AR080301A1_D0261.tif" />

217

Met Thr Ser Ala Cys Met Cys Ala Arg Trp Cys Thr Met Cys Ala

25 30 &lt;210&gt; 54 &lt;211&gt; 46 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; HindKL &lt;400&gt; 54

Thr Ala Thr Ala Gly Ala Gly Cys Thr Cys Ala Ala Gly Cys Thr Thr

10 15

Gly Gly Ala Thr Gly Gly Thr Gly Gly Gly Ala Ala Gly Ala Thr Gly

25 30

Gly Ala Thr Ala Cys Ala Gly Thr Thr Gly Gly Thr Gly Cys

<img file="AR080301A1_D0262.tif" />

218 &lt;210&gt; 55 &lt;211&gt; 39 &lt;212&gt; ADN &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; cd37-lLClead &lt;400&gt; 55 ttttgaattc gccaccatga agtttccttc tcaacttct &lt;210&gt; 56 &lt;211&gt; 23 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; CDR2 Movl9 vHC humano y quimérico compuesto &lt;220&gt;

&lt;221&gt; MISC_FEATURE &lt;222&gt; (14)..(14) &lt;223&gt; Xaal = Q, H, K, o R

<img file="AR080301A1_D0263.tif" />

219 &lt;220&gt;

&lt;221&gt; MISC_FEATURE &lt;222&gt; (15)..(15) &lt;223&gt; Xaa2 = R, Q, H, o N &lt;220&gt;

&lt;221&gt; MISC_FEATURE &lt;222&gt; (16)..(16) &lt;223&gt; Xaa3 = E, T, S, G, A, ο V &lt;220&gt;

&lt;221&gt; misc_feature &lt;222&gt; (18)..(18) &lt;223&gt; Xaa puede ser cualquier aminoâcido de origen natural &lt;220&gt;

&lt;221&gt; misc_feature &lt;222&gt; (21)..(21) &lt;223&gt; Xaa puede ser cualquier aminoâcido de origen natural &lt;400&gt; 56

Arg Ile His Pro Tyr Asp Gly Asp Thr Phe Tyr Asn Gin Xaa Ala Ala

10 15

Phe Xaa Ala Ala Xaa Ala Ala

<img file="AR080301A1_D0264.tif" />

220 &lt;210&gt; 57 &lt;211&gt; 11 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; FRl-48vL CDRl &lt;400&gt; 57

Arg Ala Ser Glu Asn Ile Tyr Ser Asn Leu Ala

10 &lt;210&gt; 58 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; FRl-48vL CDR2 &lt;400&gt; 58

Ala Ala Thr Asn Leu Ala Asp

<img file="AR080301A1_D0265.tif" />

221 &lt;210&gt; 59 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; FRl-48vL CDR3 &lt;400&gt; 59

Gin His Phe Trp Ala Ser Pro Tyr Thr

5 &lt;210&gt; 60 &lt;211&gt; 6 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; FRl-48vH CDR1 &lt;400&gt; 60

Thr Asn Tyr Trp Met Gin

<img file="AR080301A1_D0266.tif" />

222 &lt;210&gt; 61 &lt;211&gt; 10 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; FR1-48vH CDR2 &lt;400&gt; 61

Ala Ile Tyr Pro Gly Asn Gly Asp Ser Arg &lt;210&gt; 62 &lt;211&gt; 8 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; FRl-48vH CDR3 &lt;400&gt; 62

Arg Asp Gly Asn Tyr Ala Ala Tyr

<img file="AR080301A1_D0267.tif" />

223 &lt;210&gt; 63 &lt;211&gt; 11 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; FRl-49vL CDR1 &lt;400&gt; 63

Arg Ala Ser Glu Asn Ile Tyr Thr Asn Leu Ala

10 &lt;210&gt; 64 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; FRl-49vL CDR2 &lt;400&gt; 64

Thr Ala Ser Asn Leu Ala Asp

224

<img file="AR080301A1_D0268.tif" />

&lt;210&gt; 65 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; FRl-49vL CDR3 &lt;400&gt; 65

Gin His Phe Trp Val Ser Pro Tyr Thr &lt;210&gt; 66 &lt;211&gt; 6 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; FRl-49vH CDRl &lt;400&gt; 66

Thr Asn Tyr Trp Met Tyr

<img file="AR080301A1_D0269.tif" />

225

<img file="AR080301A1_D0270.tif" />

&lt;210&gt; 67 &lt;211&gt; 10 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; FRl-49vH CDR2 &lt;400&gt; 67

Ala Ile Tyr Pro Gly Asn Ser Asp Thr Thr

10 &lt;210&gt; 68 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; FRl-49vH CDR3 &lt;400&gt; 68

Arg His Asp Tyr Gly Ala Met Asp Tyr

<img file="AR080301A1_D0271.tif" />

226 &lt;210&gt; 69 &lt;211&gt; 11 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; FRl-57vL CDR1 &lt;400&gt; 69

Arg Ala Ser Gin Asn Ile Asn Asn Asn Leu His

10 &lt;210&gt; 70 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; FRl-57vL CDR2 &lt;400&gt; 70

Tyr Val Ser Gin Ser Val Ser

<img file="AR080301A1_D0272.tif" />

227 &lt;210&gt; 71 &lt;211&gt; 10 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; FRl-57vL CDR3 &lt;400&gt; 71

Gin Gin Ser Asn Ser Trp Pro His Tyr Thr

10 &lt;210&gt; 72 &lt;211&gt; 6 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; FRl-57vH CDR1 &lt;400&gt; 72

Ser Ser Phe Gly Met His

<img file="AR080301A1_D0273.tif" />

228 i

<td></td><td> &lt;210&gt;</td><td> 73</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;211&gt;</td><td> 10</td><td></td><td></td><td></td><td></td>

<td> 5</td><td> &lt;212&gt;</td><td>PRT</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;213&gt;</td><td>Secuencia</td><td>Artificial</td><td></td><td></td><td></td>

<td></td><td> &lt;220&gt;</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;223&gt;</td><td>FRl-57vH</td><td>CDR2</td><td></td><td></td><td></td>

<td> 10</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;400&gt;</td><td> 73</td><td></td><td></td><td></td><td></td>

<td></td><td colspan="2">Tyr Ile Ser Ser</td><td>Gly Ser Ser</td><td>Thr</td><td>Ile</td><td>Ser</td>

<td></td><td> 1</td><td></td><td> 5</td><td></td><td></td><td> 10</td>

<td> 15</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;210&gt;</td><td> 74</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;211&gt;</td><td> 9</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;212&gt;</td><td>PRT</td><td></td><td></td><td></td><td></td>

<td> 20</td><td> &lt;213&gt;</td><td>Secuencia</td><td>. Artificial</td><td></td><td></td><td></td>

<td></td><td> &lt;220&gt;</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;223&gt;</td><td>FR1-57VH</td><td>CDR3</td><td></td><td></td><td></td>

<td> 25</td><td> &lt;400&gt;</td><td> 74</td><td></td><td></td><td></td><td></td>

<td></td><td colspan="2">Glu Ala Tyr Gly</td><td>Ser Ser Met</td><td>Glu</td><td>Tyr</td><td></td>

<img file="AR080301A1_D0274.tif" />

229

<img file="AR080301A1_D0275.tif" />

&lt;210&gt; 75 &lt;211&gt; 11 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; FRl-65vL CDRl &lt;400&gt; 75

Lys Ala Ser Gin Asn Val Gly Pro Asn Val Ala &lt;210&gt; 76 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; FR1-65VL CDR2 &lt;400&gt; 76

Ser Ala Ser Tyr Arg Tyr Ser

<img file="AR080301A1_D0276.tif" />

230 &lt;210&gt; 77 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; FRl-65vL CDR3 &lt;400&gt; 77

Gin Gin Tyr Asn Ser Tyr Pro Tyr Thr

5 &lt;210&gt; 78 &lt;211&gt; 6 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; FRl-65vH CDR1 &lt;400&gt; 78

Thr Ser Tyr Thr Met His

<img file="AR080301A1_D0277.tif" />

231 &lt;210&gt; 79 &lt;211&gt; 10 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; FRl-65vH CDR2 &lt;400&gt; 79

Tyr Ile Asn Pro Ile Ser Gly Tyr Thr Asn &lt;210&gt; 80 &lt;211&gt; 11 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; FRl-65vH CDR3 &lt;400&gt; 80

Gly Gly Ala Tyr Gly Arg Lys Pro Met Asp Tyr

232

<img file="AR080301A1_D0278.tif" />

<img file="AR080301A1_D0279.tif" />

&lt;210&gt; 81 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; CDR2 muFRl-48 HC definido de Kabat &lt;400&gt; 81

Ala Ile Tyr Pro Gly Asn Gly Asp Ser Arg Tyr Thr Gin Lys Phe Lys

10 15

Gly &lt;210&gt; 82 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; CDR2 huFRl-48 HC definido de Kabat

<img file="AR080301A1_D0280.tif" />

&lt;400&gt; 82

Ala Ile Tyr Pro Gly Asn Gly Asp Ser Arg Tyr Thr Gin Lys Phe Gin

5 10 15

Gly &lt;210&gt; 83 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; CDR2 huFRl-49 HC definido de Rabat &lt;400&gt; 83

Ala Ile Tyr Pro Gly Asn Ser Asp Thr Thr Tyr Asn Gin Lys Phe Gin

10 15

Gly

<img file="AR080301A1_D0281.tif" />

234 &lt;210&gt; 84 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; CDR2 muFRl-57 HC definido de Rabat &lt;400&gt; 84

Tyr Ile Ser Ser Gly Ser Ser Thr Ile Ser Tyr Ala Asp Thr Val Lys

Gly &lt;210&gt; 85 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; CDR2 huFRl-57 HC definido de Rabat

<img file="AR080301A1_D0282.tif" />

235

<img file="AR080301A1_D0283.tif" />

&lt;400&gt; 85

Tyr Ile Ser Ser Gly Ser Ser Thr Ile Ser Tyr Ala Asp Ser Val Lys

10 15

Gly &lt;210&gt; 86 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; CDR2 muFRl-65 HC definido de Kabat &lt;400&gt; 86

Tyr Ile Asn Pro Ile Ser Gly Tyr Thr Asn Tyr Asn Gin Lys Phe Lys

10 15

Asp

<img file="AR080301A1_D0284.tif" />

236 &lt;210&gt; 87 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; CDR2 huFRl-65 HC definido de Kabat &lt;400&gt; 87

Tyr Ile Asn Pro Ile Ser Gly Tyr Thr Asn Tyr Asn Gin Lys Phe Gin

10 15

Gly &lt;210&gt; 88 &lt;211&gt; 108 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; muFRl-48vL &lt;400&gt;

<img file="AR080301A1_D0285.tif" />

237

Asp Ile Gin Met Thr Gin Ser Pro Ala Ser Leu Ser Val Ser Val Gly

10 15

Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn

25 30

Leu Ala Trp Tyr Gin Gin Lys Gin Gly Lys Ser Pro Gin Leu Leu Val

40 45

Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

55 60

Ser Glu Ser Gly Thr Gin Tyr Ser Leu Lys Ile Asn Ser Leu Gin Ser

70 75 80

Glu Asp Phe Gly Ser Tyr Tyr Cys Gin His Phe Trp Ala Ser Pro Tyr

90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<img file="AR080301A1_D0286.tif" />

238 &lt;210&gt; 89 &lt;211&gt; 117 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; muFRl-48vH &lt;400&gt; 89

Gin Val Gin Leu Gin Gin Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

10 15

Ser Val Lys Leu Ser Cys Arg Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

25 30

Trp Met Gin Trp Ile Lys Gin Arg Pro Gly Gin Gly Leu Glu Trp Ile

40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Ser Arg Tyr Thr Gin Lys Phe

<img file="AR080301A1_D0287.tif" />

239

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

70 75 80

Met Gin Val Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

90 95

Ala Arg Arg Asp Gly Asn Tyr Ala Ala Tyr Trp Gly Gin Gly Thr Leu

100 105 110

Val Thr Val Ser Ala

115 &lt;210&gt; 90 &lt;211&gt; 108 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; muFRl-49vL &lt;400&gt; 90

<img file="AR080301A1_D0288.tif" />

240

Asp Ile Gin Met Thr Gin Ser Pro Ala Ser Leu Ser Val Ser Val Gly

10 15

Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Thr Asn

25 30

Leu Ala Trp Tyr Gin Gin Lys Gin Gly Lys Ser Pro Gin Leu Leu Val

40 45

Tyr Thr Ala Ser Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

55 60

Ser Gly Ser Gly Thr Gin Tyr Ser Leu Lys Ile Asn Ser Leu Gin Ser

70 75 80

Glu Asp Phe Gly Thr Tyr Tyr Cys Gin His Phe Trp Val Ser Pro Tyr

90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<img file="AR080301A1_D0289.tif" />

241 &lt;210&gt; 91 &lt;211&gt; 118 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; muFRl-49vH &lt;400&gt; 91

Glu Val Gin Leu Gin Gin Ser Gly Thr Val Leu Ala Arg Pro Gly Ala

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Lys Phe Thr Asn Tyr

25 30

Trp Met Tyr Trp Ile Lys Gin Arg Pro Gly Gin Gly Leu Glu Leu Ile

Gly Ala Ile Tyr Pro Gly Asn Ser Asp Thr Thr Tyr Asn Leu Lys Phe

242

<img file="AR080301A1_D0290.tif" />

Lys Gly Lys Ala Lys Leu Thr Ala Val Thr Ser Ala Asn Thr Val Tyr

70 75 80

Met Glu Val Ser Ser Leu Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys

90 95

Thr Lys Arg His Asp Tyr Gly Ala Met Asp Tyr Trp Gly Gin Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115 &lt;210&gt; 92 &lt;211&gt; 109 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; muFRl-57vL &lt;400&gt; 92

Asp Ile Val Leu Thr Gin Ser Pro Ala Thr Leu Ser Val Thr Pro Gly

<img file="AR080301A1_D0291.tif" />

Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gin Asn Ile Asn Asn Asn

25 30

Leu His Trp Tyr Gin Gin Lys Ser His Glu Ser Pro Arg Leu Leu Ile

40 45

Lys Tyr Val Ser Gin Ser Val Ser Gly Ile Pro Ser Arg Phe Ser Gly

55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Thr

70 75 80

Glu Asp Phe Gly Met Tyr Phe Cys Gin Gin Ser Asn Ser Trp Pro His

85 90 95

Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 &lt;210&gt; 93 i

L

I î;

ί

I i

<img file="AR080301A1_D0292.tif" />

244 &lt;211&gt; 118 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; muFRl-57vH &lt;400&gt; 93

Asp Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly

10 15

Ser Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

25 30

Gly Met His Trp Val Arg Gin Ala Pro Glu Lys Gly Leu Glu Trp Val

40 45

Ala Tyr Ile Ser Ser Gly Ser Ser Thr Ile Ser Tyr Ala Asp Thr Val

55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Leu Leu

70 75 80

245

<img file="AR080301A1_D0293.tif" />

<img file="AR080301A1_D0294.tif" />

Leu Gin Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

90 95

Ala Arg Glu Ala Tyr Gly Ser Ser Met Glu Tyr Trp Gly Gin Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115 &lt;210&gt; 94 &lt;211&gt; 108 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; muFRl-65vL &lt;400&gt; 94

Asp Ile Val Met Thr Gin Ser Gin Lys Phe Met Ser Thr Ser Val Gly

10 15

<img file="AR080301A1_D0295.tif" />

246

<img file="AR080301A1_D0296.tif" />

Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gin Asn Val Gly Pro Asn

25 30

Val Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ser Pro Lys Ala Leu Ile

40 45

Tyr Ser Ala Ser

Ser Gly Ser Gly

65

Ala Asp Leu Ala

Thr Phe Gly Gly

100

Tyr Arg Tyr Ser Giu Val

Thr Asp Phe Thr Leu Thr

Giu Tyr Phe Cys Gin Gin

90

Gly Thr Lys Leu Giu Ile

105

Pro Asp Arg Phe Thr Gly

Ile Ser Asn Met Gin Ser

80

Tyr Asn Ser Tyr Pro Tyr

Lys Arg f

i

F

I’ ì

ΙΕ f

f &lt;210&gt; 95 ί &lt;211&gt; 120

<img file="AR080301A1_D0297.tif" />

247 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; muFRl-65vH &lt;400&gt; 95

Gin Val Gin Leu Gin Gin Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

25 30

Thr Met His Trp Val Lys Gin Arg Pro Gly Gin Gly Leu Ala Trp Ile

40 45

Gly Tyr Ile Asn Pro Ile Ser Gly Tyr Thr Asn Tyr Asn Gin Lys Phe

55 60

Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

70 75 80

I

<img file="AR080301A1_D0298.tif" />

248

Met Gin Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

90 95

Ala Ser Gly Gly Ala Tyr Gly Arg Lys Pro Met Asp Tyr Trp Gly Gin

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120 &lt;210&gt; 96 &lt;211&gt; 108 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; huFRl-48vL &lt;400&gt; 96

Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Val Ser Val Gly

1

249

<img file="AR080301A1_D0299.tif" />

SV&lt;i

Glu Arg Val Thr Ile Thr Cys Arg Ala Ser Giu Asn Ile Tyr Ser Asn

25 30

Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ser Pro Lys Leu Leu Val

40 45

Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

55 50

Ser Giu Ser Gly Thr Asp Tyr Ser Leu Lys Ile Asn Ser Leu Gin Pro

70 75 80

Giu Asp Phe Gly Ser Tyr Tyr Cys Gin His Phe Trp Ala Ser Pro Tyr

90 95

Thr Phe Gly Gin Gly Thr Lys Leu Glu Ile Lys Arg

100 105 &lt;210&gt; 97 &lt;211&gt; 117 &lt;212&gt; PRT

<img file="AR080301A1_D0300.tif" />

I t

250

<img file="AR080301A1_D0301.tif" />

&lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; huFRl-48vH &lt;400&gt; 97

Gin Val Gin Leu Val Gin Ser Gly Ala Glu Val Ala Lys Pro Gly Ala

10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

25 30

Trp Met Gin Trp Ile Lys Gin Arg Pro Gly Gin Gly Leu Glu Trp Ile

40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Ser Arg Tyr Thr Gin Lys Phe

55 60

Gin Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80 i

<img file="AR080301A1_D0302.tif" />

251

Met Gin Val Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

90 95

Ala Arg Arg Asp Gly Asn Tyr Ala Ala Tyr Trp Gly Gin Gly Thr Leu

100 105 110

Val Thr Val Ser Ala

115 &lt;210&gt; 98 &lt;211&gt; 108 &lt;212&gt; PRT &lt;213&gt; Secuencia Artificial &lt;220&gt;

&lt;223&gt; huFRl-49vL &lt;400&gt; 98

Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Val Ser Val Gly

10 15