Humanized antibodies to liv-1 and use of same to treat cancer.
21 claims: 3 independent, 18 dependent
- 1REIVINDICACIONES 1. Un anticuerpo humanizado que se enlaza específicamente a LIV-1 humano, caracterizado porque comprende una región variable de cadena pesada madura que comprende tres CDR de SEQ ID NO:53 y que tiene una secuencia de aminoácidos por lo menos 95% idéntica a SEQ ID NO: 53 a condición de que la posición H27 este ocupada por L, la posición H29 este ocupada por I, H30 por E y H94 por V y una región variable de cadena ligera madura que comprende tres CDR de SEQ ID NO: 60 por lo menos 95% idéntica a SEQ ID NO: 60 a condición de que la posición L36 este ocupada por Y y la posición L46 por P.
- 2El anticuerpo humanizado de la reivindicación 1, que además es a condición de que la posición H76 este ocupada por N.
- 3El anticuerpo humanizado de la reivindicación 1, en donde la región variable de cadena pesada madura es fusionada a una región constante de cadena pesada y la región variable de cadena ligera madura es fusionada a una región constante de cadena ligera.
- 4El anticuerpo humanizado de la reivindicación 3, en donde la región constante de cadena pesada es una forma 120 IMPI INSTITUTO MEXICANO oe la pxoheoaü INOUSTRiAL muíante de la región constante humana natural que tiene enlace reducido a un receptor Fe gamma en relación con la ( región constante humana natural.
- 5El anticuerpo humanizado de la reivindicación 3, en donde la región constante de cadena pesada es de isotipo es de IgGl. /
- 6El anticuerpo humanizado de la reivindicación 3, en donde la región constante de cadena pesada tiene una secuencia de aminoácidos que comprende SEQ ID NO:44 y la región constante de cadena ligera tiene una secuencia de aminoácidos que comprende SEQ ID NO:42.
- 7El anticuerpo humanizado de la reivindicación 3, en donde la región constante de cadena pesada tiene una secuencia de aminoácidos que comprende SEQ ID NO:46 (S239C) y la región constante de cadena ligera tiene una secuencia de aminoácidos que comprende SEQ ID NO:42. .
- 8El anticuerpo humanizado de la reivindicación 1, en donde la región variable de cadena pesada madura tiene una secuencia de aminoácidos designada SEQ ID NO:52 o 53 y la región variable de cadena ligera madura tiene una secuencia de aminoácidos designada SEQ ID NO: 59 o 60. '
- 9El anticuerpo humanizado de la reivindicación 1, en donde la región variable de cadena pesada madura tiene una 121 IMPI INSTITUTO MF.XICAN; DE LA MOHEDA O INDUSTRIAL secuencia de aminoácidos designada SEQ ID NO:53 y la región variable de cadena ligera madura tiene una secuencia de aminoácidos designada SEQ ID NO: 60.
- 10El anticuerpo humanizado de la reivindicación 1, en donde el anticuerpo es conjugado a un agente citotóxico o agente citostático.
- 11El anticuerpo humanizado de la reivindicación 1, que tiene una constante de asociación por LIV-1 de humano o de mono cinomolgus de 0.5 a 2 xlO 9 M 1 .
- 12Un ácido nucleico caracterizado porque codifica una región variable de cadena pesada madura y/o una región variable de cadena ligera madura de la reivindicación 1.
- 13Uso de un anticuerpo humanizado de la reivindicación 1 para la preparación de un medicamento para el tratamiento de cáncer que expresa LIV-1 en un paciente, donde el medicamento está adaptado para ser administrable en un régimen efectivo para tratar dicho padecimiento.
- 14El uso de la reivindicación 13, en donde el cáncer es cáncer de mama, cáncer de próstata, cáncer 'cervical o un melanoma.
- 15Una composición farmacéutica caracterizada porque comprende el anticuerpo humanizado de la reivindicación 1. '
- 16El uso de la reivindicación 13, en donde el / IMPI instituto moccuq DE LA rRCi’IEIMD INDUSTRIAL citotóxico o agente 13, en donde el del reivindicación 1 para 122 anticuerpo es conjugado a un agente citostático.
- 17El uso de la reivindicación anticuerpo es IgGl humano.
- 18Un anticuerpo humanizado de la usarse en el tratamiento de un cáncer que expresa LIV-1 en un paciente.
- 19El anticuerpo humanizado para usarse de acuerdo a la reivindicación 18, en donde el cáncer es cáncer de mama, cáncer de próstata, cáncer cervical o un melanoma.
- 20El anticuerpo humanizado para usarse de acuerdo a la reivindicación 18, en donde el anticuerpo es conjugado a un agente citotóxico o agente citostático.
- 21El anticuerpo humanizado para usarse de acuerdo a la reivindicación 18, en donde el del anticuerpo es IgGl humano. 123 IMPI INSTITUTO MEXICANO DE LA PROPIEDAD industrial
Independent claims21
244 paragraphs in 32 sections, as filed
(54) Title: ANTIBODIES HUMANIZED TO LIV-1 AND THE USE OF THEM TO TREAT CANCER.
(54) Title: HUMANIZED ANTIBODIES TO LIV-1 AND USE OF SAME TO TREAT CANCER.
(57) Summary
The present invention relates to humanized antibodies that specifically bind to LIV-1. Antibodies are useful for the treatment and diagnosis of various cancers as well as detecting LIV-1.
(57) Abstract
The invention provides humanized antibodies that specifically bind to LIV-1. The antibodies are useful for treatment and diagnoses of various cancers as well as detecting LIV-1.
IMPIÍ ^
PATENT TITLE No. 351027
Headlines):
SEATTLE GENETICS, INC.
Home:
21823-30th Drive SE, Bothell, Washington, 98021, USA
D nomination:
ANTIBODIES HUMANIZED TO LIV-1 AND USE OF THE SAME TO TREAT CANCER.
Classification:
CIP:
CPC:
C07K16 / 30; A61K39 / 395
C07K16 / 30; A61K39 / 395; C07K16 / 3015; C07K16 / 3053; C07K16 / 3069
Inventor (s)
MARIA LEIA SMITH; DJANGO SUS®MAh¡ · WILLIAM ARTHUR; ALBINA NESTEROVA
REQUEST
Number:
MX / a / 2013/006365
Fécüá PrAft ^ n ^^ n Internacional, 'í' 'oé de BícieWtere de 2011
PRIORITY
US US from December ¢¢ -2010 ..
February 2011
<img file="MX351027B_D0001.tif" />
ero:
,291 ,990
<img file="MX351027B_D0002.tif" />
Validity: Twenty years
Expiration Date: December 6
Date of Expedition! September 28, 2017
The patent of referenehneenaterga with fundafheAo in the aSSoílos 1%, 2 * fraM ^ ', and 59 * the Law of Industrial Property.
In accordance with article 2b of the Property Law-lr ^ tatriáf; The present is a twenty-year-old Agency * pft> renewable, counted from the date of submission * the international application and payment of the amount in force for manpower in force.
Whoever signs this title does so based on the provisions of the provisions of the 6th fraction III and 7 bis 2 of the Industrial Property Law (Official Gazette of the Federation (OIOE.) 06/27/1991, * orm®ia eí «2®8 / 19'94,» 1Ó / J »96, ^ ®12 / 1097, '05/17/1999 01/26/2004, 06/16/2005, 01/25/2006 , 06/05 / 2009,06 / 01/2010, 1M) 672ÍMO. 06/28/2 £, W27 / <fl / 2012l (ÍW ^ 3<sup>or</sup> Frajefón V subsection a). 4 »and 12» fractions I and III of the
Regulations of the Instituto Mexicano te la Pfapieíed IntkitnM (DQF 14/1 ^ 1999, r¡foftnadó »ÍM 01IB7 / 2B02, 1S / 07 / 2O04, 07/28/2004 and 09/07/2007); Articles 1, 3, 4, 5 fraction V subsection aj, 1B fraction ,? I * III yáO'WEstaiuto®rgánico del, Wlexfcano Institute of Industrial Property (DOF
12/27/1999, reformed on 10/10/2002, 07/29 ^ 04 ^ 14 »! ^ Deputy Generals, Coordinator, Departmental Directors and other subordinates of the Institute 08/04/2004 and 09/13/2007).
KAot ^ do which delegates powers to the Directors, Divisional Deputy Directors, Coordinators * 5712/1999, amended on 02/04/2000, 07/29/2004,
This document is signed with an advanced electronic signature (FIEL), based on articles 7 BIS 2 of the Industrial Property Law; 3 of its Regulations, and 1 section III, 2 section V, 26 BIS and 26 TER of the Agreement establishing the guidelines for the use of the Payment and Electronic Services Portal (PASE) of the Mexican Institute of Industrial Property, in the procedures indicated.
THE DIVISIONAL DIRECTOR OF PATENTS
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NAHANNY CANAL REYES
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NAHANNY MARISOL CANAL REYES | 00001000000403252793 | Administration Service
Tax | f695 || MX / 2017/80203 | MX / a / 2013/006365 | PCT patent title | 1220 | RRGO | Page (s) | CGqHPbybkRnfRcchdbkORoWkrR8 =
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Ld / rKUwAdXLjhDa / n45yFjW6J5K6 ZhMOXT1ddBCCuT1KqV8 + + + A + SV2BnfVbe2f1qMdN4ExGinFoLFOOcOXqpto8B77 ANV TzxEXW3q7KACU + qKOiO43DgySgwnB5 / IWFkAzlYypPPqdrtdlv3avE5IXVNbeN0pmWSbGDkLRJ8gnuEeEWvv48 DcNQ56B7DxD4byW0yyvxhYN3wbtJMnRvKtcVqJ6u4hFChAEr / DZsEkjEFfpnaVxMd0 6Uj6 utGD1uTSO7D1IC4UJSQXQJt + + + v0DJTyYJeknKsLXo4 C8y0ff / ZeVuFU0QMt8bX7JwizEcUqzQ / toQjG7t3zEkis / G3W ==
Arenal No. 550. Floor 1, Pueblo Santa Mana Tepepan, Xochimilco, 16020.
Mexico City.
(55) 53340700 www.gob.mx/impi
IIIIIIIHIIIIII
MX / 2017/80203 ¢, 51024
<img file="MX351027B_D0004.tif" />
HUMANIZED ANTIBODIES TO LIV-1 AND USE OF HDD MTSMr<sup>g</sup> POPE TREATING CANCER BACKGROUND OF THE INVENTION
LIV-1 is a member of the LZT (LIV-1-ZIP zinc transporters) family of zinc transporter proteins. Taylor et al., Biochim. Biophys. Acta 1611: 16-30 (2003). Computer analysis of the LIV-1 protein reveals a potential metalloprotease portion, which conforms to the consensus sequence for the catalytic zinc binding site portion of the zinc metalloprotease. The LIV-1 mRNA is expressed primarily in breast, prostate, pituitary gland, and brain tissue.
The LIV-1 protein has also been implicated in certain cancerous conditions, for example breast cancer and prostate cancer. Detection of LIV-1 is associated with estrogen receptor positive breast cancer, McClelland et al., Br. J. Cancer 77: 1653-1656 (1998) and metastatic spread of these cancers to regional lymph nodes. Manninget al., Eur. J. Cancer 30A: 675-678 (1994).
BRIEF DESCRIPTION OF THE INVENTION
The invention provides a humanized antibody comprising a mature heavy chain variable region having an amino acid sequence at least 90% identical to SEQ ID NO: 53 provided that position H27 is occupied by L, position H29 is occupied by I, H30 and E and H94 by V and a mature light chain variable region at least 90% identical to SEQ ID NO: 60 provided that position L36 is occupied by Y and position L46 by P. Optionally, the humanized antibody comprises three CDRs of SEQ ID NO: 53 and three CDRs of SEQ ID NO: 60. Those CDRs are shown in Figure 16. Optionally, position H76 is occupied by N. Optionally, the humanized antibody comprises one Mature heavy chain variable region having an amino acid sequence at least 95% identical to SEQ ID NO: 53 and a mature light chain variable region at least 95% identical to SEQ ID NO: 60. Optionally, the mature heavy chain variable region is fused to a heavy chain constant region and the mature light chain constant region is fused to a light chain constant region. Optionally, the heavy chain constant region is a mutant form of the natural human constant region that is down-bound to a Fe gamma receptor relative to the natural human constant region. Optionally, the heavy chain constant region is of the IgGl isotype. Optionally, the heavy chain constant region has an amino acid sequence comprising SEQ ID NO: 44 and the light chain constant region has an amino acid sequence comprising SEQ ID NO: 42.
<img file="MX351027B_D0005.tif" />
IMPI
MEXICAN INSTITUTE
M OF THE PROPERTY • j <sub>#</sub> INDUSTRIAL
Optionally, the heavy chain constant region has an amino acid sequence comprising SEQ ID NO: 46 (S239C) and the light chain constant region has an amino acid sequence comprising SEQ ID NO: 42. In some such humanized antibodies, Any differences in the CDRs of the mature heavy chain variable region and mature light variable region of SEQ ID NO: 52 and 60, respectively, reside at positions H60-H65. In some such humanized antibodies, the human heavy chain variable region has an amino acid sequence designated SEQ ID NO: 52 and 53 and the mature light chain variable region has an amino acid sequence designated SEQ ID NO: 59 or 60. In some such humanized antibodies, the mature heavy chain variable region has an amino acid sequence designated SEQ ID NO: 53 and the mature light chain variable region has an amino acid sequence designated SEQ ID NO: 60. Some of such humanized antibodies they are conjugated to a cytotoxic agent or cytostatic agent. Some of such humanized antibodies are conjugated to a cytotoxic agent or cytostatic agent. Some of such humanized antibodies have an association constant for human cynomolgus monkey LI-1 of 0.5 x 2 x 10<sup>9</sup> M "<sup>1</sup>.
The invention also provides a humanized antibody comprising a mature heavy chain variable region comprising the three Kabat CDRs of SEQ ID NO: 52, wherein position H27 is occupied by L, position H29 is occupied by I, H30 by E, H76 for N and H94 for V and a mature light chain variable region comprising the three Kabat CDRs of SEQ ID NO: 60 provided that position L36 is occupied by Y and position L46 by P. The invention also provides a nucleic acid encoding a mature heavy chain variable region and / or a mature light chain variable region of any of the humanized antibodies defined above. The invention further provides a method of treating a patient who has or is at risk for cancer, comprising administering to the patient an effective regimen of any of the humanized antibodies defined above. The cancer can be for example breast cancer, cervical cancer, melanoma or prostate cancer.
The invention further provides a pharmaceutical composition comprising a humanized antibody as defined above.
The invention further provides methods of treating a subject afflicted with a melanoma expressing the LIV-1 protein by administering to the subject a LIV-1 specific antibody or a LIV-I antibody-drug conjugate in an amount sufficient to inhibit growth. of melanoma cancer cells.
<img file="MX351027B_D0006.tif" />
<img file="MX351027B_D0007.tif" />
INSTITHTt · MEXICANO DE LA FR ·. PIEDAD INDUS<sup>1</sup> KIa.L
The invention further provides methods for subject afflicted with cervical cancer that expresses the LIV-1 protein by administering to the subject a LIV-1 specific antibody or a LIV-1 drug antibody conjugate in an amount sufficient to inhibit the growth of cancer cells. cervical.
The invention further provides a humanized antibody comprising a mature heavy chain variable region having an amino acid sequence at least 90% identical to HB (SEQ ID NO: 10) and a mature light chain variable region at least 90% identical to LB (SEQ ID NO: 15). Optionally, the antibody comprises a mature heavy chain variable region having an amino acid sequence at least 95% identical to HB and a mature light chain variable region at least 95% identical to LB. Optionally, in any of the antibodies, positions H29, H30 and H76 are occupied by I, E, and N and L36 is occupied by Y. Optionally, any differences in the variable region structures of the mature heavy chain variable region and SEQ ID NO: 10 is / are selected from the group consisting of H27 occupied by F, H28 occupied by N, H48 occupied by I, H66 by K, H67 by A, H71 occupied by A, H76 occupied by N, H93 occupied by N, H94 occupied by V, L37 occupied by L, L39 occupied by K, L45 occupied by K and L46 occupied by L. The three of the string variable region
<img file="MX351027B_D0008.tif" />
The mature heavy chain are those of SEQ ID NO: lcT and the three CÚRs of the mature chain variable region are those of SEQ ID NO: 15. The CDRs are shown in Figure 1. Optionally, the mature heavy chain variable region is fused to a heavy chain constant region and the mature light chain constant region is fused to a light chain constant region. Optionally, the heavy chain constant region is a mutant form of the natural human constant region that is down-bound to a Fe gamma receptor relative to the natural human constant region. Optionally, the IgGl isotype heavy chain constant region. Optionally, the heavy chain constant region has an amino acid sequence comprising SEQ ID NO: 6 and the light chain constant region has an amino acid sequence comprising SEQ ID NO: 4. Optionally, the heavy chain constant region has an amino acid sequence comprising SEQ ID NO: 8 (S239C) and the light chain constant region has an amino acid sequence comprising SEQ ID NO: 4. Optionally, any differences in CDR of the mature heavy chain variable region and mature light chain variable region of SEQ ID NO: 10 and 15 respectively reside at positions H60-H65. Optionally, the mature heavy chain variable region has an amino acid sequence comprising SEQ ID NO:
and the light chain variable region ssrr * ·.
WICKED
MEXICAN INSTITUTE £
OF MATURE INDUSTRIAL PROPERTY has a sequence of amino acids that comprise He ^^ T ^<sup>: </sup>ID NO: 15. Optionally, the antibody is conjugated to a cytotoxic agent or cytostatic agent. Preferred humanized antibodies have higher affinity for LIV-1 than the BR2-14a antibody. In another embodiment, the humanized antibody has an association constant for human or cynomolgus monkey LIV-1 of 0.5 to 2 x 10<sup>9</sup> M<sup>-1</sup>.
The invention further provides a humanized antibody comprising a mature heavy chain variable region comprising the 3 CDRs of SEQ ID NO: 10 and wherein the H20, H30 and H76 positions are occupied by I, E and N respectively and a variable region of mature light chain that comprises the 3 CDRs of SEQ ID NO: 15 and where the position L36 is occupied by Y.
The invention further provides a nucleic acid encoding a mature heavy chain variable region and / or a mature light chain variable region of any of the humanized antibodies described above.
The invention further provides a method of treating a patient who has or is at risk for cancer, comprising administering to the patient an effective regimen of a humanized antibody as described above. Optionally, the cancer is breast cancer, cervical cancer, melanoma, or prostate cancer.
The invention further provides a pharmaceutical composition
<img file="MX351027B_D0009.tif" />
or. · • IMPI 'INSTITUTO MEXICANO DE LA ER <ΤΙΕΓ / Α, INDUSTRIAL comprising a humanized antibody as described above.
The invention further provides a method of treating a patient who has or is at risk for triple negative breast cancer, comprising administering to the patient an effective regimen of an antibody that effectively binds to LIV1. Optionally, in such methods, the antibody is conjugated to a cytotoxic agent or cytostatic agent.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows an alignment of the amino acid sequences of the parental murine mAb (designated as BR2-14a) with the humanized LIV-1 heavy chain (two upper panels) and light chain (two lower panels) variable regions.
Figure 2 shows the binding curves for the humanized LIV-1 mAbs and the parental murine antibody (designated as BR2-14a).
Figure 3 shows results of competition binding studies of the humanized LIV-1 mAbs and the parental murine antibody (designated as BR2-14a). The numbers in parentheses after each variant indicate the number of back mutations.
Figure 4 shows the results of MCDF7 cell saturation binding studies. BR2-14a-AF refers to
<img file="MX351027B_D0010.tif" />
parental AF-labeled murine antibody. hLIV-14 refers to the AF-labeled HBLB antibody, a humanized antibody that specifically binds to LIV-1.
Figure 5 shows the results of competition binding studies on CHO cells expressing recombinant LIV-1 protein. BR2-14a refers to the parental murine antibody. hLIV-14 HBLB WT refers to the HBLB antibody. hLIV-14 HBLB S239C refers to the HBLB antibody that has serine to cysteine substitutions at every position on the heavy chain.
Figure 6 shows an analysis of LIV-1 protein expression by IHC on post-hormone treated breast cancer patient samples.
Figure 7 shows an analysis of LIV-1 protein expression by IHC on samples from the patient with hormone-refractory metastatic prostate cancer.
Figure 8 shows an analysis of LIV-1 protein expression by IHC on samples from the triple negative breast cancer patient.
Figure 9 shows the results of cytotoxicity analysis on the hLIV-14 drug antibody conjugates, that is, the mAb HBLB conjugated to vcMMAE (1006) or mcMMAF (1269), also as control and human murine (mlgG) antibody conjugates ( hlgG). hLIV-14-SEA-1006 refers to a non-fucosylated form of the HBLB mAb conjugated to vcMMAE
<img file="MX351027B_D0011.tif" />
IMPI
MEXICAN INSTITUTE
OF THE FRCPIEDAO
INDUSTRIAL (1006). _____________
Figure 10 shows the results of an in vitro ADCC analysis on MCF7 cells using human NK cells (donor; V / V). hLIV-14 WT refers to mAb HBLB. hLIV-14
SEA refers to the non-fucosylated form of the HBLB mAb. hLIV mcMMAF refers to an antibody-drug conjugate of the HBLB mAb conjugated to mcMMAF. hLIV-14 vcMMAE refers to an antibody-drug conjugate of mAb HBLB conjugated to vcMMAE. hLIV-14 SEA vcMMAE refers to a non-fucosylated form of the HBLB mAb-antibody vcMMAEdrug conjugate.
Figure 11 shows the results of an in vitro ADCC assay on MCF7 cells using human NK cells (donor 2). hLIV-14 WT refers to mAb HBLB. hLIV-14 SEA refers to the non-fucosylated form of the mAb HBLB. cLIV14 SEA refers to the non-fucosylated form of the chimeric parental murine antibody. hLIV-14 mcF (4) refers to an antibody-drug conjugate of the HBLB mAb with an average of 4 mcMMAF drug linker molecules per antibody. hLIV-14 vcE (4) refers to an antibody-drug conjugate of the HBLB mAb with an average of 4 vcMMAE drug linker molecules per antibody.
hLIV-14 vcE (4) SEA refers to a non-fucosylated form of the mAb HBLB-antibody vcMMAE-drug conjugate having an average of 4 molecules of vcMMAE drug linker per antibody. hlgG refers to IgG huirraiTá mcF (4) refers to a test drug antibody conjugate of an unbound antibody with an average of 4 mcMMAF drug linker molecules per antibody. H00-vcE (4) refers to a test drug antibody conjugate of an unbound antibody with an average of 4 vcMMAE drug linker molecules per antibody.
Figure 12 shows the results of a xenograft study of the MCF7 breast cancer line in nude mice. cLIV-14-mcMMAF (4) refers to an antibody-drug conjugate of the chimeric form of the parental murine antibody having an average of 4 mcMMAF drug linker molecules per antibody. cLIV-14vcMMAE (4) refers to an antibody-drug conjugate of the chimeric form of the parent murine antibody having an average of 4 molecules of vcMMAE drug linker per antibody. HOO-mcMMAF (4) refers to an antibody-drug conjugate of a non-binding control antibody having an average of 4 mcMMAF drug linker molecules per antibody. HOO-vcMMAE (4) refers to an antibody-drug conjugate of a non-binding control antibody having an average of 4 molecules of mcMMAF drug linker per antibody. The dose and time of administration are indicated in the figure.
Figure 13 shows the results of a study of
<img file="MX351027B_D0012.tif" />
IMPI
MEXICAN INSTITUTE
DE LA MüUt'DAO INDUSTRIAL xenograft of the PC3 prostate cancer line in male nude mice. cLIV-14-vcMMAE (4) refers to an antibody-drug conjugate of the chimeric form of the parent murine antibody having an average of 4 molecules of vcMMAE drug linker per antibody. hBU12vcMMAE (4) refers to an antibody-drug conjugate of an anti-CD19 antibody that has an average of 4 molecules of vcMMAE drug linker per antibody. The dose and time of administration are indicated in the figure.
Figure 14 shows the results of a xenograft study of the MCF7 breast cancer line in nude mice. hLIV-14-vcMMAE (4) refers to an HBLB antibody-antibody drug conjugate having an average of 4 vcMMAE drug linker molecules per antibody. hLIV-14d-vcMMAE (2) refers to an antibody-drug conjugate of the HBLB antibody having an average of 2 vcMMAE drug linker molecules per antibody, each conjugated at position S239C of each heavy chain. H00vcMMAE (4) refers to an antibody-drug conjugate of a non-binding control antibody having an average of 4 molecules of vcMMAE drug linker per antibody. The dose and time of administration are indicated in the figure.
Figure 15 shows the results of a xenograft study of the PC3 prostate cancer line in male nude mice. hLIV-14-vcMMAE (4) refers to a conjugate
<img file="MX351027B_D0013.tif" />
of antibody-drug of the HBLB antibody having an i Branch UV «vaί a - * ΜΚ— average of 4 molecules of drug linker vcMMAE per antibody. hLIV-14-mcMMAF (4) refers to an antibody-drug conjugate of the HBLB antibody having an average of 4 mcMMAF drug linker molecules per antibody. hLIV-14d-vcMMAE (2) refers to an antibody-drug conjugate of the HBLB antibody having an average of 2 vcMMAE drug linker molecules per antibody each conjugated at the S239C position of each heavy chain. hLIV-14d-mcMMAF (2) refers to an antibody-drug conjugate of the HBLB antibody having an average of 2 mcMMAF drug linker molecules per antibody, each conjugated at position S239C of each heavy chain. H00vcMMAE (4) refers to an antibody-drug conjugate of a non-binding control antibody having an average of 4 molecules of vcMMAE drug linker per antibody. HOO-mcMMAF (4) refers to an antibody-drug conjugate of a non-binding control antibody having an average of 4 mcMMAF drug linker molecules per antibody. The dose and time of administration are indicated in the figure.
Figures 16Ά and 16B show alignments of humanized heavy chain (Figure ISA) and light chain (16B) mature variable regions with those of the mouse BR2-22a cell line.
Figure 17 shows competition link analysis
<img file="MX351027B_D0014.tif" />
of different permutations of HA-HF humanized heavy chains and LA-LF humanized light chains derived from the murine monoclonal anti-LIV-1 antibody BR2-22a. The total number of murine back mutations in each heavy or light chain is shown in parentheses. Only HELF showed sufficient binding retention.
Figure 18 shows systemic variation of the HE and LF chains to test the contribution of individual back mutations to antigen binding. The sites of potential somatic hypermutation are in parentheses. Mouse residues are underlined. The remaining residues are human germline residues.
Figure 19 shows the competition analysis of the LF variants in the upper part of the figure. The tested backmutations are shown at the bottom of the figure. Mouse residues are underlined. The remaining residues are human germline residues.
Figure 20 shows the competition link of the HE variants at the top of the figure. The tested backmutations are shown at the bottom of the figure. Mouse residues are underlined. The remaining residues are human germline residues.
Figure 21 shows the competition binding of different permutations of HE, HF, HG and LF and LG.
Figure 22 shows the saturation link of the
INSTITUTO MEXICANO DE LA PM i'IEDAP INDUSTRIAL humanized LIV14 antibody and humanized LIV22 antibody Mil ~ TT-- ~ .9 «00»> ^ W00.
on human LIV-1 and cynomolgus expressed from CHO cells.
Figure 23 shows the cytotoxic activity of humanized LIV22-vcMMAE on MCF-7 cells after 144 hours of treatment. hOO-1269 is a control drug-conjugate antibody.
Figure 24 shows the cytotoxic activity of hLIV22mcMMAF on MCF-7 cells after 144 hours of treatment. hOO-1269 is a control conjugated drug-antibody.
Figure 25 shows the activity of the hLIV22 antibody on PC3 prostate carcinoma model (DSMZ) in nude female mice. Dose days are indicated by triangles on the X-axis.
Figure 26 shows the activity of the Hliv22 antibody on MCF7 breast carcinoma tumors (NCI) in nude mice.
Figure 27 compares the activity of hLIV22 and hLIV14 in the same model as Figure 26.
Figure 28 shows an analysis of LIV-1 protein expression by IHC on samples from melanoma cancer patients.
DEFINITIONS
Monoclonal antibodies are commonly provided in
IMPI
INSTITUTO MEXICANO IX LA / MOHEDAL · INDUSTRIAL in isolation. This means that an antibody is commonly at least 50% pure w / w of interfering proteins and other contaminants arising from its production or purification but does not exclude the possibility that the monoclonal antibody is combined with an excess of carrier (s). acceptable pharmaceutical (s) or other vehicle intended to facilitate its use. Sometimes monoclonal antibodies are at least 60%, 70%, 80%, 90%, 95 or 99% w / w pure of interfering proteins and contaminants from production or purification.
The specific binding of a monoclonal antibody to its target antigen means an affinity of at least 10<sup>6</sup>, 10<sup>7</sup>, ΙΟ<sup>8</sup>, ΙΟ<sup>9</sup> or 10<sup>10</sup> M<sup>-1</sup>. The specific binding is detectably higher in magnitude and distinguishable from the non-specific binding that is prese nted to at least one unrelated target. Specific bonding can be the result of bonding between particular functional groups or particular spatial adjustment (eg, key and lock type) while non-specific bonding is usually the result of van der Waals forces. Specific binding does not, however, necessarily imply that a monoclonal antibody binds to one and only one target.
The basic antibody structural unit is a tetramer of subunits. Each tetramer includes two identical pairs of polypeptide chains, each pair has a light chain (around 25 KDa) and a heavy chain (around 50-70 KDa). The amino terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. This variable region is initially expressed linked to a cleavable signal peptide. The variable region without the signal peptide is sometimes like a mature variable region. Thus, for example, a mature light chain variable region means a light chain varia ble region without the light chain signal peptide. The carboxy-terminal portion of each chain defines a constant region primarily responsible for the effector function.
Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon and define the antibody isotype as IgG, IgM, IgA, IgD, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a J region of about 12 or more amino acids, with the heavy chain also including a D region of about 10 or more amino acids. (See generally, Fundamental Tmmunology (Paul, W., ed., 2nd ed. Raven Press, NY, 1989, Ch. 7, incorporated by reference in its entirety for all purposes).
The mature variable regions of each light / heavy chain pair form the antibody binding site. Thus, an intact antibody has two binding sites. Except in bifunctional or bis specific antibodies the two binding sites are the same. All chains exhibit the same general frameworks of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs of the two chains of each pair are aligned by framework regions, allowing binding to a specific epitope. From N-terminal to C-terminal, both heavy and light chains comprise the FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 domains. The assignment of amino acids to each domain is in accordance with the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991) or Chothia & Lesk, J. Mol. Biol. 196: 901-917 (1987); Chothia et al., Nature 342: 878-883 (1989). Kabat also provides a widely used numbering convention (Kabat numbering) in which corresponding residues between different heavy chains or between different light chains are assigned the same number.
The term "antibody" includes intact antibodies and binding fragments thereof. Commonly, antibody fragments compete with intact antibody '
IMPI INSTITUTO MEXICANO DE LA PROPIEDAD INDUSTRIAL from which they were derived for the specific binding to the target including separate heavy chains, Fab, Fab ', F (ab') 2, F (ab) c light chains, diabodies, Dab, nanobodies and Fv. The fragments can be produced by recombinant DNA techniques or by enzymatic separation or chemical separation of intact immunoglobulins. The term antibody also includes a diabody (homodimeric Fv fragment) or a mini body (VL-VH-CH3), a bis specific antibody, or the like. A bis specific or bifunctional antibody is an artificial hybrid antibody that has two different heavy / light chain pairs and two different binding sites (see, for example Songsivilai and Lachmann, Clin. Exp. Immunol., 79: 315321 (1990); Kostelny et al., J. Immunol., 148: 1547-53 (1992)). The term "antibody" includes an antibody on its own (naked antibody) or an antibody conjugated to a cytotoxic or cytostatic drug.
The term "epitope" refers to a site on an antigen to which an antibody binds. An epitope can be formed from contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids are commonly retained on exposure to denaturing solvents, while epitopes formed by tertiary folding are commonly lost in the
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denaturing solvent treatment. An epitope commonly includes at least 3 and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods for determining the special conformation of epitopes include for example X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).
Antibodies that recognize the same epitopes or overlapping epitopes can be identified in a simple immunoassay that shows the ability of an antibody to compete with the binding of another antibody to a target antigen. The epitope of an antibody can also be defined by X-ray crystallography of the antibody bound to its antigen to identify contact residues. Alternatively, two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other.
Antibody competition is determined by an assay in which an antibody under test inhibits the specific binding of a reference antibody to a
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common antigen (see, for example Junghans et al., Cancer Res. 50: 1495, 1990). A test antibody competes with a reference antibody if an excess of a test antibody (at least 2x, 5x, 10x, 20x or 100x) inhibits the binding of the reference antibody by at least 50% but preferably 75% or 90 % or 99%, as measured in a competitive binding analysis. Antibodies identified by competition analysis (competent antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to an adjacent epitope close enough to the epitope bound by the reference antibody for spherical hindrance to occur or be present.
The term "patient" includes human subjects and other mammalian subjects receiving either prophylactic or therapeutic treatment.
For purposes of classifying amino acid substitutions as conservative or non-conservative, amino acids are grouped as follows: Group I (hydrophobic side chains):): met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acid side chains): asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V (residues that influence chain orientation): gly, pro and Group VI (aromatic side chains): trp, tyr, phe.
Conservative substitutions involve substitutions between amino acids of the same class. Non-conservative substitutions constitute an exchange of a member of one of these classes for a member of another.
The percentage of sequence identities is determined with maximally aligned antibody sequences by the Kabat numbering convention. After alignment, if a subject antibody region (eg, the entire mature variable region of a heavy or light chain) is compared to the same region of a reference antibody, the percent sequence identity between the subject regions y reference is the number of positions occupied by the same amino acid in both the subject and reference antibody region divided by the total number of aligned positions of the two regions, not counting separations or spaces, multiplied by 100 to convert to a percentage.
Compositions or methods comprising one or more elements mentioned may include other elements not specifically mentioned. For example, a composition comprising an antibody may contain the antibody alone or in combination with other ingredients.
The designation of a range of values includes all the numbers within or that define the range.
An antibody effector function refers to a
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ΓΗ- LA FROHE. AD INDUSTRIAL function contributed by a domain (Sj de 're be a Yg. Such functions can be, for example, antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis or complement-dependent cytotoxicity. Such a function can be carried out, for example, by binding an Fe effector domain (s) to an Fe receptor on an immune cell via phagocytic or lytic activity or by binding an effector domain (s). ) of Fe on an immune cell with phagocytic activity or lytic activity or by binding of an effector domain (s) of Fe to components of the complement system. Commonly, the moderate effect (s) by Fe-binding cells or complement components results in inhibition and / or depletion of the targeted LIV-1 cell. Antibody Fe regions can recruit cells that express Fe receptor (FcR) and juxtapose them with antibody-coated target cells. Cells expressing surface FcRs for IgG include FcyRIII (CD16), FcyRII (CD32) and FcyRIII (CD64) can act as effector cells for the killing of IgG-coated cells. Such effector cells include monocytes, macrophages, natural killer (NK) cells, neutrophils, and eosinophils. Coupling of FcyR by IgG activates anti-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP). ADCC is moderated by CD16 + effector cells through the secretion of
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Membrane pore-forming proteins and proteases7 "" ^ Tr so much that phagocytosis is moderated by CD32 + and CD34 + effector cells (see Fundamental Immunology, 4<sup>to</sup>. ed., Paul ed. , Lippincott-Raven, NY, 1997, Chapters 3, 17 and 30; Uchida et al., 2004, J. Exp. Med. 199: 1659-69; Akewanlop et al., 2001, Cancer Res. 61: 4061-65; Watanabe et al., 1999, Breast Cancer Res. Treat. 53: 199-207). In addition to ADCC and ADCP, the Fe regions of cell-bound antibodies can also activate the classical complement pathway to produce complement-dependent cytotoxicity (CDC). Clq from the complement system binds to the Fe regions of antibodies when they are complexed with antigens. The binding of Clq to cell-linked antibodies can initiate a cascade of events involving proteolytic activation of C4 and C2 to generate C3 convertase. The cleavage of C3 to C3b by C3 convertase allows the activation of terminal complement components including C5b, C6, C7, C8 and C9. Collectively, these proteins form complex membrane attack pores on antibody-coated cells. These pores disrupt cell membrane integrity, killing the target cell (see Immunobiology, 6th ed., Janeway et al., Garland Science, NY, 2005, Chapter 2).
The term antibody-dependent cellular cytotoxicity, or ADCC, is a mechanism for inducing cell death that depends on the interaction of target cells.
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INSTITUTO MEXICANO DE LA HtOHEDAD INDUSTRIAL antibody-coated with immune cells that possess lithic activity (also known as elector cells). Such elector cells include natural killer cells, monocyte / macrophage, and neutroilia. Elector cells attach to an Ig Fe elector domain (s) bound to target cells via their antigen combining sites. The death of the antibody-coated target cell occurs as a result of elector cell activity.
The term antibody-dependent cellular lakecytosis, or ADCP, refers to the process by which antibody-coated cells are internalized, either in whole or in part, by phagocytic immune cells (eg, macrophages, neutroils, and dendritic cells) that bind. to one (s) elector domain (s) of Fe de Ig.
The term complement-dependent cytotoxicity, or CDC, refers to a mechanism to induce cell death in which an Fe effector domain (s) of a target-linked antibody activates a series of enzymatic reactions that culminate in the formation of target cell membrane holes. Commonly, antibody-antigen complexes such as those on antibody-coated target cells bind and activate the complement component Clq which in turn activates the complement cascade leading to target cell death. Activation of
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OF THE PROPERTY
1N DUSTRI AL _ ™ LJZ> ^ complement may also result in 11a — Ía · - ^ poe-ÍOion — of— components of complement on the surface of the target cell that facilitates ADCC by binding of complement receptors (eg, CR3) on leukocytes.
A cytotoxic effect refers to the depletion, elimination and / or killing of a target cell. A cytotoxic agent refers to an agent that has a cytotoxic effect on a cell. Cytotoxic agents can be conjugated to an antibody or administered in combination with an antibody.
A cytostatic effect refers to the inhibition of cell proliferation. A cytostatic agent refers to an agent that has a cytostatic effect on a cell, thereby inhibiting the growth and / or expansion of a specific subset of cells. Cytostatic agents can be conjugated to an antibody or administered or in combination with an antibody.
The term "pharmaceutically acceptable" means approved or that may be approved by a regulatory agency of the federal or state government or listed in the United States Pharmacopeia or other recognized pharmacopoeia for use in general in animals and more particularly in humans. The term "pharmaceutically compatible ingredient" refers to a pharmaceutically acceptable diluent, adjuvant, excipient, or carrier with which
IMPI INSTITUTO MEXICAN. , DE LA MONEDAD industrial an anti-LIV-1 antibody.
The phrase "pharmaceutically acceptable salt" refers to pharmaceutically acceptable organic or inorganic salts of an anti-LIV-1 antibody or conjugate thereof or agent administered with an anti-LIV-1 antibody. Exemplary salts include salts of sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate , maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (that is, 1,1'-methylene bis- (2-hydroxy-3-naphthoate)). A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion, or another counter ion. The counter ion can be any organic or inorganic portion that establishes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt can have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt may have multiple counter ions. Hence, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counter ions.
Unless otherwise evident from the context,
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LO'JPIFOAL MEXICAN IIUÍO DE LA lO'JPIFOAL) industrial the term around encompasses values within the standard deviation of the stated value.
DETAILED DESCRIPTION
I. General
The invention provides monoclonal antibodies that specifically bind to LIV-1. The antibodies are useful for the treatment and diagnosis of various cancers as well as the detection of LIV-1.
II. Target molecules
Unless otherwise indicated, LIV-1 means human LIV-1. An exemplary human sequence is assigned no. Accession Swiss ProtQ13433. Q13433 is included herein as SEQ ID NO: 83. Three variant isoforms and one polymorphism are known. A second version of the human LIV-1 protein, accession number AAA96258.2, is included herein as SEQ ID NO: 84. Four extracellular domains are linked by residues 29-325, 377423, 679-686 and 746-755 of Q13433 respectively.
Unless otherwise apparent from the context, reference LIV-1 means at least one extracellular domain of the protein and usually the whole protein other than a cleavable signal peptide (amino acids 1-28 of Q13433).
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MEXICAN INSTITUTE OF ΙΛ INDUSTRIAL PROPERTY
III. Antibodies of the invention
A. Binding specificity and functional properties
The invention provides humanized antibodies derived from two mouse antibodies, BR2-14a and BR2-22a. Unless specifically stated otherwise, the present disclosure is concerned with both antibodies. The two mouse antibodies show 94% and 91% sequence identity to each other in the mature heavy chain and light chain variable regions. The two antibodies bind to the same epitopes or overlapping epitopes on human LIV-1. However, the BR2-22a antibody has about ten times higher affinity for human LIV-1 and about 3 times higher affinity for cynomolgus monkey LIV-1 than BR2-14a as shown in Figure 22.
The affinity of the humanized forms of the mouse BR2-14a antibody (ie, Ka) is preferably within a factor of five or a factor of two than that of the mouse BR2-14a antibody to human LIV-1. Humanized BR2-14a antibodies specifically bind to human LIV-1 naturally and / or recombinantly expressed from CHO cells as does the mouse antibody from which they are derived. Preferred humanized BR2-14a antibodies have an affinity the same or greater than (that is, greater than beyond the margin of error in measurement) that of BR2-14a for
<img file="MX351027B_D0024.tif" />
INDUSTRIAL
Human LIV-1 (for example, 1.1-5 times, 1.1 a ... 3 ..vpr.pg, 1.5 a ... 3 times, 1.7 to 2.3 times or 1.7-2.1 times the affinity or about two times the affinity of BR2-14a). Preferred humanized BR2-14a antibodies bind to the same epitope and / or compete with BR2-14a for binding to human LIV-1. Preferred humanized BR2-14a antibodies also bind to the LIV-1 zino-homologue thus allowing pre-clinical testing in non-human primates.
The affinity of humanized forms of the mouse BR2-22a antibody (i.e., Ka) for human LIV-1, expressed naturally or expressed from CHO cells, is preferably within a factor of five or a factor of two of aguel of BR2-22 mouse antibody. Some humanized BR2-22a antibodies have an association constant that is essentially the same as that of BR222a (that is, within experimental error). Some BR2-22a antibodies have an association constant within a range of 0.5 to 0.5-1.5 that of the association constant for the BR2-22a antibody. Preferred humanized BR2-22a antibodies have an association constant greater than 5 x 10<sup>8</sup> M "<sup>1</sup> or in a range of 0.5 to 2 x 10<sup>9</sup> M<sup>-1</sup> or about 0.8 x10<sup>9</sup> M<sup>-1</sup> (+/- error in measurement) for human LIV1 expressed from CHO cells. Here as elsewhere in this application, the affinities can be measured according to the methods of the examples. BR2-
<img file="MX351027B_D0025.tif" />
Preferred humanized 22a bind to the same epitope and / or compete with BR2-22a for binding to human LIV-1. Humanized BR2-22a antibodies bind to the cyno-homolog of LIV-1, also like human LIV-1. Preferred humanized BR2-22a antibodies bind essentially the same association constant to human LIV-1 and cynomolgus monkey LIV-1 both expressed from CHO cells (within experimental error) thus allowing and increasing the predictive accuracy of pretests. clinics in nonhuman primates.
Preferred antibodies (both humanized BR2-14a and humanized BR2-22a) inhibit cancer (eg, cell growth, metastasis, and / or mortality to organisms) as shown in cancer cells that were tested in culture, in a model animal or clinical test. Animal models can be formed by implanting human tumor cell lines expressing LIV-1 into appropriate immune-deficient rodent strains, for example athymic nude mice or SCID mice. These tumor cell lines can be established in immune deficient rodent hosts either as solid tumors by subcutaneous injections or as disseminated tumors by intravenous injections. Once established within the host, these tumor models can be applied to evaluate the therapeutic efficacy of antiLIV-1 antibodies or conjugated forms thereof as described in
<img file="MX351027B_D0026.tif" />
the examples.
B. Humanized antibodies
A humanized antibody is a genetically engineered antibody in which the CDRs of a human donor antibody are grafted onto human acceptor antibody sequences (see, for example, Queen, US Patents 5,530,101 and 5,585,089; Winter, US Patent 5,225,539; Carter, US Patent 6,407,213 ; Adair, US Patent 5,859,205 and Foote, US Patent 6,881,557). The acceptor antibody sequences can be for example a mature human antibody sequence, a combination of such sequences, a human antibody consensus sequence or a germline region sequence. A preferred acceptor sequence for the heavy chain is exon V<sub>H</sub> V<sub>H</sub>l-2 germline V<sub>H </sub>(also referred to in the literature as HV1-2) (Shin et al., 1991, EMBO J. 10: 3641-3645) and for the hinge region (J<sub>H</sub>), exon J<sub>h</sub>-6 (Mattila et al., 1995, Eur. J. Immunol. 25: 2578-2582). For the light chain, a preferred acceptor sequence is exon VK2-30 (also referred to in the literature as KV2-30) and for the hinge region exon Jk-4 (Hieter et al., 1982, J. Biol. Chem. 257: 1516-1522). Thus, a humanized antibody is an antibody that has some or all of the CDRs completely or substantially of a donor antibody and variable region framework sequences and
INSTITUTO MtXICA, - »· Γ, υε The rtóLff.in ré-ríí J '^ í indu; t, li.al constant regions, if present, completely or substantially of human antibody sequences. Similarly, a humanized heavy chain has at least one, two, and usually all three CDRs completely or substantially from a donor antibody heavy chain and a heavy chain variable region and heavy chain constant region framework sequence, if present. , substantially from human heavy chain variable region framework sequences and constant region sequences. Similarly, a humanized light chain has at least one, two, and usually all three CDRs completely or substantially from a donor antibody light chain and a light chain variable region and light chain constant region framework sequence, if present. , substantially from human light chain variable region framework sequences and constant region sequences. Unlike nanobodies and dAbs, a humanized antibody comprises a humanized heavy chain and a humanized light chain. A CDR in a humanized antibody is substantially a corresponding CDR in a non-human antibody, when at least 60%, 85%, 90%, 95% or 100% of corresponding residues (as defined by Kabat) are identical among the respective CDR. The structural sequences of the variable region of an antibody or the constant region of a chain
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of antibody are substantially of human variable region or human constant region framework sequence, respectively, when at least 85%, 90%, 95% or
100% of the corresponding residues defined by Kabat are identical.
Although humanized antibodies frequently incorporate all six CDR 4s (preferably as defined by Kabat) of a mouse antibody, they can also be made with fewer than all CDRs (eg, at least 3, 4 or 5) CDRs of a mouse antibody (eg, Pascalis et al., J. Immunol. 169: 3076, 2002; Vajdos et al., Journal of Molecular Biology, 320: 415-428, 2002; Iwahashi et al., Mol. Immunol. 36: 1079-1091, 1999; Tamura et al, Journal of Immunology, 164: 1432-1441, 2000).
Certain amino acids of human variable region framework residues can be selected for substitution based on their possible influence on CDR conformation and / or antigen binding. the investigation of such possible influences is through modeling, examination of the characteristics of amino acids at particular sites, or empirical observation of the effects of substitution or muta genesis of particular amino acids.
For example, when an amino acid differs between a murine variable region structure residue and a selected human variable region structure residue, Instituto Mexicano
OF THE PROPERTY
INDUSTRIAL Vg-gHPjjS * the amino acid of human structure can the amino acid of equivalent structure of mouse antibody when the amino acid is reasonably expected to:
(1) non-covalently binds antigen directly, (2) is adjacent to a CDR region, (3) otherwise interacts with a CDR region (e.g., is within about 6 Á of a CDR region ) or (4) moderates the interaction between heavy and light chains.
The invention provides humanized forms of the mouse BR2-14a antibody including five exemplified humanized heavy chain mature variable regions (HA-HE) and six exemplified humanized light chain mature variable regions (LA-LF). The permutations of these chains that have the strongest bond (lowest EC50) are HBLB, HBLF, HCLB, HCLF, HDLB, HDLF, HELE, and HELF. Of these permutations, HBLB (also known as hLIV14) is preferred because it has the strongest binding, about twice as strong as the mouse donor antibody, and has the fewest back mutations (four).
The invention provides variants of the humanized HBLB antibody in which the mature humanized heavy chain variable regions show at least 90%, 95% or 99% of
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INSTITUTO MEXICANO DE LA PRCFIEOAi »¡NLUSTRIAL identity to SEQ ID NO: 10 and the variable region” mature ** ^ ”'”' humanized light chain shows at least 90%, 95% or 99% sequence identity to SEQ ID NO: 15. Preferably, in such antibodies, some or all of the HBLB back mutations are retained. In other words, at least 1, 2 or preferably all 3 heavy chain positions H29, H30 and H7 6 are occupied by I and E and N, respectively. Also, position L36 is preferably occupied by Y. The CDR regions of such humanized antibodies are preferably substantially identical to the CDR regions of HBLB, which are the same as those of the mouse donor antibody. The CDR regions can be defined by any conventional definition (eg Chothia) but are preferably as defined by Kabat. In one embodiment, the humanized antibody comprises a heavy chain comprising the three CDRs of SEQ ID NO: 10 and variable region structures with at least 95% identity to the variable region structures of SEQ ID NO:
10. In another embodiment, the humanized antibody comprises a light chain comprising the 3 CDRs of SEQ ID NO: 15 and variable region structures with at least 95% identity to the variable region structure of SEQ ID NO: 15. In a In a further embodiment, the humanized antibody comprises a heavy chain comprising the 3 CDRs of SEQ ID
NO: 10 and variable region structures with at least 95% identity to the variable region structures of SEQ ID NO: 10 and a light chain comprising the 3 CDRs of SEQ ID NO: 15 and variable region structures with for at least 95% identity to the variable region structures of SEQ ID NO: 15.
Since humanized antibodies show some variation from the exemplified humanized HBLB antibody, one possibility for such additional variation is additional back mutations in the variable region structures. Any or all back mutated positions in other exemplified mature humanized heavy chain or light chain variable regions can also be elaborated (i.e., 1, 2, 3, 4, 5, 6, 7, 8 or all 9 of occupied H27 by F, H28 occupied by N, H48 occupied by I, H66 occupied by K, H67 occupied by A, H71 occupied by A, H7 6 occupied by N, H93 occupied by N and H94 occupied by V in the heavy chain and 1, 2, 3, 4 or all 5 of L37 occupied by L, L39 occupied by K, L45 occupied by K and L46 occupied by L in the light chain. However, such additional back mutations are not preferred because they generally do not improve affinity and the introduction of more mouse residues can give increased risk of immunogenicity.
The invention provides humanized forms of the mouse BR-22a antibody including three exemplified humanized mature heavy chain variable regions (HER, HF, and HG) and two
IMPI mexican institute DE LA MONEDA D
INDUSTRIAL * 5 * exemplified humanized light chains (LF and LF) that can be combined in different permutations with appropriate linkage (see Figure 21). Of these permutations HGLG (also known as hLIV22) is preferred because it has the best combination of binding properties (essentially the same as the mouse BR2-22a antibody within experimental error) and fewer back-mutations (seven).
The invention provides humanized antibody variants of HGLG in which the humanized heavy chain mature variable region shows at least 90%, 95%, 98% or 99% identity to SEQ ID NO: 53 and the mature chain variable region Humanized light shows at least 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 60. Preferably, in such antibodies, some or all of the back mutations in HGLG are retained. In other words, at least 1, 2, 3, 4 or preferably all 5 heavy chain positions H27, H29, H30, H76 and H94 are occupied by L, I, E, N and V (here, as in any part of this application, Kabat numbering is used to describe positions in the mature variable heavy and light chain variable regions). Of these backmutations, H94 contributes the most to retention of binding affinity and H76 the least. Also, positions L36 and L46 are preferably occupied by Y and P respectively. The CDR regions of such humani 7, λdQS -, - S antibodies are substantially identical in preference to the CDR regions of HGLG, which are the same as those of the mouse donor antibody. The CDR regions can be defined by any conventional definition (eg Chothia) but are preferably as defined by Kabat. In one embodiment, the humanized antibody comprises a heavy chain comprising the 3 CDRs of SEQ ID NO: 53 and variable region structure with at least 95% identity to the variable region structures of SEQ ID NO: 53. In another embodiment, the humanized antibody comprises a light chain comprising the 3 CDRs of SEQ ID NO: 60 and variable region structures with at least 95% identity to the variable region structures of SEQ ID NO: 60. In a further embodiment, the humanized antibody comprises a heavy chain comprising the 3 CDRs of SEQ ID NO: 53 and variable region structure with at least 95% identity to the variable region structures of SEQ ID NO: 53 and a light chain comprising the 3 CDRs of SEQ ID NO: 60 and variable region structure with at least 95% identity to the variable region structures of SEQ ID NO: 60.
Since the humanized BR2-22a antibodies show some variation from the exemplified humanized HGLG antibody, one possibility for such additional variation is additional backmutations in the variable region structures. Any or all back mutated positions in other exemplified humanized heavy or light chain mature variable regions can also be made (i.e. 1, 2, 3, 4, 5 or all 6 of H28 occupied by N, H48 occupied by I , H66 occupied by K, H67 occupied by A, H71 occupied by A, H93 occupied by T in the heavy chain and 1 or 2 of L37 occupied by L37 occupied by L and L45 occupied by K. however, such additional backmutations are not preferred because they generally do not improve affinity and the introduction of more mouse residues can give increased risk of immunogenicity.
Another possible variation is to replace certain residues in the mouse antibody CDRs with corresponding residues from human CDR sequences, commonly from the CDRs of the human acceptor sequences used in designing the exemplified humanized antibodies. In some antibodies, only part of the CDRs, ie the subset of CDR residues required for binding, termed the SDRs, are necessary to retain the binding in a humanized antibody. CDR residues that do not contact the antigen and are not in the SDRs can be identified based on previous studies (e.g., H60-H65 residues in H2 of CDRs are often not required), from CDR regions of Kabat that fall outside
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INSTITUTO MEXICANO DE LA PROPIEDAD INDUSTRIAL the hypervariable loops of Chothia (Chothia, J. Mol. Biol. 196: 901, 1987) by molecular modeling and / or empirically or as described in Gonzales et al., Mol. Immunol. 41: 863 (2004). In such humanized antibodies, at positions in which one or more donor CDR residues is absent or in which either an entire donor CDR is omitted, the amino acid occupying the position may be an amino acid occupying the corresponding position (by numbering de Kabat) in the acceptor antibody sequence. The number of such donor amino acid acceptor substitutions in the CDR to include reflects a balance of competent considerations. Such substitutions are potentially advantageous in lowering the number of mouse amino acids in a humanized antibody and consequently lowering potential immunogenicity. However, substitutions can also cause affinity changes and significant reductions in affinity are preferably avoided. In a further variation, one or more residues in a CDR of a humanized BR2-22a antibody (which would otherwise be the same as the CDR of the mouse BR2-22a antibody) can be replaced by corresponding residues of a CDR of the BR2 antibody. -14a mouse (or vice versa). Positions for substitution in CDRs and amino acids to substitute can also be selected empirically.
Although not preferred, others can be made
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D £ LA ΜΟίΊΙΞΟΑΟ V * wrtB INDUSTRIAL amino acid substitutions, for example, at framework residues not in contact with CDRs or even some amino acids from potential CDR contact residues within CDRs. Frequently, the replacements made to variant humanized sequences are conservative with respect to the replaced HBLB amino acids (in the case of humanized BR2-14a) or HGLG amino acids (in the case of humanized BR2-22). Preferably, replacements relative to HBLB or HGLG (whether conservative or not) have no substantial effect on the binding affinity or potency of the humanized mAb, i.e., smoothness to bind to human LIV-1 and inhibit cell growth. Of cancer.
Variants commonly differ from HBLB (HLIV14) or HGLG (hLIV22) heavy and light chain mature variable region sequences by a small number (e.g., commonly no more than 1, 2, 3, 5, or 10 in either the mature variable region of light chain or heavy chain or both) of replacements, cancellations or insertions.
C. Constant region selection
The heavy and light chain variable regions of humanized antibodies can be linked to at least a portion of a human constant region. The choice of constant region depends in part, if antibody-dependent cell-moderate cytotoxicity is desired, cellular phagocytosis
<img file="MX351027B_D0030.tif" />
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antibody-dependent and / or cytotoxicity-dependent-icnt-o- -decomplement. For example, the human IgGl and IgG3 isotypes have strong complement-dependent cytotoxicity, the human IgG2 isotype weak complement-dependent cytotoxicity, and human IgG4 lacks complement-dependent cytotoxicity. Human IgGl and IgG3 also induce stronger cell-moderated effector functions than human IgG2 and IgG4. The light chain constant regions can be lambda or kappa. Antibodies can be expressed as tetramers containing two light chains and two heavy chains, as separate light chains, light chains, such as Fab, Fab ', F (ab') 2 and Fv, or as single chain antibodies in which the Heavy and light chain variable domains are linked together by means of a spacer.
Human constant regions show allotypic variation and isoalotypic variation between different individuals, that is, constant regions can differ between different individuals in one or more polymorphic positions. Isoalotypes differ from allotypes in that sera that recognize an isoalotype bind to a non-polymorphic region of one or more other isotypes.
One or more amino acids at the amino or carboxy terminus of the light chain and / or the heavy chain, such as the C-terminal lysine of the heavy chain, may be missing
<img file="MX351027B_D0031.tif" />
<img file="MX351027B_D0032.tif" />
LNSTJTU ΤΟ ΜΛΧ! C a é O DE La PííOO; '?. \ D INDUSTRIAL or be derived in a proportion or all · ίά · α<sup>1</sup> molecule ^ ST ^ 'S ^ ”· can make substitutions in constant regions to reduce or increase effector function such as moderate complement cytotoxicity or ADCC (see, for example, Winter et al., US Patent No. 5,624,821; Tso et al. ., US Patent No. 5,834,597 and Lazar et al., Proc. Nati. Acad. Sci. USA 103: 4005, 2006) or to prolong the half-life in humans (see, for example, Hinton et al., J. Biol Chem. 279: 6213, 2004).
Exemplary substitution that includes the amino acid substitution of the natural amino acid to a cysteine residue is introduced at amino acid position 234, 235, 237, 239, 267, 298, 299, 326, 330 or 332, preferably an S239C mutation at a human IgGl isotype (US patent 20100158909). The presence of an additional cysteine residue allows for interchain disulfide bond formation. Such interchain disulfide bond formation can cause spherical hindrance, thereby reducing the affinity of the Fc-Fc and R region bonding interaction. The cysteine residue (s) introduced into or in proximity to the Fe region of an IgG constant region may also serve as a site for conjugation to therapeutic agents (i.e., coupling cytotoxic drugs using specific reagents of thiols such as maleimide derivatives of drugs). The
<img file="MX351027B_D0033.tif" />
<img file="MX351027B_D0034.tif" />
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THE INDUSTRIAL PROn £ -'AD presence of a therapeutic agent causes steric hindrance, thereby further reducing the affinity of the Fe-FcyR region-binding interaction. Other substitutions at any of positions 234, 235, 236, and / or 237 reduce affinity for Fcy receptors, particularly the FcyRI receptor (see, for example, US Pat. No. 6,624,821, US Pat.
5,624,821).
The in vivo half-life of an antibody can also impact its effector functions. The half-life of an antibody can be increased or decreased to modify its therapeutic activities. FcRn is a receptor that is structurally similar to MHC class I antigen that associates non-covalently with p2-microglobulin. FcRn regulates IgG metabolism and its transcytosis through tissues (Ghetie and Ward, 2000, Annu. Rev. Immunol. 18: 739-766; Ghetie and Ward, 2002, Immunol. Res. 25: 97-113). The IgG-FcRn interaction takes place at pH 6.0 (pH of intracellular vesicles) but not at pH 7.4 (pH of blood); this interaction allows IgG to be recycled back into the circulation (Ghetie and Ward, 2000, Ann. Rev. Immunol. 18: 739-766; Ghetie and Ward, 2002, Immunol. Res. 25: 97-113). The region on human IgGl involved in FcRn binding has been mapped (Shields et al., 2001, J. Biol. Chem. 276: 6591-604). Alanine substitutions in positions
<img file="MX351027B_D0035.tif" />
INDUSTRIAL
Pro238, Thr256, Thr307, Gln311, Asp312, -G'1L138 (T7 —— or Asn434 from human IgGl enhance the binding of FcRn (Shields et al., 2001, J. Biol. Chem. 276: 6591-604). Molecules of IgGl harboring these substitutions have longer serum half-lives. Consequently, these modified IgGl molecules may be able to carry out their effector functions and thereby exert their therapeutic efficacy over a longer period of time compared to IgGl. unmodified. Other exemplary substitutions to increase binding to FcRn include a Gln at position 250 and / or a Leu at position 248. EU numbering is used for all positions in the constant region.
The oligosaccharides covalently attached to conserved Asn297 are involved in the ability of the Fe region of an IgG to bind to FcyR (Lund et al., 1996, J. Immunol. 157: 4963-69; Wright and Morrison, 1997, Trends Biotechnol 15: 26-31). Designing this glycoform on IgG can significantly improve IgG-moderate ADCC. The addition of bisecting N-acetylglucosamine modifications (Umana et al., 1999, Nat. Biotechnol. 17: 176-180; Davies et al., 2001, Biotech. Bioeng. 74: 288-94) to this glycoform or fucose removal (Shields et al., 2002, J. Biol. Chem. 277: 26733-40; Shinkawa et al., 2003, J. Biol. Chem. 278: 6591- 604; Niwa et al., 2004, Cancer Res. 64: 212733) of this glycoform are two examples of Fe design of
IgG that enhances the binding between IgG Fe and FcyR, thereby enhancing Ig-moderate ADCC activity.
A systemic solvent-exposed amino acid substitution of the Fe region of human IgGl has generated IgG variants with altered FcyR binding affinities (Shields et al., 2001, J. Biol. Chem. 276: 6591-604). When compared to parental IgGl, a subset of these variants involving substitutions at Thr256 / Ser298, Ser298 / Glu333, Ser298 / Lys334, or Ser298 / Glu333 / Lys334 to Ala demonstrate both increased binding affinity for FcyR and ADCC activity (Shields et al. al., 2001, J. Biol. Chem. 276: 6591-604; Okazaki et al., 2004, J. Mol. Biol. 336: 123949).
The complement fixation activity of antibodies (both Clq binding and CDC activity) can be enhanced by substitutions in Lys326 and Glu333 (Idusogie et al., 2001, J. Immunol. 166: 2571-2575). The same substitutions on a human IgG2 backbone can convert an antibody isotype that binds poorly to Clq and is severely deficient in complement activation activity to one that can both bind Clq and moderate CDC (Idusogie et al., 2001, J. Immunol. 166: 2571-75). Several other methods have also been applied to enhance the complement fixation activity of antibodies. For example, the grafting of
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INSTITUTO mexican ..
FROM THE INDUSTRIAL MOntriAl 'xA- ”μΓ an 18 amino acid carboxyl-terminal tail piece of IgM to the carboxyl termini of IgG greatly enhances its CDC activity. This is observed with IgG4, which normally does not have any detectable CDC activity (Smith et al., 1995, J. Immunol. 154: 2226-36). Also, the substitution of Ser444 located near the carboxy-terminus of the IgGl heavy chain with Cys induced tail-to-tail dimerization of IgGl with a 200-fold increase in CDC activity relative to monomeric IgGl (Shopes et al., 1992 , J. Immunol. 148: 2918-22). Furthermore, a specific bis diabody construct with specificity for Clq also confers CDC activity (Kontermann et al., 1997, Nat. Biotech. 15: 629-31).
Complement activity can be reduced by mutating at least one of amino acid residues 318, 320 and 322 of the heavy chain to a residue having a different side chain, such as Ala. Other alkyl-substituted nonionic residues, such as Gly, lie, Leu, or Val, or such aromatic non-polar residues such as Phe, Tyr, Trp, and Pro instead of any one of the three residues also reduces or abolishes the bond of Clq. Ser, Thr, Cys and Met can be used at residues 320 and 322, but not 318, to reduce or abolish the binding activity of Clq. Replacement of residue 318 (Glu) by a polar residue can modify but not abolish the binding activity of Clq. Replacement of residues 297 (Asn) with Ala, results in removal of lytic activity but only slightly reduces (about three times weaker) the affinity for Clq. this alteration destroys the glycosylation site and the presence of carbohydrate that is required for complement activation. Any other substitution at this site also destroys the glycosylation site. The following mutations and any combination of them also reduce Clq binding: D270A, K322A, P329A and P311S (see WO 06/036291).
Reference to a human constant region includes a constant region with any natural allotypes or any permutation of residues occupying polymorphic positions in natural allotypes. Also, up to 1, 2, 5, or 1.0 mutations may be present relative to a natural human constant region, such as those listed above to reduce Fcy receptor binding or increase FcRn binding.
D. Expression of recombinant antibodies
Humanized antibodies are commonly produced by recombinant expression. Recombinant polynucleotide constructs or constructs commonly include an expression control sequence operably linked to antibody chain coding sequences, including associated promoter regions.
INSTITUTO MEXICANO DE LA FROPIEI'Ai INDUSTRIAL naturally or heterologous. Preferably. the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under appropriate conditions for high-level expression of the nucleotide sequences and the collection and purification of cross-reactive antibodies.
Mammalian cells are a preferred host for expressing immunoglobulin-encoding nucleotide segments or fragments thereof. See Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). A number of appropriate host cell lines capable of secreting intact heterologous proteins have been developed in the art and include CEO cell lines (eg, DG44), various COS cell lines, HeLa cells, HEK293 cells, L cells, and myelomas that do not produce antibodies. including Sp2 / 0 and NSO. Preferably the cells are non-human. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and information processing sites. necessary, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. The sequences of
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98 members in 27 offices
Priority claims14
| Document | Office | Kind | Date |
|---|---|---|---|
| 42029110 | United States of America | P | |
| 42029110 | United States of America | P | |
| 61420291 | United States of America | – | |
| 201161446990 | United States of America | P | |
| 201161446990 | United States of America | P | |
| 61446990 | United States of America | – | |
| 2011063612 | United States of America | W | |
| 2011063612 | United States of America | W | |
| 61420291 | – | – | – |
| 61446990 | – | – | – |
| PCTUS2011063612 | – | – | – |
| US20100420291P | – | – | – |
| US201161446990P | – | – | – |
| WO2011US63612 | – | – | – |
Members98
| Document | Office | Kind | |
|---|---|---|---|
| CA2819038A1 | Canada | A1 | |
| CA3211246A1 | Canada | A1 | |
| WO2012078688A2 | World Intellectual Property Organization (WIPO) | A2 | |
| WO2012078688A3 | World Intellectual Property Organization (WIPO) | A3 | |
| TW201241181A | Taiwan Province of China | A | |
| AU2011338480A1 | Australia | A1 | |
| SG190938A1 | Singapore | A1 | |
| MX2013006365A | Mexico | A | |
| US2013259860A1 | United States of America | A1 | |
| EP2648752A2 | European Patent Office (EPO) | A2 | |
| KR20130135884A | Republic of Korea | A | |
| CN103533957A | China | A | |
| JP2014506120A | Japan | A | |
| ZA201303803B | South Africa | B | |
| RU2013130609A | Russian Federation | A | |
| EP2648752A4 | European Patent Office (EPO) | A4 | |
| NZ611468A | New Zealand | A | |
| US9228026B2 | United States of America | B2 | |
| SG10201510041QA | Singapore | A | |
| CN103533957B | China | B | |
| US2016185858A1 | United States of America | A1 | |
| BR112013013781A2 | Brazil | A2 | |
| RU2608646C2 | Russian Federation | C2 | |
| EP2648752B1 | European Patent Office (EPO) | B1 | |
| AU2011338480B2 | Australia | B2 | |
| AU2011338480A8 | Australia | A8 | |
| AU2011338480B8 | Australia | B8 | |
| DK2648752T3 | Denmark | T3 | |
| PT2648752T | Portugal | T | |
| JP6105481B2 | Japan | B2 | |
| LT2648752T | Lithuania | T | |
| EP3156420A1 | European Patent Office (EPO) | A1 | |
| HRP20170567T1 | Croatia | T1 | |
| AU2017203851A1 | Australia | A1 | |
| ES2620264T3 | Spain | T3 | |
| SI2648752T1 | Slovenia | T1 | |
| TWI591176B | Taiwan Province of China | B | |
| HUE031726T2 | Hungary | T2 | |
| PL2648752T3 | Poland | T3 | |
| JP2017149717A | Japan | A | |
| RS55843B1 | Serbia | B1 | |
| MX351027BThis record | Mexico | B | |
| US9783608B2 | United States of America | B2 | |
| CY1118874T1 | Cyprus | T1 | |
| US2018079810A1 | United States of America | A1 | |
| IL226754A | Israel | A | |
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| IL261900A | Israel | A | |
| IL261900D0 | Israel | D0 | |
| JP6453924B2 | Japan | B2 | |
| AU2017203851B2 | Australia | B2 | |
| EP3156420B1 | European Patent Office (EPO) | B1 | |
| EP3461847A1 | European Patent Office (EPO) | A1 | |
| DK3156420T3 | Denmark | T3 | |
| AU2019202530A1 | Australia | A1 | |
| JP2019073515A | Japan | A | |
| TR201905240T4 | Türkiye | T4 | |
| PT3156420T | Portugal | T | |
| SI3156420T1 | Slovenia | T1 | |
| KR101993921B1 | Republic of Korea | B1 | |
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| ES2719548T3 | Spain | T3 | |
| HUE043355T2 | Hungary | T2 | |
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| US2020165335A1 | United States of America | A1 | |
| CY1121546T1 | Cyprus | T1 | |
| IL273607A | Israel | A | |
| IL273607D0 | Israel | D0 | |
| EP3461847B1 | European Patent Office (EPO) | B1 | |
| MX2020010639A | Mexico | A | |
| DK3461847T3 | Denmark | T3 | |
| PT3461847T | Portugal | T | |
| KR20200145867A | Republic of Korea | A | |
| KR102198189B1 | Republic of Korea | B1 | |
| EP3786185A1 | European Patent Office (EPO) | A1 | |
| SI3461847T1 | Slovenia | T1 | |
| AU2019202530B2 | Australia | B2 | |
| EP3461847A8 | European Patent Office (EPO) | A8 | |
| JP6869218B2 | Japan | B2 | |
| HUE052806T2 | Hungary | T2 | |
| PL3461847T3 | Poland | T3 | |
| ES2842895T3 | Spain | T3 | |
| AU2021204473A1 | Australia | A1 | |
| JP2021106599A | Japan | A | |
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| USRE48959E | United States of America | E | |
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1 legal event, as the office reported them to INPADOC
Events
| Event | Code | |
|---|---|---|
| Grant or registrationFG | FG |
Numbers
- Publication
- 351027
- Publication, DOCDB
- 351027
- Publication, EPODOC
- MX351027
- Application
- 2013006365
- Application, DOCDB
- 2013006365
- Application, EPODOC
- MX20130006365
Titles2
- Spanish
- ANTICUERPOS HUMANIZADOS A LIV-1 Y USO DE LOS MISMOS PARA TRATAR CANCER.
- English
- ANTIBODIES HUMANIZED TO LIV-1 AND USE OF THE SAME TO TREAT CANCER.
Classification
- CPC, 17
- C07K16/28
- A61K2039/505
- C07K16/3015
- C07K16/3069
- C07K2317/565
- C07K2317/92
- C07K2317/24
- A61K47/6865
- C07K16/465
- A61P13/08
- A61P15/00
- A61P17/00
- A61P35/00
- C07K2317/56
- C07K2317/732
- C07K16/3053
- C07K2317/52
- IPC, 2
- C07K16 30
- A61K39 395
