JP5778700B2

Folate receptor 1 antibodies and immunoconjugates and uses thereof

Abstract

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JP5778700B2, drawing sheet 1 — Sheet 1 of 57

Term

4.4 yearsleft in the term

Expires 24 February 2031.

43 claims: 34 independent, 9 dependent

1
ヒトフォレート受容体1に特異的に結合するヒト化抗体又はその抗原結合フラグメントであって、該抗体が下記:(a)GYFMN(配列番号1)を含む重鎖CDR1;RIHPYDGDTFYNQKFQG(配列番号2) を含む重鎖CDR2;及びYDGSRAMDY(配列番号3)を含む重鎖CDR3;及び、 (b)KASQSVSFAGTSLMH(配列番号7)を含む軽鎖CDR1;RASNLEA(配列番号8)を含む軽鎖CDR2;及びQQSREYPYT(配列番号9)を含む軽鎖CDR3;を含む、ヒト化抗体又はその抗原結合フラグメント。
2
配列番号4の重鎖可変ドメイン、および配列番号10又は11の軽鎖可変ドメインを含む、請求項1に記載のヒト化抗体又はその抗原結合フラグメント。
3
配列番号6の重鎖、および配列番号12又は13の軽鎖を含む、請求項1に記載のヒト化抗体又はその抗原結合フラグメント。
4
2010年4月7日にATCCに寄託され、そしてATCC受託番号PTA-10772 を有するプラスミドDNAによりコードされる重鎖、および2010年4月7日にATCCに寄託され、そしてATCC受託番号 PTA-10773又は PTA- 10774を有するプラスミドDNAによりコードされる軽鎖を含むヒト化抗体又はその抗原結合フラグメント。
5
完全長抗体である請求項1~ 4 の何れか1項に記載のヒト化抗体又はその抗原結合フラグメント。
6
抗原結合フラグメントである請求項1 または2の何れか1項に記載のヒト化抗体又はその抗原結合フラグメント。
7
該ヒト化抗体又はその抗原結合フラグメントがFab、Fab’、F(ab’) 2 、Fd、単鎖Fv即ちscFv、ジスルフィド連結Fv、 v NARドメイン、IgNar、イントラボディー、IgG-CH2、ミニボディー、F(ab') 3 、テトラボディー、トリアボディー、ダイアボディー、単ドメイン抗体、DVD-Ig、Fcab、mAb 2 、(scFv) 2 、又はscFv-Fcを含む、請求項 6 記載のヒト化抗体又はその抗原結合フラグメント。
8
1 .0 ~1 0nMのKdでヒトフォレート受容体1に結合する、請求項1~ 7 の何れか1項に記載のヒト化抗体又はその抗原結合フラグメント。
9
0.06~1.0nMのKdでヒトフォレート受容体1に結合する、請求項1~7の何れか1項に記載のヒト化抗体又はその抗原結合フラグメント。
10
(a)請求項1~ 9 の何れか1項に記載のヒト化抗体を発現する細胞を培養すること;及び (b)該培養細胞からヒト化抗体を単離することを含む該ヒト化抗体を作成する方法。
11
式(A)-(L)-(C)を有し、式中:(A)はヒト化抗体又は抗原結合フラグメントであり;(L)はリンカーであり;そして、(C)は細胞傷害剤であり;そして該リンカー(L)は(A)を(C)に連結し、該ヒト化抗体又は抗原結合フラグメント(A)は、GYFMN(配列番号1)を含む重鎖CDR1;RIHPYDGDTFYNQKFQG(配列番号2)を含む重鎖CDR2;YDGSRAMDY(配列番号3)を含む重鎖CDR3;KASQSVSFAGTSLMH(配列番号7)を含む軽鎖CDR1;RASNLEA(配列番号8)を含む軽鎖CDR2;及びQQSREYPYT(配列番号9)を含む軽鎖CDR3を含む、イムノコンジュゲート。
12
該リンカーが切断可能なリンカー、切断不可能なリンカー、親水性リンカー、及びジカルボン酸系リンカーよりなる群から選択される、請求項1 1 記載のイムノコンジュゲート。
13
該リンカーがN-スクシンイミジル4-(2-ピリジルジチオ)ペンタノエート(SPP)又はN-スクシンイミジル4-(2-ピリジルジチオ)-2-スルホペンタノエート(スルホ-SPP);N-スクシンイミジル4-(2-ピリジルジチオ)ブタノエート(SPDB)又はN-スクシンイミジル4-(2-ピリジルジチオ)-2-スルホブタノエート(スルホ-SPDB);N-スクシンイミジル4-(マレイミドメチル)シクロヘキサンカルボキシレート(SMCC);N-スルホスクシンイミジル4-(マレイミドメチル)シクロヘキサンカルボキシレート(スルホSMCC);N-スクシンイミジル4-(ヨードアセチル)-アミノベンゾエート(SIAB);及びN-スクシンイミジル-[(N-マレイミドプロピオンアミド)-テトラエチレングリコール]エステル(NHS-PEG4-マレイミド)よりなる群から選択される、請求項1 2 記載のイムノコンジュゲート。
14
該リンカーがN-スクシンイミジル4-(2-ピリジルジチオ)ブタノエート(SPDB)又はN-スクシンイミジル4-(2-ピリジルジチオ)-2-スルホブタノエート(スルホ-SPDB)である、請求項1 3 記載のイムノコンジュゲート。
15
該細胞傷害剤がマイタンシノイド、マイタンシノイド類縁体、ベンゾジアゼピン、タキソイド、CC-1065、CC-1065類縁体、デュオカルマイシン、デュオカルマイシン類縁体、カリケアマイシン、ドラスタチン、ドラスタチン類縁体、アリスタチン、トマイマイシン誘導体、及びレプトマイシン誘導体又は該剤のプロドラッグよりなる群から選択される、請求項1 1 ~1 4 の何れか1項に記載のイムノコンジュゲート。
16
該細胞傷害剤がマイタンシノイドである、請求項1 5 記載のイムノコンジュゲート。
17
該細胞傷害剤がN(2’)-デアセチル-N(2’)-(3-メルカプト-1-オキソプロピル)-マイタンシン又はN(2’)-デアセチル-N ( 2 ’) -(4-メルカプト-4-メチル-1-オキソペンチル)-マイタンシンである、請求項1 6 記載のイムノコンジュゲート。
18
下記:(A)配列番号4の重鎖可変ドメイン及び配列番号10又は配列番号11の軽鎖可変ドメインを含むヒト化抗体;(L)N-スクシンイミジル-[(N-マレイミドプロピオンアミド)-テトラエチレングリコール]エステル(NHS-PEG4-マレイミド);N-スクシンイミジル4-(2-ピリジルジチオ)ブタノエート(SPDB);又はN-スクシンイミジル4-(2-ピリジルジチオ)-2-スルホブタノエート(スルホ-SPDB);及び、(C)N(2’)-デアセチル-N ( 2 ’) -(4-メルカプト-4-メチル-1-オキソペンチル)-マイタンシン;を含み、ここで(L)は(A)を(C)に連結する、イムノコンジュゲート。
19
下記:(A)配列番号4の重鎖可変ドメイン及び配列番号10又は配列番号11の軽鎖可変ドメインを含むヒト化抗体;(L)N-スクシンイミジル4-(2-ピリジルジチオ)-2-スルホブタノエート(スルホ-SPDB);及び、(C)N(2’)-デアセチル-N ( 2 ’) -(4-メルカプト-4-メチル-1-オキソペンチル)-マイタンシン;を含み、ここで(L)は(A)を(C)に連結する、イムノコンジュゲート。
20
該ヒト化抗体は、配列番号4の重鎖可変ドメイン、および配列番号11の軽鎖可変ドメインを含む、請求項 1 1~ 19の何れかに記載のイムノコンジュゲート。
21
(A)が、ヒトフォレート受容体1への特異的結合に関してchMov19と競合する、抗体又はその抗原結合フラグメントを含む、請求項1 1 ~2 0の何れかに記載のイムノコンジュゲート。
22
該ヒト化抗体又は抗原結合フラグメントが、10nM以下のKdでヒトフォレート受容体1に結合する、請求項1 1 ~2 1の何れかに記載のイムノコンジュゲート。
23
2~6個の(C)をさらに含む、請求項1 1 ~2 2 の何れか1項に記載のイムノコンジュゲート。
24
請求項1~ 9 の何れか1項に記載のヒト化抗体又はその抗原結合フラグメント、或いは請求項1 1 ~2 3 の何れか1項に記載のイムノコンジュゲート及び製薬上許容しうる担体を含む医薬組成物。
25
請求項1~ 9 の何れか1項に記載のヒト化抗体又はその抗原結合フラグメント、或いは請求項1 1 ~2 3 の何れか1項に記載のイムノコンジュゲートを含み、第2の抗癌剤をさらに含む、医薬組成物。
26
該イムノコンジュゲートが(A)当たり平均で3~4 個の(C)を有する請求項2 4 又は2 5 に記載の医薬組成物。
27
請求項1~ 9 の何れか1項に記載のヒト化抗体又はその抗原結合フラグメントを含む診断試薬。
28
標識をさらに含む、請求項27に記載の診断試薬。
29
該標識が放射標識、蛍光団、発色団、画像化剤及び金属イオンよりなる群から選択される、請求項 28 記載の診断試薬。
30
請求項1~ 9 の何れか1項に記載のヒト化抗体又はその抗原結合フラグメント、或いは請求項1 1 ~2 3 の何れか1項に記載のイムノコンジュゲートを含むキット。
31
腫瘍生育の阻害における使用のための、請求項2 4 ~ 26 のいずれか一項に記載の医薬組成物。
32
腫瘍生育の阻害のための医薬の製造における、請求項1~ 9 の何れか1項に記載のヒト化抗体又はその抗原結合フラグメント、請求項1 1 ~2 3 の何れか1項に記載のイムノコンジュゲート、或いは請求項2 4 ~ 26 のいずれか一項に記載の医薬組成物の使用。
33
癌の治療における使用のための、請求項2 4 ~ 26 のいずれか一項に記載の医薬組成物。
34
癌の治療のための医薬の製造における、請求項1~ 9 の何れか1項に記載のヒト化抗体又はその抗原結合フラグメント、請求項1 1 ~2 3 の何れか1項に記載のイムノコンジュゲート、或いは請求項2 4 ~ 26 のいずれか一項に記載の医薬組成物の使用。
35
該腫瘍又は癌が、卵巣腫瘍又は癌、脳腫瘍又は癌、乳房腫瘍又は癌、子宮腫瘍又は癌、子宮内膜腫瘍又は癌、膵臓腫瘍又は癌、腎臓腫瘍又は癌、及び肺腫瘍又は癌よりなる群から選択される、請求項3 1 又は3 3 に記載の医薬組成物。
36
該腫瘍が卵巣腫瘍又は肺腫瘍である、請求項 35 に記載の医薬組成物。
37
癌の該治療において腫瘍生育が阻害される、請求項3 3 に記載の医薬組成物。
38
該腫瘍又は癌が、卵巣腫瘍又は癌、脳腫瘍又は癌、乳房腫瘍又は癌、子宮腫瘍又は癌、子宮内膜腫瘍又は癌、膵臓腫瘍又は癌、腎臓腫瘍又は癌、及び肺腫瘍又は癌よりなる群から選択される、請求項3 2 又は3 4 に記載の使用。
39
該腫瘍が卵巣腫瘍又は肺腫瘍である、請求項 38 に記載の使用。
40
癌の該治療において腫瘍生育が阻害される、請求項3 4 に記載の使用。
41
該医薬は、第2の抗癌剤を含む、請求項3 2 又は3 4 に記載の使用。
42
該第2の抗癌剤が化学療法剤である、請求項4 1 記載の使用。
43
請求項1~ 9 の何れか1項に記載のヒト化抗体、又は抗原結合フラグメントを生産する単離された細胞。

Independent claims43

192 paragraphs, as filed

0001Cross-reference of related applications This application is filed on February 24, 2010, US Provisional Patent Application No. 61 / 307,797, US Provisional Patent Application No. 61 / 346,595, filed on May 20, 2010, and November 12, 2010. Claim the priority of US Provisional Patent Application No. 61 / 413,172, each of which is incorporated herein by reference in its entirety.

0002The field of the present invention generally relates to antibodies and immunoconjugates that bind to human forate receptor 1 and methods of using antibodies and immunoconjugates for the treatment of diseases such as cancer.

0003Cancer is one of the leading causes of death in developed countries, with more than one million people diagnosed with cancer annually in the United States and 500,000 deaths. Overall, more than one in three will develop some form of cancer during their lifetime. There are more than 200 different types of cancer, four of which, breast cancer, lung cancer, colorectal cancer and prostate cancer, account for the majority of all new cases (Jemal et al., 2003, Cancer J. Clin. 53). : 5-26).

0004Forate receptor (FOLR1), also known as forate receptor alpha or forate-binding protein, is an N-glycosylation protein expressed on the plasma membrane of cells. FOLR1 has a high affinity for folic acid and for several reduced folic acid derivatives. FOLR1 mediates the delivery of physiological forate, 5-methyltetrahydrofolate, into the cell.

0005FOLR1 is overexpressed in most ovarian cancers and in many uterine cancers, endometrial cancers, pancreatic cancers, kidney cancers, lung cancers and breast cancers, but FOLR1 expression on normal tissues is proximal tubules, Confined to the apical membrane of epithelial cells in the alveolar, bladder, testis, choroidal flora, and thyroid (Weitman SD, et al., Cancer Res 52: 3396-3401 (1992); Antony AC, AnnuRev Nutr 16: 501-521 ( 1996); Kalli KR, et al., Gynecol Oncol 108: 619-626 (2008)). This pattern of expression of FOLR1 makes it a desirable target for the treatment of FOLR1-oriented cancers.

0006Because ovarian cancer is typically non-symptomatic until advanced stage, it is often diagnosed late and with currently available procedures, typically chemotherapeutic agents after surgical weight loss. Poor prognosis when treated (von Gruenigen V et al., Cancer 112: 2221-2227 (2008); Ayhan A et al., Am JObstet Gynecol 196: 81 e81-86 (2007); Harry VN et al., Obstet GynecolSurv 64 : 548-560 (2009)). That is, there is a clear medical need for a more effective therapeutic agent for ovarian cancer.

0007Three anti-FOLR1 antibodies are being investigated as potential anti-cancer agents. The murine monoclonal antibodies Mov18 and Mov19 were isolated in the late 1980s (Miotti S et al., Int J Cancer 39: 297-303 (1987)) and confirmed to target FOLR1 (ConeyLR et al., Cancer Res 51: 6125- 6132 (1991)), and their ability to eradicate antigen-expressing cancer cells as conjugates with cytotoxic ribosome inactivating proteins has been tested in preclinical studies (CondeFP et al., Eur J Biochem 178: 795-802). (1989)).

0008Mov19 is a bispecific antibody that targets cytotoxic T cells and natural killer cells (Mezzanzanica D et al., Int J Cancer 41: 609-615 (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 fusion protein of Mov19 single-stranded Fv with interleukin-2 in vivo (Melani C et al., Cancer Res 58: 4146-4154 (1998)). Being tested. Chimeric (variable murine / human constant) anti-FOLR1 antibodies Mov18 and Mov19 have been preclinically investigated for their ability to mediate cytotoxic immune cell-dependent killing of FOLR1-expressing tumors in vitro (ConeyLR et al., Cancer Res). 54: 2448-2455 (1994)), and the chimeric Mov18-IgE has been tested in an IgE-dependent pre-immunotherapy clinical model (Karagiannis SN et al., J Immunol 179: 2832-2843 (2007); Gould HJ et al., Eur J Immunol 29: 3527-3537 (1999)).

0009Mov18 was studied in preclinical studies and then in clinical trials in the early 1990s (Zacchetti A et al., Nucl Med Biol 36: 759-770 (2009)) in the form of conjugates with various radionuclides, which drug Has also been discontinued without approval for clinical use.

0010The humanized form of MORAb003, the murine monoclonal anti-FOLR1 antibody LK26, is as an unmodified antibody (Ebel W et al., Cancer Immun 7: 6 (2007)), and111It has been preclinically evaluated as a conjugate with In radionuclides (Smith-Jones PM et al., Nucl Med Biol 35: 343-351 (2008)) and is currently in clinical trials as an unmodified antibody (Smith-Jones PM et al., Nucl Med Biol 35: 343-351 (2008)). DK Armstrong et al., J. Clin. Oncol. 26: Addendum, May 20, 2008; Abstract 5500).

<nplcit num="1"><text>Jemal et al., 2003, Cancer J. Clin. 53: 5-26</text></nplcit><nplcit num="2"><text>WeitmanSD, et al., Cancer Res 52: 3396-3401 (1992)</text></nplcit><nplcit num="3"><text>AntonyAC, Annu Rev Nutr 16: 501-521 (1996)</text></nplcit><nplcit num="4"><text>Kalli KR, et al., Gynecol Oncol 108: 619-626 (2008)</text></nplcit><nplcit num="5"><text>vonGruenigen V et al., Cancer 112: 2221-2227 (2008)</text></nplcit><nplcit num="6"><text>AyhanA et al., Am J Obstet Gynecol 196: 81 e81-86 (2007)</text></nplcit><nplcit num="7"><text>HarryVN et al., Obstet Gynecol Surv 64: 548-560 (2009)</text></nplcit><nplcit num="8"><text>MiottiS et al., Int J Cancer 39: 297-303 (1987)</text></nplcit><nplcit num="9"><text>ConeyLR et al., Cancer Res 51: 6125-6132 (1991)</text></nplcit><nplcit num="10"><text>Conde FP et al., Eur J Biochem 178: 795-802 (1989)</text></nplcit><nplcit num="11"><text>Mezzanzanica D et al., Int J Cancer 41: 609-615 (1988)</text></nplcit><nplcit num="12"><text>Ferrini S et al., Int J Cancer Suppl 4: 53-55 (1989)</text></nplcit><nplcit num="13"><text>Ferrini S et al., Int J Cancer 48: 227-233 (1991)</text></nplcit><nplcit num="14"><text>Melani C et al., Cancer Res 58: 4146-4154 (1998)</text></nplcit><nplcit num="15"><text>Coney LR et al., Cancer Res 54: 2448-2455 (1994)</text></nplcit><nplcit num="16"><text>Karagiannis SN et al., J Immunol 179: 2832-2843 (2007)</text></nplcit><nplcit num="17"><text>Gould HJ et al., Eur J Immunol 29: 3527-3537 (1999)</text></nplcit><nplcit num="18"><text>Zacchetti A et al., Nucl Med Biol 36: 759-770 (2009)</text></nplcit><nplcit num="19"><text>Ebel W et al., Cancer Immun 7: 6 (2007)</text></nplcit><nplcit num="20"><text>Smith-Jones PM et al., Nucl Med Biol 35: 343-351 (2008)</text></nplcit><nplcit num="21"><text>DK Armstrong et al., J. Clin. Oncol. 26: May 20, 2008 Addendum; Summary 5500</text></nplcit>

The present invention provides novel antibodies that bind to human forate receptor 1, immunoconjugates containing these antibodies, and methods of using them. The present invention also provides novel polypeptides, such as antibodies that bind to human forate receptor 1, fragments of such antibodies, and other polypeptides associated with such antibodies. Polynucleotides containing nucleic acid sequences encoding polypeptides, as well as vectors containing polynucleotides are also provided. Cells containing the polypeptides and / or polynucleotides of the invention are also provided. Compositions comprising novel forate receptor 1 antibodies or immunoconjugates (eg, pharmaceutical compositions) are also provided. Furthermore, there is also provided a method for producing and using a novel forate receptor 1 antibody or immunoconjugate, for example, a method for using a novel forate receptor 1 antibody or conjugate for inhibiting tumor growth and / or treating cancer. Will be done. That is, in one aspect, the invention provides a humanized antibody or antigen-binding fragment thereof that specifically binds to human forate receptor 1, where the antibody is heavy, comprising: (a) GYFMN (SEQ ID NO: 1): Chain CDR1; RIHPYDGDTFYNQXaa1FXaa2Xaa3Heavy chain CDR2 containing (SEQ ID NO: 56) and heavy chain CDR3 containing YDGS RAMDY (SEQ ID NO: 3); and (b) Light chain CDR1 containing KASQSVSFAGTSLMH (SEQ ID NO: 7); Light containing RASNLEA (SEQ ID NO: 8). Chain CDR2; and light chain CDR3 containing QQSREYPYT (SEQ ID NO: 9); however, where Xaa1Is selected from K, Q, H and R; Xaa2Is selected from Q, H, N and R; and Xaa3Includes those selected from G, E, T, S, A and V; In certain embodiments, the humanized antibody or antigen-binding fragment thereof binds to human forate receptor 1 with substantially the same affinity as antibody chimera Mov19. In certain embodiments, the humanized antibody or antigen-binding fragment thereof comprises the heavy chain CDR2 sequence RIHPYDGDTFYNQKFQG (SEQ ID NO: 2). In certain embodiments, binding affinity is measured by flow cytometry, Biacore or radioimmunoassay. In another embodiment, the invention comprises the following: (a) heavy chain CDR1 comprising GYFMN (SEQ ID NO: 1), or a variant thereof comprising a conservative amino acid substitution of 1, 2, 3 or 4; RIHPYDGDT FYNQKFQG. Heavy chain CDR2 containing (SEQ ID NO: 2), or a variant thereof containing a conservative amino acid substitution of 1, 2, 3 or 4; and heavy chain CDR3 containing YDG SRAMDY (SEQ ID NO: 3), or 1, 2, 3 or Its variants containing 4 conservative amino acid substitutions; and / or (b) light chain CDR1 containing KASQSVSFAGTSLMH (SEQ ID NO: 7), or its variants containing 1, 2, 3 or 4 conservative amino acid substitutions; Light chain CDR2 containing RASNLEA (SEQ ID NO: 8), or a variant thereof containing a conservative amino acid substitution of 1, 2, 3 or 4; and light chain CDR3 containing QQSREYPYT (SEQ ID NO: 9), or 1, 2, 3 Alternatively, a humanized antibody that specifically binds to human forate receptor 1, or an antigen-binding fragment thereof, which comprises a variant thereof containing 4 conservative amino acid substitutions; is provided. In certain embodiments, the present invention provides a humanized antibody or antigen-binding fragment thereof that specifically binds to human forate receptor 1, which comprises the heavy chain of SEQ ID NO: 6. In another embodiment, the humanized antibody or antigen-binding fragment thereof was deposited with the ATCC on April 7, 2010 and is encoded by a plasmid DNA bearing ATCC Accession Nos. PTA-10772, PTA-10773 or 10774. In certain embodiments, the present invention relates to (a) heavy chain CDR1 comprising GYFMN (SEQ ID NO: 1); RIHPYDGDTFYNQXaa for specific binding to FOLR1.1FXaa2Xaa3Heavy chain CDR2 containing (SEQ ID NO: 56) and heavy chain CDR3 containing YDGS RAMDY (SEQ ID NO: 3); and (b) Light chain CDR1 containing KASQSVSFAGTSLMH (SEQ ID NO: 7); Light containing RASNLEA (SEQ ID NO: 8). Chain CDR2; and light chain CDR3 containing QQSREYPYT (SEQ ID NO: 9); however, where Xaa1Is selected from K, Q, H and R; Xaa2Is selected from Q, H, N and R; and Xaa3Provides a humanized antibody or antigen-binding fragment thereof that competes with an antibody comprising one selected from G, E, T, S, A and V; In certain embodiments, the humanized antibody comprises the heavy chain CDR2 sequence RIHPYDGDTFYNQKFQG (SEQ ID NO: 2). In certain embodiments, the invention comprises a heavy chain variable domain that is at least about 90% identical to SEQ ID NO: 4 and a light chain variable domain that is at least about 90% identical to SEQ ID NO: 10 or SEQ ID NO: 11. Provided is a polypeptide which is a humanized antibody or an antigen-binding fragment thereof. In another embodiment, the humanized antibody or antigen binding fragment is a heavy chain variable domain that is at least about 95% identical to SEQ ID NO: 4, and a light chain variable that is at least about 95% identical to SEQ ID NO: 10 or SEQ ID NO: 11. Includes domain. In other embodiments, the humanized antibodyThe antigen binding fragment comprises a heavy chain variable domain that is at least about 99% identical to SEQ ID NO: 4 and a light chain variable domain that is at least about 99% identical to SEQ ID NO: 10 or SEQ ID NO: 11. In certain embodiments, the humanized antibody comprises a heavy chain variable domain of SEQ ID NO: 4, and a light chain variable domain of SEQ ID NO: 10 or SEQ ID NO: 11 for the antigen binding fragment. In certain embodiments, the invention provides a polypeptide, antibody, or antigen binding fragment that is at least about 90% identical to SEQ ID NOs: 88-119. In certain embodiments, the invention provides a polypeptide, antibody, or antigen binding fragment that is at least about 95% identical to SEQ ID NOs: 88-119. In certain embodiments, the invention provides a polypeptide, antibody, or antigen binding fragment that is at least about 99% identical to SEQ ID NOs: 88-119. In certain embodiments, the present invention provides a humanized antibody or antigen-binding fragment thereof expressed at least 10-fold higher than chMov19 in eukaryotic cells. In certain embodiments, the eukaryotic cell is a HEK-293T cell. In certain embodiments, the present invention comprises the following: (a) heavy chain CDR1 containing SSYGMS (SEQ ID NO: 30); heavy chain CDR2 containing TISSGGSYTY (SEQ ID NO: 31); and / or DGEGGL YAMDY (SEQ ID NO: 32). Heavy chain CDR3 containing: and / or (b) light chain CDR1 containing KASDHINNWLA (SEQ ID NO: 27); light chain CDR2 containing GATSLET (SEQ ID NO: 28); and light chain CDR3 containing QQYWSTPFT (SEQ ID NO: 29); Provided is an antibody that specifically binds to human forate receptor 1, or an antigen-binding fragment thereof. In another embodiment, the invention comprises the following: (a) heavy chain CDR1 containing TNYWMQ (SEQ ID NO: 60); heavy chain CDR2 containing AIYPGNGDSR (SEQ ID NO: 61); and / or RDGNYAAY (SEQ ID NO: 62). Heavy chain CDR3 containing: and / or (b) light chain CDR1 containing RASENIYSNLA (SEQ ID NO: 57); light chain CDR2 containing AATNLAD (SEQ ID NO: 58); and light chain CDR3 containing QHFWASPYT (SEQ ID NO: 59); Provided is an antibody that specifically binds to human forate receptor 1, or an antigen-binding fragment thereof. In another embodiment, the invention comprises the following: (a) heavy chain CDR1 containing TNYWMY (SEQ ID NO: 66); heavy chain CDR2 containing AIYPGNSDTT (SEQ ID NO: 67); and / or RHDYGAMDY (SEQ ID NO: 68). ) Containing heavy chain CDR3; and / or (b) light chain CDR1 containing RASENIYTNLA (SEQ ID NO: 63); light chain CDR2 containing TASNLAD (SEQ ID NO: 64); and light chain CDR3 containing QHFWVSPYT (SEQ ID NO: 65); Provided is an antibody that specifically binds to human forate receptor 1, or an antigen-binding fragment thereof. In another embodiment, the invention comprises the following: (a) heavy chain CDR1 containing SSFGMH (SEQ ID NO: 72); heavy chain CDR2 containing YISSGSSTIS (SEQ ID NO: 73); and / or EAYGSSMEY (SEQ ID NO: 74). Heavy chain CDR3; And / or (b) light chain CDR1 containing RASQNINNNLH (SEQ ID NO: 69); light chain CDR2 containing YVSQSVS (SEQ ID NO: 70); and light chain CDR3 containing QQSNSWPHYT (SEQ ID NO: 71) to human forate receptor 1. Provided is an antibody that specifically binds or an antigen-binding fragment thereof. In another embodiment, the invention comprises the following: (a) heavy chain CDR1 containing TSYTMH (SEQ ID NO: 78); heavy chain CDR2 containing YINPISGYTN (SEQ ID NO: 79); and / or GGAYGRKPMDY (SEQ ID NO: 80). Heavy chain CDR3 containing: and / or (b) light chain CDR1 containing KASQNVGPNVA (SEQ ID NO: 75); light chain CDR2 containing SASYRYS (SEQ ID NO: 76); and light chain CDR3 containing QQYNSYPYT (SEQ ID NO: 77); Provided is an antibody that specifically binds to human forate receptor 1, or an antigen-binding fragment thereof. In certain embodiments, the polypeptides of the invention are full-length antibodies or antigen-binding fragments. In certain embodiments, the antibody or antigen binding fragment is Fab, Fab', F (ab').2, Fd, single chain Fv or scFv, disulfide linked Fv, V-NAR domain, IgNar, intrabody, IgG-CH2, minibody, F (ab')3, Tetrabody, Triabody, Diabody, Monoclonal antibody, DVD-Ig, Fcab, mAb2, (ScFv)2, Or scFv-Fc. In certain embodiments, the antibodies or polypeptides of the invention bind to human forate receptor 1 at about 1.0 to about 10 nM Kd. In one embodiment, the antibody or polypeptide binds to human forate receptor 1 at about 1.0 nM or better Kd. In certain embodiments, binding affinity is measured by flow cytometry, Biacore or radioimmunoassay. The present invention also provides a method of producing the antibody, comprising culturing cells expressing the antibody of the invention; and (b) isolating the antibody from the cultured cells. In certain embodiments, the cell is a eukaryotic cell. The invention also has formulas (A)-(L)-(C), where (A) is the antibody or antigen binding fragment or polypeptide of the invention; (L) is the linker; and , (C) is a cytotoxic agent; and the linker (L) provides an immunoconjugate that links (A) to (C). In certain embodiments, the linker is selected from the group consisting of cleavable linkers, non-cleavable linkers, hydrophilic linkers, and dicarboxylic acid-based linkers. In other embodiments, the linker is N-succinimidyl 4- (2-pyridyldithio) pentanoate (SPP) or N-succinimidyl 4- (2-pyridyldithio) -2-sulfopentanoate (sulfo-SPP); N- Succinimidyl 4- (2-pyridyldithio) butanoate (SPDB) or N-succinimidyl 4- (2-pyridyldithio) -2-sulfobutanoate (sulfo-SPDB); N-succinimidyl 4- (maleimidemethyl) cyclohexanecarboxylate (SMCC); N-Sulfosuccinimidyl 4- (maleimidemethyl) cyclohexanecarboxylate (sulfoSMCC); N-Succinimidyl 4- (iodoacetyl) -aminobenzoate (SIAB); and N-succinimidyl-[(N-) It is selected from the group consisting of maleimide propionamide) -tetraethylene glycol] ester (NHS-PEG4-maleimide). In certain embodiments, the linker is N-succinimidyl-[(N-maleimidepropionamide) -tetraethylene glycol] ester (NHS-PEG4-maleimide). In one embodiment, the immunoconjugates are maytansinoids, maytansinoid analogs, benzodiazepines, taxoids, CC-1065, CC-1065 analogs, duocarmycin, duocarmycin analogs, calikeamycin, drastatin, Includes cytotoxic agents selected from the group consisting of drastatin analogs, aristatin, tomymycin derivatives, and leptomycin derivatives or prodrugs of the agents. In other embodiments, the cytotoxic agent is maytansinoid. In another embodiment, the cytotoxic agent is N (2')-deacetyl-N (2')-(3-mercapto-1-oxopropyl) -maitansine or N (2')-deacetyl-N2- (4). -Mercapto-4-methyl-1-oxopentyl) -maitansine. In one embodiment, the invention is a humanized antibody comprising (A) a heavy chain variable domain of SEQ ID NO: 4 and a light chain variable domain of SEQ ID NO: 10 or SEQ ID NO: 11; (L) N-succinimidyl-[(N-). Maleimide propionamide) -tetraethylene glycol] ester (NHS-PEG4-maleimide); and (C) N (2')-deacetyl-N2- (4-mercapto-4-methyl-1-oxopentyl) -maytancin; Where (L) provides an immunoconjugate that connects (A) to (C). In one embodiment, the invention is a humanized antibody comprising (A) a heavy chain variable domain of SEQ ID NO: 4 and a light chain variable domain of SEQ ID NO: 10 or SEQ ID NO: 11; (L) N-succinimidyl 4- (2-). Pyridyldithio) butanoate (SPDB); and (C) N (2')-deacetyl-N2- (4-mercapto-4-methyl-1-oxopentyl) -maytancin; where (L) is (L) Provided is an immunoconjugate that connects A) to (C). In one embodiment, the invention is a humanized antibody comprising (A) a heavy chain variable domain of SEQ ID NO: 4 and a light chain variable domain of SEQ ID NO: 10 or SEQ ID NO: 11; (L) N-succinimidyl 4- (2-). Includes pyridyldithio) -2-sulfobutanoate (sulfo-SPDB); and (C) N (2') -deacetyl-N2- (4-mercapto-4-methyl-1-oxopentyl) -mitancin; , Where (L) provides an immunoconjugate that connects (A) to (C). In one embodiment, the invention is a humanized antibody comprising (A) a heavy chain variable domain of SEQ ID NO: 4 and a light chain variable domain of SEQ ID NO: 10 or SEQ ID NO: 11; (L) N-succinimidyl 4- (2-). Pyridyldithio) -2-sulfopentanoate (sulfo-SPP); and (C); N (2')-deacetyl-N (2')-(3-mercapto-1-oxopropyl) -maytancin; Including, where (L) provides an immunoconjugate connecting (A) to (C). In one embodiment, the invention is a humanized antibody comprising (A) a heavy chain variable domain of SEQ ID NO: 4 and a light chain variable domain of SEQ ID NO: 10 or SEQ ID NO: 11; (L) N-succinimidyl 4- (2-). Pyridyldithio) pentanoate (SPP); and (C); N (2')-deacetyl-N (2')-(3-mercapto-1-oxopropyl) -maitansine; where (L) is Provided is an immunoconjugate that connects (A) to (C). The present invention also provides a pharmaceutical composition comprising the antibody, antigen binding fragment, polypeptide or immunoconjugate of the present invention and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition further comprises a first anti-cancer agent. The invention also provides diagnostic reagents comprising labeled antibodies of the invention, antigen binding fragments, polypeptides or immunoconjugates. In one embodiment, the label is selected from the group consisting of radiolabels, fluorophores, chromophores, imaging agents and metal ions. The present invention also provides a kit comprising the antibody, antigen binding fragment, polypeptide or immunoconjugate of the present invention. The invention also provides a method of inhibiting tumor growth in a subject, comprising administering to the subject a therapeutically effective amount of an antibody, antigen binding fragment, polypeptide, immunoconjugate or pharmaceutical composition of the invention. In certain embodiments, the invention has formulas (A)-(L)-(C), where (A) is an antibody or antigen-binding fragment thereof that specifically binds to human forate receptor 1. (L) is a linker; and (C) is a cytotoxic agent selected from the group consisting of maytansinoids and maytansinoid analogs; where (L) is (A) ( C) involves administering a therapeutically effective amount of an immunoconjugate linked to C); where the immunoconjugate reduces mean tumor volume by at least 2-fold in a KB xenograft model, a method of inhibiting tumor growth in a subject. provide. In certain embodiments, the methods are (a) heavy chain CDR1 containing GYFMN (SEQ ID NO: 1); heavy chain CDR2 containing RIHPYDGDTFYNQXaa1FXaa2Xaa3 (SEQ ID NO: 56); and heavy chain CDR3 containing YDGS RAMDY (SEQ ID NO: 3); , (B) Light chain CDR1 containing KASQSVSFAGTSLMH (SEQ ID NO: 7); Light chain CDR2 containing RASNLEA (SEQ ID NO: 8); and Light chain CDR3 containing QQSREYPYT (SEQ ID NO: 9);1Is selected from K, Q, H and R; Xaa2Is selected from Q, H, N and R; and Xaa3Includes administration of an antibody or antigen-binding fragment thereof, including those selected from G, E, T, S, A and V; In other embodiments, the antibody comprises heavy chain CDR2 comprising RIHPYDGDTFYNQKFQG (SEQ ID NO: 2). In certain embodiments, the present invention has been deposited with the ATCC on April 7, 2010, and is an antibody or antigen-binding fragment thereof encoded by a plasmid DNA having ATCC accession numbers PTA-10772, PTA-10773 or 10774. To provide a method for inhibiting tumor growth, including administration of. In another embodiment, the method is a humanized antibody comprising a heavy chain variable domain of SEQ ID NO: 4 and a light chain variable domain of SEQ ID NO: 10 or SEQ ID NO: 11; (L) N-succinimidyl-[(N-maleimidepropionamide). )-Tetraethylene glycol] ester (NHS-PEG4-maleimide); and (C) N (2')-deacetyl-N2- (4-mercapto-4-methyl-1-oxopentyl) -mytancin; Provided to administer a conjugate. In another embodiment, the method is a humanized antibody comprising (A) the heavy chain variable domain of SEQ ID NO: 4 and the light chain variable domain of SEQ ID NO: 10 or SEQ ID NO: 11; (L) N-succinimidyl 4- (2-). Pyridyldithio) butanoate (SPDB); and (C) N (2')-deacetyl-N2- (4-mercapto-4-methyl-1-oxopentyl) -maytancin; where (L) is (L) It is provided to administer an immunoconjugate that links A) to (C). In another embodiment, the method is a humanized antibody comprising (A) the heavy chain variable domain of SEQ ID NO: 4 and the light chain variable domain of SEQ ID NO: 10 or SEQ ID NO: 11; (L) N-succinimidyl 4- (2-). Includes pyridyldithio) -2-sulfobutanoate (sulfo-SPDB); and (C) N (2') -deacetyl-N2- (4-mercapto-4-methyl-1-oxopentyl) -mitancin; Here (L) provides administration of an immunoconjugate that links (A) to (C). In another embodiment, the method is a humanized antibody comprising (A) the heavy chain variable domain of SEQ ID NO: 4 and the light chain variable domain of SEQ ID NO: 10 or SEQ ID NO: 11; (L) N-succinimidyl 4- (2-). Pyridyldithio) -2-sulfopentanoate (sulfo-SPP); and (C); N (2')-deacetyl-N (2')-(3-mercapto-1-oxopropyl) -maytancin; Included; where (L) provides administration of an immunoconjugate that links (A) to (C). In another embodiment, the method is a humanized antibody comprising (A) the heavy chain variable domain of SEQ ID NO: 4 and the light chain variable domain of SEQ ID NO: 10 or SEQ ID NO: 11; (L) N-succinimidyl 4- (2-). Includes pyridyldithio) pentanoate (SPP); and (C); N (2')-deacetyl-N (2')-(3-mercapto-1-oxopropyl) -mitancin; where (L) is It is provided to administer an immunoconjugate that links (A) to (C). In another embodiment, the method was deposited with the ATCC on April 7, 2010, and administered an immunoconjugate comprising the huFR-1-21 antibody with ATCC accession numbers PTA-10775 and PTA-10776. Including. In certain embodiments, the huFR1-21 antibody comprises (a) heavy chain CDR1 containing SSYGMS (SEQ ID NO: 30); heavy chain CDR2 containing TISSGGSYTY (SEQ ID NO: 31); and DGEGGL YAMDY (SEQ ID NO: 32). Includes heavy chain CDR3; including heavy chain CDR3; and (b) light chain CDR1 containing KASDHINNWLA (SEQ ID NO: 27); light chain CDR2 containing GATSLET (SEQ ID NO: 28); and light chain CDR3; containing QQYWSTPFT (SEQ ID NO: 29). In certain embodiments, the methods are (a) heavy chain CDR1 containing TNYWMQ (SEQ ID NO: 60); heavy chain CDR2 containing AIYPGNGDSR (SEQ ID NO: 61); and heavy chain CDR3 containing RDGNYAAY (SEQ ID NO: 62); , (B) Light chain CDR1 containing RASENIYSNLA (SEQ ID NO: 57); Light chain CDR2 containing AATNLAD (SEQ ID NO: 58); Light chain CDR3 containing QHFWASPYT (SEQ ID NO: 59); Includes administration of an immunoconjugate that is. In certain embodiments, (a) heavy chain CDR1 containing TNYWMY (SEQ ID NO: 66); heavy chain CDR2 containing AIYPGNSDTT (SEQ ID NO: 67); and heavy chain CDR3 containing RHDYGAMDY (SEQ ID NO: 68); and ( b) The antibody is a huFR1-49 antibody, including light chain CDR1 containing RASENIYTNLA (SEQ ID NO: 63); light chain CDR2 containing TASNLAD (SEQ ID NO: 64) and light chain CDR3 containing QHFWVSPYT (SEQ ID NO: 65). Includes administration of immunoconjugates. In certain embodiments, (a) heavy chain CDR1 containing SSFGMH (SEQ ID NO: 72); heavy chain CDR2 containing YISSGSSTIS (SEQ ID NO: 73); And heavy chain CDR3 containing EAYGSSMEY (SEQ ID NO: 74); and (b) light chain CDR1 containing RASQNINNNLH (SEQ ID NO: 69); light chain CDR2 containing YVSQSVS (SEQ ID NO: 70); and QQSNSWPHYT (SEQ ID NO: 71). Includes administration of an immunoconjugate whose antibody is a huFR1-57 antibody, including light chain CDR3 ;. In certain embodiments, (a) heavy chain CDR1 containing TSYTMH (SEQ ID NO: 78); heavy chain CDR2 containing YINPISGYTN (SEQ ID NO: 79); and heavy chain CDR3 containing GGAYGRKPMDY (SEQ ID NO: 80); and ( b) The antibody is a huFR1-65 antibody, including light chain CDR1 containing KASQNVGPNVA (SEQ ID NO: 75); light chain CDR2 containing SASYRYS (SEQ ID NO: 76) and light chain CDR3 containing QQYNSYPYT (SEQ ID NO: 77). Includes administration of immunoconjugates. In one embodiment, the method inhibits the growth of ovarian tumors, brain tumors, breast tumors, uterine tumors, endometrial tumors, pancreatic tumors, kidney tumors, and lung tumors. In certain embodiments, the method inhibits ovarian tumor growth. In another embodiment, the invention inhibits lung tumor growth. In certain embodiments, inhibition of tumor growth is used to treat cancer. In other embodiments, the method comprises administering a second anti-cancer agent to the subject. In certain embodiments, the second anti-cancer agent is a chemotherapeutic agent. The invention also provides isolated cells that produce the antibodies, antigen binding fragments or polypeptides of the invention. The present invention has also been isolated comprising a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 5, 14, 15, 37, 38, 43, 44, 47, 48 and 120-127. Provide a polynucleotide. In certain embodiments, the isolated polypeptide is at least 95% into a sequence selected from the group consisting of SEQ ID NOs: 5, 14, 15, 37, 38, 43, 44, 47, 48 and 120-127. It is the same. In another embodiment, the isolated polypeptide is at least 99% into a sequence selected from the group consisting of SEQ ID NOs: 5, 14, 15, 37, 38, 43, 44, 47, 48 and 120-127. It is the same. The present invention also provides vectors containing any of the polynucleotides of SEQ ID NOs: 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 the polynucleotides of SEQ ID NOs: 5, 14, 15, 37, 38, 43, 44, 47, 48 and 120-127.The present invention provides, for example, the following items.(Item 1) Antibodies are below: (a) Heavy chain CDR1 containing GYFMN (SEQ ID NO: 1); RIHPYDGDTFYNQXaa1FXaa2Xaa3Heavy chain CDR2 containing (SEQ ID NO: 56) and heavy chain CDR3 containing YDG SRAMDY (SEQ ID NO: 3); (b) Light chain CDR1 containing KASQSVSFAGTSLMH (SEQ ID NO: 7); light chain CDR2 containing RASNLEA (SEQ ID NO: 8); and light chain CDR3 containing QQSREYPYT (SEQ ID NO: 9); But here, Xaa1Is selected from K, Q, H and R; Xaa2Is selected from Q, H, N and R; and Xaa3Is selected from G, E, T, S, A and V;A humanized antibody or antigen-binding fragment thereof that specifically binds to human forate receptor 1.(Item 2) The humanized antibody or antigen-binding fragment thereof according to item 1, which binds to human forate receptor 1 with substantially the same affinity as the antibody chimera Mov19.(Item 3) Item 2. The humanized antibody or antigen-binding fragment thereof according to Item 2, wherein the binding affinity is measured by flow cytometry, Biacore or radioimmunoassay.(Item 4) The humanized antibody or antigen-binding fragment thereof according to any one of items 1 to 3, wherein the heavy chain CDR2 sequence comprises RIHPYDGDTFYNQKFQG (SEQ ID NO: 2).(Item 5) Antibodies are below: (a) Heavy chain CDR1 containing GYFMN (SEQ ID NO: 1), or a variant thereof containing a conservative amino acid substitution of 1, 2, 3 or 4; Heavy chain CDR2 containing RIHPYDGDTFYNQKFQG (SEQ ID NO: 2), or 1, 2 , A variant thereof containing a conservative amino acid substitution of 3 or 4; and a heavy chain CDR3 containing YDGS RAMDY (SEQ ID NO: 3), or a variant thereof containing a conservative amino acid substitution of 1, 2, 3 or 4; and / or , (b) Light chain CDR1 containing KASQSVSFAGTSLMH (SEQ ID NO: 7), or a variant thereof containing a conservative amino acid substitution of 1, 2, 3 or 4; Light chain CDR2 containing RASNLEA (SEQ ID NO: 8), or 1, 2 , 3 or 4 conservative amino acid substitutions; and light chain CDR3 containing QQSREYPYT (SEQ ID NO: 9), or 1, 2, 3 or 4 conservative amino acid substitutions;A humanized antibody or antigen-binding fragment thereof that specifically binds to human forate receptor 1.(Item 6) A humanized antibody or antigen-binding fragment thereof that specifically binds to human forate receptor 1 containing the heavy chain of SEQ ID NO: 6.(Item 7) A humanized antibody or antigen-binding fragment thereof deposited with ATCC on April 7, 2010 and encoded by plasmid DNA having ATCC accession numbers PTA-10772, PTA-10773 or 10774.(Item 8) A humanized antibody or an antigen-binding fragment thereof that competes with the antibody according to any one of items 1 to 7 for specific binding to human forate receptor 1.(Item 9) A humanized antibody or antigen-binding fragment thereof comprising a heavy chain variable domain that is at least about 90% identical to SEQ ID NO: 4 and a light chain variable domain that is at least about 90% identical to SEQ ID NO: 10 or SEQ ID NO: 11.(Item 10) The humanized antibody of item 9, wherein the heavy chain variable domain is at least about 95% identical to SEQ ID NO: 4, and the light chain variable domain is at least about 95% identical to SEQ ID NO: 10 or SEQ ID NO: 11. Antibodies binding fragment.(Item 11) The humanized antibody of item 10, wherein the heavy chain variable domain is at least about 99% identical to SEQ ID NO: 4, and the light chain variable domain is at least about 99% identical to SEQ ID NO: 10 or SEQ ID NO: 11. Antibodies binding fragment.(Item 12) The humanized antibody or antigen-binding fragment thereof according to item 11, wherein the heavy chain variable domain is identical to SEQ ID NO: 4, and the light chain variable domain is identical to SEQ ID NO: 10 or SEQ ID NO: 11.(Item 13) The humanized antibody or antigen-binding fragment thereof according to any one of Items 1 to 12, wherein the antibody is expressed in eukaryotic cells at least 10 times higher than that of chMov19.(Item 14) The humanized antibody according to item 13, wherein the eukaryotic cell is a HEK-293T cell.(Item 15) The polypeptide is below:(a) A polypeptide having at least about 90% sequence identity to SEQ ID NO: 4; and / or、(b) A polypeptide having at least about 90% sequence identity to SEQ ID NO: 10 or SEQ ID NO: 11;A polypeptide that specifically binds to human forate receptor 1, including.(Item 16) The polypeptide is as follows:(a) A polypeptide having at least about 95% sequence identity to SEQ ID NO: 4; and / or、(b) A polypeptide having at least about 95% sequence identity to SEQ ID NO: 10 or SEQ ID NO: 11;15. The polypeptide of item 15.(Item 17) The polypeptide is as follows:(a) A polypeptide having at least about 99% sequence identity to SEQ ID NO: 4; and / or、(b) A polypeptide having at least about 99% sequence identity to SEQ ID NO: 10 or SEQ ID NO: 11;16. The polypeptide of item 16.(Item 18) The polypeptide is as follows:(a) Polypeptide of SEQ ID NO: 4; and / or(b) Polypeptide of SEQ ID NO: 10 or SEQ ID NO: 11;17. The polypeptide of item 17.(Item 19) The polypeptide is as follows:(a) Polypeptide of SEQ ID NO: 4; and(b) Polypeptide of SEQ ID NO: 10 or SEQ ID NO: 11;18. The polypeptide of item 18.(Item 20) Antibodies are below: (a) Heavy chain CDR1 containing SSYGMS (SEQ ID NO: 30); Heavy chain CDR2 containing TISSGGSYTY (SEQ ID NO: 31); and / or Heavy chain CDR3 containing DGEGGL YAMDY (SEQ ID NO: 32); and / or (b) Light chain CDR1 containing KASDHINNWLA (SEQ ID NO: 27); Light chain CDR2 containing GATSLET (SEQ ID NO: 28); and light chain CDR3 containing QQYWSTPFT (SEQ ID NO: 29);An antibody that specifically binds to human forate receptor 1, or an antigen-binding fragment thereof.(Item 21) Antibodies are below: (a) Heavy chain CDR1 containing TNYWMQ (SEQ ID NO: 60); Heavy chain CDR2 containing AIYPGNGDSR (SEQ ID NO: 61); and / or Heavy chain CDR3 containing RDGNYAAY (SEQ ID NO: 62); and / or (b) Light chain CDR1 containing RASENIY SNLA (SEQ ID NO: 57); Light chain CDR2 containing AATNLAD (SEQ ID NO: 58); and light chain CDR3 containing QHFWASPYT (SEQ ID NO: 59);An antibody that specifically binds to human forate receptor 1, or an antigen-binding fragment thereof.(Item 22) Antibodies are below: (a) Heavy chain CDR1 containing TNYWMY (SEQ ID NO: 66); Heavy chain CDR2 containing AIYPGNSDTT (SEQ ID NO: 67); and / or Heavy chain CDR3 containing RHDYGAMDY (SEQ ID NO: 68); and / or (b) Light chain CDR1 containing RASENIYTNLA (SEQ ID NO: 63); Light chain CDR2 containing TASNLAD (SEQ ID NO: 64); and light chain CDR3 containing QHFWVSPYT (SEQ ID NO: 65);An antibody that specifically binds to human forate receptor 1, or an antigen-binding fragment thereof.(Item 23) Antibodies are below: (a) Heavy chain CDR1 containing SSFGMH (SEQ ID NO: 72); Heavy chain CDR2 containing YISSGSSTIS (SEQ ID NO: 73); and / or Heavy chain CDR3 containing EAYGSSMEY (SEQ ID NO: 74); and / or (b) Light chain CDR1 containing RASQNINNNLH (SEQ ID NO: 69); Light chain CDR2 containing YVSQSVS (SEQ ID NO: 70); and light chain CDR3 containing QQSNSWPHYT (SEQ ID NO: 71);An antibody that specifically binds to human forate receptor 1, or an antigen-binding fragment thereof.(Item 24) Antibodies are below: (a) Heavy chain CDR1 containing TSYTMH (SEQ ID NO: 78); Heavy chain CDR2 containing YINPISGYTN (SEQ ID NO: 79); and / or Heavy chain CDR3 containing GGAYGRKPMDY (SEQ ID NO: 80); and / or (b) Light chain CDR1 containing KASQNVGPNVA (SEQ ID NO: 75); Light chain CDR2 containing SASYRYS (SEQ ID NO: 76); and Light chain CDR3 containing QQYNSYPYT (SEQ ID NO: 77);An antibody that specifically binds to human forate receptor 1, or an antigen-binding fragment thereof.(Item 25) The polypeptide is below:(a) A polypeptide having at least about 90% sequence identity to SEQ ID NO: 97; and / or(b) less in SEQ ID NO: 96 with a polypeptide having about 90% sequence identity;A polypeptide that specifically binds to human forate receptor 1, including.(Item 26) The polypeptide is as follows:(a) A polypeptide having at least about 95% sequence identity to SEQ ID NO: 97; and / or(b) A polypeptide having at least about 95% sequence identity to SEQ ID NO: 96;25. The polypeptide of item 25.(Item 27) The polypeptide is as follows:(a) A polypeptide having at least about 99% sequence identity to SEQ ID NO: 97; and / or(b) A polypeptide having at least about 99% sequence identity to SEQ ID NO: 96;26. The polypeptide of item 26.(Item 28) The polypeptide is as follows:(a) Polypeptide of SEQ ID NO: 97; and / or(b) Polypeptide of SEQ ID NO: 96;27. The polypeptide of item 27.(Item 29) 28. The polypeptide of item 28, wherein the polypeptide comprises the polypeptide of SEQ ID NO: 97 and the polypeptide of SEQ ID NO: 96.(Item 30) The polypeptide is below:(a) A polypeptide having at least about 90% sequence identity to SEQ ID NO: 99; and / or(b) A polypeptide having at least about 90% sequence identity to SEQ ID NO: 98;A polypeptide that specifically binds to human forate receptor 1, including.(Item 31) The polypeptide is as follows:(a) A polypeptide having at least about 95% sequence identity to SEQ ID NO: 99; and / or(b) A polypeptide having at least about 95% sequence identity to SEQ ID NO: 98;30. The polypeptide according to item 30.(Item 32) The polypeptide is as follows:(a) A polypeptide having at least about 99% sequence identity to SEQ ID NO: 99; and / or(b) A polypeptide having at least about 99% sequence identity to SEQ ID NO: 98;31. The polypeptide of item 31.(Item 33) The polypeptide is as follows:(a) Polypeptide of SEQ ID NO: 99; and / or(b) Polypeptide of SEQ ID NO: 98;32. The polypeptide of item 32.(Item 34) 33. The polypeptide of item 33, wherein the polypeptide comprises the polypeptide of SEQ ID NO: 99 and the polypeptide of SEQ ID NO: 98.(Item 35) The polypeptide is below:(a) A polypeptide having at least about 90% sequence identity to SEQ ID NO: 101; and / or(b) A polypeptide having at least about 90% sequence identity to SEQ ID NO: 100;A polypeptide that specifically binds to human forate receptor 1, including.(Item 36) The polypeptide is as follows:(a) A polypeptide having at least about 95% sequence identity to SEQ ID NO: 101; and / or(b) A polypeptide having at least about 95% sequence identity to SEQ ID NO: 100;35. The polypeptide of item 35.(Item 37) The polypeptide is as follows:(a) A polypeptide having at least about 99% sequence identity to SEQ ID NO: 101; and / or(b) A polypeptide having at least about 99% sequence identity to SEQ ID NO: 100;36. The polypeptide of item 36.(Item 38) 37. The polypeptide of item 37, wherein the polypeptide comprises (a) the polypeptide of SEQ ID NO: 101; and / or (b) the polypeptide of SEQ ID NO: 100.(Item 39) 38. The polypeptide of item 38, wherein the polypeptide comprises the polypeptide of SEQ ID NO: 101 and the polypeptide of SEQ ID NO: 100.(Item 40) The polypeptide is below:(a) A polypeptide having at least about 90% sequence identity to SEQ ID NO: 103; and / or(b) A polypeptide having at least about 90% sequence identity to SEQ ID NO: 102;A polypeptide that specifically binds to human forate receptor 1, including.(Item 41) The polypeptide is as follows:(a) A polypeptide having at least about 95% sequence identity to SEQ ID NO: 103; and / or(b) A polypeptide having at least about 95% sequence identity to SEQ ID NO: 102;40. The polypeptide of item 40.(Item 42) The polypeptide is as follows:(a) A polypeptide having at least about 99% sequence identity to SEQ ID NO: 103; and / or(b) A polypeptide having at least about 99% sequence identity to SEQ ID NO: 102;41. The polypeptide of item 41.(Item 43) 42. The polypeptide of item 42, wherein the polypeptide comprises (a) the polypeptide of SEQ ID NO: 103; and / or (b) the polypeptide of SEQ ID NO: 102.(Item 44) 43. The polypeptide of item 43, wherein the polypeptide comprises the polypeptide of SEQ ID NO: 103 and the polypeptide of SEQ ID NO: 102.(Item 45) The polypeptide is below:(a) A polypeptide having at least about 90% sequence identity to SEQ ID NO: 113; and / or(b) A polypeptide having at least about 90% sequence identity to SEQ ID NO: 112;A polypeptide that specifically binds to human forate receptor 1, including.(Item 46) The polypeptide is as follows:(a) A polypeptide having at least about 95% sequence identity to SEQ ID NO: 113; and / or(b) A polypeptide having at least about 95% sequence identity to SEQ ID NO: 112;45. The polypeptide of item 45.(Item 47) The polypeptide is as follows:(a) A polypeptide having at least about 99% sequence identity to SEQ ID NO: 113; and / or(b) A polypeptide having at least about 99% sequence identity to SEQ ID NO: 112;46. The polypeptide of item 46.(Item 48) 47. The polypeptide of item 47, wherein the polypeptide comprises (a) the polypeptide of SEQ ID NO: 113; and / or (b) the polypeptide of SEQ ID NO: 112.(Item 49) 48. The polypeptide of item 48, wherein the polypeptide comprises the polypeptide of SEQ ID NO: 113 and the polypeptide of SEQ ID NO: 112.(Item 50) The polypeptide is below:(a) A polypeptide having at least about 90% sequence identity to SEQ ID NO: 115; and / or(b) A polypeptide having at least about 90% sequence identity to SEQ ID NO: 114;A polypeptide that specifically binds to human forate receptor 1, including.(Item 51) The polypeptide is as follows:(a) A polypeptide having at least about 95% sequence identity to SEQ ID NO: 115; and / or(b) A polypeptide having at least about 95% sequence identity to SEQ ID NO: 114;50. The polypeptide according to item 50.(Item 52) The polypeptide is as follows:(a) A polypeptide having at least about 99% sequence identity to SEQ ID NO: 115; and / or(b) A polypeptide having at least about 99% sequence identity to SEQ ID NO: 114;51. The polypeptide of item 51.(Item 53) 52. The polypeptide of item 52, wherein the polypeptide comprises (a) the polypeptide of SEQ ID NO: 115; and / or (b) the polypeptide of SEQ ID NO: 114.(Item 54) 53. The polypeptide of item 53, wherein the polypeptide comprises the polypeptide of SEQ ID NO: 115 and the polypeptide of SEQ ID NO: 114.(Item 55) The polypeptide is below:(a) A polypeptide having at least about 90% sequence identity to SEQ ID NO: 117; and / or(b) A polypeptide having at least about 90% sequence identity to SEQ ID NO: 116;A polypeptide that specifically binds to human forate receptor 1, including.(Item 56) The polypeptide is as follows:(a) A polypeptide having at least about 95% sequence identity to SEQ ID NO: 117; and / or(b) A polypeptide having at least about 95% sequence identity to SEQ ID NO: 116;55. The polypeptide of item 55.(Item 57) The polypeptide is as follows:(a) A polypeptide having at least about 99% sequence identity to SEQ ID NO: 117; and / or(b) A polypeptide having at least about 99% sequence identity to SEQ ID NO: 116;56.(Item 58) 57. The polypeptide of item 57, wherein the polypeptide comprises (a) the polypeptide of SEQ ID NO: 117; and / or (b) the polypeptide of SEQ ID NO: 116.(Item 59) 58. The polypeptide of item 58, wherein the polypeptide comprises the polypeptide of SEQ ID NO: 117 and the polypeptide of SEQ ID NO: 116.(Item 60) The polypeptide is below:(a) A polypeptide having at least about 90% sequence identity to SEQ ID NO: 119; and / or(b) A polypeptide having at least about 90% sequence identity to SEQ ID NO: 118;A polypeptide that specifically binds to human forate receptor 1, including.(Item 61) The polypeptide is as follows:(a) A polypeptide having at least about 95% sequence identity to SEQ ID NO: 119; and / or(b) A polypeptide having at least about 95% sequence identity to SEQ ID NO: 118;60. The polypeptide according to item 60.(Item 62) The polypeptide is as follows:(a) A polypeptide having at least about 99% sequence identity to SEQ ID NO: 119; and / or(b) A polypeptide having at least about 99% sequence identity to SEQ ID NO: 118;61. The polypeptide of item 61.(Item 63) 62. The polypeptide of item 62, wherein the polypeptide comprises (a) the polypeptide of SEQ ID NO: 119; and / or (b) the polypeptide of SEQ ID NO: 118.(Item 64) 63. The polypeptide of item 63, wherein the polypeptide comprises the polypeptide of SEQ ID NO: 119 and the polypeptide of SEQ ID NO: 118.(Item 65) The antibody according to any one of items 25 to 65, which is an antibody.(Item 66) The antibody according to any one of items 1 to 24, which is a full-length antibody.(Item 67) The antibody according to any one of items 1 to 24, which is an antigen-binding fragment.(Item 68) The antibody or antigen binding fragment is Fab, Fab', F (ab')2, Fd, single chain Fv or scFv, disulfide linked Fv, V-NAR domain, IgNar, intrabody, IgG-CH2, minibody, F (ab')3, Tetra body, TriabodeE, diabody, monoclonal antibody, DVD-Ig, Fcab, mAb2, (ScFv)267. The antibody according to item 67, which comprises, or scFv-Fc.(Item 69) The antibody or polypeptide according to any one of items 1 to 68, which binds to human forate receptor 1 at Kd of about 1.0 to about 10 nM.(Item 70) The antibody or polypeptide according to any one of items 1 to 69, which binds to human forate receptor 1 at about 1.0 nM or better Kd.(Item 71) The antibody of item 69 or 70, wherein the binding affinity is measured by flow cytometry, Biacore or radioimmunoassay.(Item 72) A method for producing the antibody, which comprises (a) culturing cells expressing the antibody according to any one of items 1 to 24; and (b) isolating the antibody from the cultured cells.(Item 73) 72. The method of item 72, wherein the cell is a eukaryotic cell.(Item 74) It has the formulas (A)-(L)-(C), and in the formula:(A) is the antibody or antigen-binding fragment or polypeptide according to any one of items 1 to 71;(L) is a linker; and(C) is a cytotoxic agent;The linker (L) is an immunoconjugate that connects (A) to (C).(Item 75) The immunoconjugate according to item 74, wherein the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a hydrophilic linker, and a dicarboxylic acid-based linker.(Item 76) The linker is N-succinimidyl 4- (2-pyridyldithio) pentanoate (SPP) or N-succinimidyl 4- (2-pyridyldithio) -2-sulfopentanoate (sulfo-SPP); N-succinimidyl 4- (2). -Pyridyldithio) butanoate (SPDB) or N-succinimidyl 4- (2-pyridyldithio) -2-sulfobutanoate (sulfo-SPDB); N-succinimidyl 4- (maleimidemethyl) cyclohexanecarboxylate (SMCC); N -Sulfosuccinimidyl 4- (maleimidemethyl) cyclohexanecarboxylate (sulfoSMCC); N-succinimidyl 4- (iodoacetyl) -aminobenzoate (SIAB); and N-succinimidyl-[(N-maleimidepropionamide)- The immunoconjugate according to item 75, selected from the group consisting of a tetraethylene glycol] ester (NHS-PEG4-maleimide).(Item 77) The immunoconjugate according to item 75, wherein the linker is N-succinimidyl-[(N-maleimide propionamide) -tetraethylene glycol] ester (NHS-PEG4-maleimide).(Item 78) Item 75, wherein the linker is N-succinimidyl 4- (2-pyridyldithio) butanoate (SPDB) or N-succinimidyl 4- (2-pyridyldithio) -2-sulfobutanoate (sulfo-SPDB). Conjugate.(Item 79) The cytotoxic agents are maytansinoids, maytansinoid analogs, benzodiazepines, taxoids, CC-1065, CC-1065 analogs, duocarmycin, duocarmycin analogs, calikeamycin, drastatin, drastatin analogs, ants The immunoconjugate according to any one of items 74 to 78, which is selected from the group consisting of statins, tomymycin derivatives, and leptomycin derivatives or prodrugs of the agents.(Item 80) 79. The immunoconjugate according to item 79, wherein the cytotoxic agent is a maytansinoid.(Item 81) The cytotoxic agent is N (2')-deacetyl-N (2')-(3-mercapto-1-oxopropyl) -maitansine or N (2') -deacetyl-N2- (4-mercapto-4-methyl). -1-oxopentyl)-Mytansine, the immunoconjugate of item 80.(Item 82) following:(A) A humanized antibody comprising a heavy chain variable domain of SEQ ID NO: 97, 99, 101 or 103 and a light chain variable domain of SEQ ID NO: 96, 98, 100 or 102;(L) N-succinimidyl 4- (maleimidemethyl) cyclohexanecarboxylate (SMCC); and(C) N (2')-deacetyl-N (2')-(3-mercapto-1-oxopropyl) -maitansine;Including, where (L) connects (A) to (C), an immunoconjugate.(Item 83) following:(A) A humanized antibody comprising a heavy chain variable domain of SEQ ID NO: 4 and a light chain variable domain of SEQ ID NO: 10 or SEQ ID NO: 11;(L) N-succinimidyl-[(N-maleimidepropionamide) -tetraethylene glycol] ester (NHS-PEG4-maleimide); and(C) N (2')-deacetyl-N2- (4-mercapto-4-methyl-1-oxopentyl) -maitansine;Including, where (L) connects (A) to (C), an immunoconjugate.(Item 84) following:(A) A humanized antibody comprising a heavy chain variable domain of SEQ ID NO: 4 and a light chain variable domain of SEQ ID NO: 10 or SEQ ID NO: 11;(L) N-succinimidyl 4- (2-pyridyldithio) butanoate (SPDB); and(C) N (2')-deacetyl-N2- (4-mercapto-4-methyl-1-oxopentyl) -maitansine;Including, where (L) connects (A) to (C), an immunoconjugate.(Item 85) following:(A) A humanized antibody comprising a heavy chain variable domain of SEQ ID NO: 4 and a light chain variable domain of SEQ ID NO: 10 or SEQ ID NO: 11;(L) N-succinimidyl 4- (2-pyridyldithio) -2-sulfobutanoate (sulfo-SPDB); and(C) N (2')-deacetyl-N2- (4-mercapto-4-methyl-1-oxopentyl) -maitansine;Including, where (L) connects (A) to (C), an immunoconjugate.(Item 86) following:(A) A humanized antibody comprising a heavy chain variable domain of SEQ ID NO: 4 and a light chain variable domain of SEQ ID NO: 10 or SEQ ID NO: 11;(L) N-succinimidyl 4- (2-pyridyldithio) -2-sulfopentanoate (sulfo-SPP); and(C) N (2')-deacetyl-N (2')-(3-mercapto-1-oxopropyl) -maitansine;Including, where (L) connects (A) to (C), an immunoconjugate.(Item 87) following:(A) A humanized antibody comprising a heavy chain variable domain of SEQ ID NO: 4 and a light chain variable domain of SEQ ID NO: 10 or SEQ ID NO: 11;(L) N-succinimidyl 4- (2-pyridyldithio) pentanoate (SPP); and(C) N (2')-deacetyl-N (2')-(3-mercapto-1-oxopropyl) -maitansine;Including, where (L) connects (A) to (C), an immunoconjugate.(Item 88) The immunoconjugate according to any one of items 74 to 87, further comprising a second (C).(Item 89) 88. The immunoconjugate according to item 88, further comprising a third (C).(Item 90) The immunoconjugate according to item 89, further comprising a fourth (C).(Item 91) The immunoconjugate according to any one of items 74 to 90, which comprises 2 to 6 (C).(Item 92) The immunoconjugate according to any one of items 74 to 90, which comprises 3 to 4 (C).(Item 93) A pharmaceutical composition comprising the antibody, antigen-binding fragment, polypeptide or immunoconjugate according to any one of items 1 to 71 and 74 to 92 and a pharmaceutically acceptable carrier.(Item 94) The pharmaceutical composition comprising the immunoconjugate according to any one of items 74 to 92, wherein the immunoconjugate has an average of about 3 to about 4 (C) per (A).(Item 95) 47. The pharmaceutical composition according to item 47, wherein the immunoconjugates have an average of about 3.5 ± 0.5 (C) per (A).(Item 96) The pharmaceutical composition according to any one of items 93 to 95, further comprising a second anticancer agent.(Item 97) A diagnostic reagent comprising the antibody, antigen-binding fragment, polypeptide or immunoconjugate according to any one of labeled items 1 to 71 and 74 to 92.(Item 98) 97. The diagnostic reagent of item 97, wherein the label is selected from the group consisting of radiolabels, fluorophores, chromophores, imaging agents and metal ions.(Item 99) A kit containing the antibody, antigen-binding fragment, polypeptide or immunoconjugate according to any one of items 1 to 71 and 74 to 92.(Item 100) Tumor growth in a subject, including administering to the subject a therapeutically effective amount of the antibody, antigen binding fragment, polypeptide, immunoconjugate or pharmaceutical composition according to any one of items 1-71 and 71-95. How to inhibit.(Item 101) It has the formulas (A)-(L)-(C), and in the formula:(A) is an antibody that specifically binds to human forate receptor 1 or an antigen-binding fragment thereof;(L) is a linker; and(C) is a cytotoxic agent selected from the group consisting of maytansinoids and maytansinoid analogs; where (L) is the therapeutically effective amount of immunoconjugate that links (A) to (C). Including administration; Immunoconjugates are a method of inhibiting tumor growth in a subject, reducing mean tumor volume by at least 2-fold in a KB xenograft model.(Item 102) The antibody or its antigen-binding fragment is described below:(a) Heavy chain CDR1 containing GYFMN (SEQ ID NO: 1); RIHPYDGDTFYNQXaa1FXaa2Xaa3Heavy chain CDR2 containing (SEQ ID NO: 56) and heavy chain CDR3 containing YDG SRAMDY (SEQ ID NO: 3); (b) Light chain CDR1 containing KASQSVSFAGTSLMH (SEQ ID NO: 7); light chain CDR2 containing RASNLEA (SEQ ID NO: 8); and light chain CDR3 containing QQSREYPYT (SEQ ID NO: 9); But here, Xaa1Is selected from K, Q, H and R; Xaa2Is selected from Q, H, N and R; and Xaa3Is selected from G, E, T, S, A and V;101. The method of item 101.(Item 103) 102. The method of item 102, wherein the heavy chain CDR2 sequence comprises RIHPYDGDTFYNQKFQG (SEQ ID NO: 2).(Item 104) Item 101. The method of item 101, wherein the antibody or antigen-binding fragment thereof was deposited with the ATCC on April 7, 2010 and is encoded by a plasmid DNA having ATCC accession numbers PTA-10772, PTA-10773 or 10774.(Item 105) The immunoconjugate is below:(A) A humanized antibody comprising a heavy chain variable domain of SEQ ID NO: 4 and a light chain variable domain of SEQ ID NO: 10 or SEQ ID NO: 11;(L) N-succinimidyl-[(N-maleimidepropionamide) -tetraethylene glycol] ester (NHS-PEG4-maleimide); and(C) N (2')-deacetyl-N2- (4-mercapto-4-methyl-1-oxopentyl) -maitansine;101. The method of item 101.(Item 106) The immunoconjugate is below:(A) A humanized antibody comprising a heavy chain variable domain of SEQ ID NO: 4 and a light chain variable domain of SEQ ID NO: 10 or SEQ ID NO: 11;(L) N-succinimidyl 4- (2-pyridyldithio) butanoate (SPDB); and(C) N (2')-deacetyl-N2- (4-mercapto-4-methyl-1-oxopentyl) -maitansine;Including;Here, (L) is the method according to item 101, in which (A) is connected to (C).(Item 107) The immunoconjugate is below:(A) A humanized antibody comprising a heavy chain variable domain of SEQ ID NO: 4 and a light chain variable domain of SEQ ID NO: 10 or SEQ ID NO: 11;(L) N-succinimidyl 4- (2-pyridyldithio) -2-sulfobutanoate (sulfo-SPDB); and(C) N (2')-deacetyl-N2- (4-mercapto-4-methyl-1-oxopentyl) -maitansine;Including;Here, (L) is the method according to item 101, in which (A) is connected to (C).(Item 108) The immunoconjugate is below:(A) A humanized antibody comprising a heavy chain variable domain of SEQ ID NO: 4 and a light chain variable domain of SEQ ID NO: 10 or SEQ ID NO: 11;(L) N-succinimidyl 4- (2-pyridyldithio) -2-sulfopentanoate (sulfo-SPP); and(C) N (2')-deacetyl-N (2')-(3-mercapto-1-oxopropyl) -maitansine;Including;Here, (L) is the method according to item 101, in which (A) is connected to (C).(Item 109) The immunoconjugate is below:(A) A humanized antibody comprising a heavy chain variable domain of SEQ ID NO: 4 and a light chain variable domain of SEQ ID NO: 10 or SEQ ID NO: 11;(L) N-succinimidyl 4- (2-pyridyldithio) pentanoate (SPP); and(C) N (2')-deacetyl-N (2')-(3-mercapto-1-oxopropyl) -maitansine;Including;Here, (L) is the method according to item 101, in which (A) is connected to (C).(Item 110) The method of item 101, wherein the antibody is a huFR-1-21 antibody deposited with ATCC on April 7, 2010 and having ATCC accession numbers PTA-10775 and PTA-10776.(Item 111) The antibody is a huFR1-21 antibody, where the antibody is described below: (a) Heavy chain CDR1 containing SSYGMS (SEQ ID NO: 30); heavy chain CDR2 containing TISSGGSYTY (SEQ ID NO: 31); and heavy chain CDR3 containing DGEGGL YAMDY (SEQ ID NO: 32); (b) Light chain CDR1 containing KASDHINNWLA (SEQ ID NO: 27); Light chain CDR2 containing GATSLET (SEQ ID NO: 28); and light chain CDR3 containing QQYWSTPFT (SEQ ID NO: 29);101. The method of item 101.(Item 112) The antibody is a huFR1-48 antibody, where the antibody is described below: (a) Heavy chain CDR1 containing TNYWMQ (SEQ ID NO: 60); Heavy chain CDR2 containing AIYPGNGDSR (SEQ ID NO: 61); and Heavy chain CDR3 containing RDGNYAAY (SEQ ID NO: 62); (b) Light chain CDR1 containing RASENIY SNLA (SEQ ID NO: 57); Light chain CDR2 containing AATNLAD (SEQ ID NO: 58); and light chain CDR3 containing QHFWASPYT (SEQ ID NO: 59);101. The method of item 101.(Item 113) The antibody is a huFR1-49 antibody, where the antibody is described below: (a) Heavy chain CDR1 containing TNYWMY (SEQ ID NO: 66); Heavy chain CDR2 containing AIYPGNSDTT (SEQ ID NO: 67); and Heavy chain CDR3 containing RHDYGAMDY (SEQ ID NO: 68); (b) Light chain CDR1 containing RASENIYTNLA (SEQ ID NO: 63); Light chain CDR2 containing TASNLAD (SEQ ID NO: 64); and light chain CDR3 containing QHFWVSPYT (SEQ ID NO: 65);101. The method of item 101.(Item 114) The antibody is a huFR1-57 antibody, where the antibody is described below: (a) Heavy chain CDR1 containing SSFGMH (SEQ ID NO: 72); heavy chain CDR2 containing YISSGSSTIS (SEQ ID NO: 73); and heavy chain CDR3 containing EAYGSSMEY (SEQ ID NO: 74); (b) Light chain CDR1 containing RASQNINNNLH (SEQ ID NO: 69); Light chain CDR2 containing YVSQSVS (SEQ ID NO: 70); and light chain CDR3 containing QQSNSWPHYT (SEQ ID NO: 71);101. The method of item 101.(Item 115) The antibody is a huFR1-65 antibody, where the antibody is described below: (a) Heavy chain CDR1 containing TSYTMH (SEQ ID NO: 78); Heavy chain CDR2 containing YINPISGYTN (SEQ ID NO: 79); and Heavy chain CDR3 containing GGAYGRKPMDY (SEQ ID NO: 80); (b) Light chain CDR1 containing KASQNVGPNVA (SEQ ID NO: 75); Light chain CDR2 containing SASYRYS (SEQ ID NO: 76); and Light chain CDR3 containing QQYNSYPYT (SEQ ID NO: 77);101. The method of item 101.(Item 116) The method according to any one of items 100 to 115, wherein the tumor is selected from the group consisting of ovarian tumor, brain tumor, breast tumor, uterine tumor, endometrial tumor, pancreatic tumor, kidney tumor, and lung tumor.(Item 117) The method of item 116, wherein the tumor is an ovarian tumor.(Item 118) The method of item 116, wherein the tumor is a lung tumor.(Item 119) The method according to any one of items 100 to 118, which inhibits tumor growth to treat cancer.(Item 120) 119. The method of item 119, further comprising administering to the subject a second anti-cancer agent.(Item 121) 120. The method of item 120, wherein the second anti-cancer agent is a chemotherapeutic agent.(Item 122) An isolated cell that produces the antibody, antigen-binding fragment, or polypeptide according to any one of items 1 to 71.(Item 123) An isolated polynucleotide comprising a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 5, 14, 15, 37, 38, 43, 44, 47, 48 and 120-127.(Item 124) The isolated of item 123, wherein the sequence is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 5, 14, 15, 37, 38, 43, 44, 47, 48 and 120-127. Polynucleotide.(Item 125) The isolated of item 124, wherein the sequence is at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 5, 14, 15, 37, 38, 43, 44, 47, 48 and 120-127. Polynucleotide.(Item 126) A vector containing the polynucleotide according to any one of items 123 to 125.(Item 127) A host cell comprising the vector according to item 126.

0046<figref num="1-1">Figure 1 shows surface residues for murine (muMov19) and humanized (huMov19) Mov19. (A) Mouse and humanized Mov19 light chain surface residues. The murine and humanized Mov19 light chain variable region frame surface residues and number of positions (Kabat system) are shown. Human residues that differ from the original murine sequence are underlined. * The 74th position is not a surface position, but in order to remove the consensus N-linked glycosylation site in version 1.00, this position was changed to threonine (the most common human residue at this position), thereby resulting in version 1.60. did. (B) Mouse and human Mov19 heavy chain surface residues. Mouse and humanized Mov19 heavy chain variable region frame surface residues and number of positions (Kabat system) are shown. Human residues that differ from the original murine sequence are underlined. Similar surface residues are given for FR1-21 (C) and (D).</figref><figref num="1-2">Figure 1 shows surface residues for murine (muMov19) and humanized (huMov19) Mov19. (A) Mouse and humanized Mov19 light chain surface residues. The murine and humanized Mov19 light chain variable region frame surface residues and number of positions (Kabat system) are shown. Human residues that differ from the original murine sequence are underlined. * The 74th position is not a surface position, but in order to remove the consensus N-linked glycosylation site in version 1.00, this position was changed to threonine (the most common human residue at this position), thereby resulting in version 1.60. did. (B) Mouse and human Mov19 heavy chain surface residues. Mouse and humanized Mov19 heavy chain variable region frame surface residues and number of positions (Kabat system) are shown. Human residues that differ from the original murine sequence are underlined. Similar surface residues are given for FR1-21 (C) and (D).</figref><figref num="1-3">Figure 1 shows surface residues for murine (muMov19) and humanized (huMov19) Mov19. (A) Mouse and humanized Mov19 light chain surface residues. The murine and humanized Mov19 light chain variable region frame surface residues and number of positions (Kabat system) are shown. Human residues that differ from the original murine sequence are underlined. * The 74th position is not a surface position, but in order to remove the consensus N-linked glycosylation site in version 1.00, this position was changed to threonine (the most common human residue at this position), thereby resulting in version 1.60. did. (B) Mouse and human Mov19 heavy chain surface residues. Mouse and humanized Mov19 heavy chain variable region frame surface residues and number of positions (Kabat system) are shown. Human residues that differ from the original murine sequence are underlined. Similar surface residues are given for FR1-21 (C) and (D).</figref><figref num="2">FIG. 2 shows the alignment of the chimeric Mov19 and huMov19 heavy and light chain variable domains and the muFR1-21 and huFR1-21 heavy and light chain variable domains. Alignment of Mov19 and FR1-21 variable region resurfacing sequences with their murine counterparts. A) and C) light chain variable domains; B) and D) heavy chain variable domains. The dash "-" indicates the identity with the murine sequence. The CDR (Kabat definition) is underlined.</figref><figref num="3">Figure 3 shows the expression of chimeric Mov19 and huMov19 in HEK cells. Chimeric and human Mov19 expression plasmids were transiently transfected into suspended HEK-293T cells, harvested 7 days later, and expressed antibodies were measured by quantitative ELISA. Light chain and heavy chain plasmids were transfected in their respective molar ratios of 3: 1 or 6: 1.</figref><figref num="4">FIG. 4 shows the binding specificity of anti-FOLR1 antibodies detected by their binding to FOLR1-expressing 300-19 cells. Binding of huMov19 to 300-19-FOLR1 cells by flow cytometry. 300-19 parental cells expressing FOLR1. The gray-filled area shows extracellular autofluorescence; the black dotted line shows cells incubated with FITC-conjugated anti-human secondary antibody, and the black solid line shows cells incubated with huMov19 antibody and FITC-conjugated anti-human secondary antibody. Shows cells.</figref><figref num="5">FIG. 5 shows the binding affinity of anti-FOLR1 antibody and immunoconjugate and the cytotoxic activity in vitro. The binding affinity of huMov19 and various murine and humanized FR-1 antibodies was measured in SKOV3 cells. The in vitro cytotoxic activity of the PEG4-Mal-DM4 conjugates of the listed antibodies was also assayed.</figref><figref num="6">FIG. 6 shows antibody-dependent cellular cytotoxicity of immunoconjugates. ADCC activity of huMov19, huFR1-21 and Mor003 was assayed on Igrov1 cells. Igrov1 was incubated with 1500 cells / well with a target: NK cell ratio of 1: 4.</figref><figref num="7">FIG. 7 shows the cytotoxic activity of continuous exposure to huFR1-21-PEG4-mal-DM4 and huMov19-PEG4-mal-DM4 in KB cells. Excessive amounts of non-conjugated antibodies suppressed immunoconjugate activity when they were co-incubated in the presence of KB cells, indicating that cytotoxic activity is antigen-dependent.</figref><figref num="8">Figure 8 shows the in vivo efficacy of huMov19 targeting conjugates in a KB xenograft model. FOLR1-targeting cuttable conjugate huMov19-SPDB-DM4 (B) contrasting with non-FOLR1-targeting huC242-SPDM-DM4 (D) and non-cuttable conjugate huMov19-PEG4-Mal-DM4 (C) Was tested using an established xenograft model of KB cells subcutaneously transplanted into SCID mice, in contrast to huC242-PEG4Mal-DM4 (E). Targeting FOLR1 by huMov19 resulted in a significant reduction in mean tumor volume.</figref><figref num="9">FIG. 9 shows the in vivo efficacy of huMov19-PEG4-Mal-DM4 when compared to the murine FR-1 anti-FOLR1 antibody in the KB xenograft model. FR-1 series antibodies conjugated to unconjugated or PEG4-Mal-DM4 were tested for their ability to reduce mean tumor volume in a KB xenograft tumor model compared to huMov19-PEG4-Mal-DM4. did. (A) FR-1-9, (B) FR-1-13, (C) FR-1-22 and (D) FR-1-23.</figref><figref num="10">FIG. 10 shows the in vivo efficacy of huMov19-PEG4-Mal-DM4 and huFR1-21-PEG4-Mal-DM4 in a KB xenograft model. On the 6th day after inoculation, a single injection of 10 mg / kg of huMov19-PEG4-Mal-DM4 and huFR1-21-PEG4-Mal-DM4 was performed. Both huMov19-PEG4-mal-DM4 and huFR1-21-PEG4-Mal-DM4 showed a significant reduction in mean tumor volume. "Average TV" refers to the average tumor volume.</figref><figref num="11">Figure 11 shows that huMov19-PEG4-mal-DM4 showed dose-dependent activity in the KB xenograft model. The dose-dependent activity of the immunoconjugate was assayed over the range of doses tested. Weekly dosing resulted in improved antitumor activity. Only high drug loading had a limited improvement in activity in the 10 mg / kg dose group and decreased activity in the lower dose group. 3.4 DAR refers to 3.7 drug molecules per antibody.</figref><figref num="12">FIG. 12 shows the in vivo efficacy of huMov19 conjugated to DM1 and DM4 using various linkers. Conjugate huMov19 to SMCC-DM1 with 3.9 drug molecules per antibody, to sulfo-mal-DM4 with 3.7 drug molecules per antibody (B), and to sulfo-mal-DM4 with 8.23 drug molecules per antibody (C). They were then assayed for their ability to reduce mean tumor volume at various concentrations in comparison to huMov19-PEG4-mal-DM4.</figref><figref num="13">FIG. 13 shows the in vivo efficacy of huMov19 conjugated to DM1 and DM4 using various linkers. huMov19 to SPP-DM1 with 4.3 drug molecules per antibody, to sulfo-SPDB-DM4 with 3.8 drug molecules per antibody, to SPDB-DM4 with 3.8 drug molecules per antibody, and to sulfo-SPDB-DM4 with 6.8 drug molecules per antibody. And assayed for their ability to reduce mean tumor volume. Mice were dosed with one of the above-mentioned conjugates of 5 mg / kg (A) and 2.5 mg / kg (B), or PBS alone.</figref><figref num="14">FIG. 14 shows the in vivo efficacy of huMov19-sulfo-SPDB-DM4 in an OVCAR-3 xenograft tumor model. Mice received 25, 50 or 100 μg / kg of huMov19-sulfo-SPDB-DM4, or PBS alone.</figref><figref num="15">FIG. 15 shows the in vivo efficacy of huMov19-sulfo-SPDB-DM4 in an IGROV-1 xenograft tumor model. Mice received 25, 50 or 100 μg / kg of huMov19-sulfo-SPDB-DM4, or PBS alone.</figref><figref num="16">FIG. 16 shows the in vivo efficacy of huMov19-sulfo-SPDB-DM4 in an OV-90 xenograft tumor model. Mice received 25, 50 or 100 μg / kg of huMov19-sulfo SPDB-DM4, or PBS alone.</figref><figref num="17">FIG. 17 shows the effect of cleavable and non-cleavable linkers on the efficacy of immunoconjugates in the KB xenograft model.</figref><figref num="18">FIG. 18 shows the effect of a cleavable linker on the efficacy of immunoconjugates in (A) KB xenograft model (B) OVCAR-3 xenograft model.</figref><figref num="19">FIG. 19 shows the in vitro and in vivo efficacy of huFR1-48, huFR1-49, huFR1-57 and huFR1-65-SMCC-DM1 in KB and xenograft tumor models. Mice received a single dose of 200 μg / kg.</figref>

0047The present invention provides new agents including, but not limited to, polypeptides that bind to human forate receptor 1 (FOLR1), such as antibodies and immunoconjugates. Also provided are relevant polypeptides and polynucleotides, compositions containing FOLR1 binders, and methods of making FOLR1 binders. Methods of using new FOLR1 binders, such as inhibiting tumor growth and / or treating cancer, are also provided.

00481. Definition To facilitate the understanding of the present invention, many terms and phrases are defined below.

0049The term "human forate receptor 1" or "FOLR1" as used herein refers to any native human FOLR1 unless otherwise stated. The term "FOLR1" also includes "full-length", unprocessed FOLR1, as well as any form of FOLR1 that results from intracellular processing. The term also includes naturally occurring FOLR1 variants such as splice variants, allelic variants and isoforms. The FOL R1 polypeptides described herein can be isolated from a variety of sources, such as from human tissue types, or from other weight loss, or can be prepared by recombinant or synthetic methods. Examples of FOLR1 sequences include, but are not limited to, NCBI reference numbers P15328, NP_001092242.1, AAX29268.1, AAX37119.1, NP_057937. And NP_057936.1.

0050The term "antibody" recognizes targets such as proteins, polypeptides, peptides, carbohydrates, polynucleotides, lipids or combinations thereof through at least one antigen recognition site within the variable region of an immunoglobulin molecule. It means an immunoglobulin molecule that specifically binds. As used herein, the term "antibody" refers to undamaged polyclonal antibodies, undamaged monoclonal antibodies, antibody fragments (eg Fab, Fab', F (ab') 2, and Fv fragments), single chain Fv. (scFv) mutants, multispecific antibodies such as bispecific antibodies formed from at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing the antigenic determining portion of the antibody, And any other modified immunoglobulin molecule, including an antigen recognition site, as long as the antibody exhibits the desired biological activity. Antibodies are based on the identities of their heavy chain constant domains, called alpha, delta, epsilon, gamma and mu, respectively: the five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, or their subclasses. It can be any (isotype) (eg IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). Different classes of immunoglobulins have different well-known subunit structures and three-dimensional arrangements. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.

0051A "blocking" or "antagonist" antibody is one that inhibits or reduces the biological activity of an antigen to which it binds, such as FOL R1. In some embodiments, the blocking or antagonist antibody substantially or completely inhibits the biological activity of the antigen. Desirably, biological activity can be reduced by 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or even 100%.

0052The term "anti-FOLR1 antibody" or "antibody that binds to FOLR1" refers to binding to FOLR1 with sufficient affinity so that the antibody is useful as a diagnostic and / or therapeutic agent when targeting FOLR1. Refers to an antibody that can produce. The degree of binding of anti-FOLR1 antibody to unrelated non-FOLR1 protein is less than about 10% of antibody binding to FOLR1, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to FOLR1 has a dissociation constant (Kd) of 1 μM, 100 nM, 10 nM, 1 nM, or 0.1 nM.

0053The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigenic determination variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F (ab') 2, and multispecific antibodies formed from Fv fragments, linear antibodies, single chain antibodies, and antibody fragments.

0054"Monoclonal antibody" refers to a single antigenic determinant, a homogeneous population of antibodies involved in the highly specific recognition and binding of epitopes. This is in contrast to polyclonal antibodies, which typically include different antibodies directed against different antigenic determinants. The term "monoclonal antibody" refers to intact and full-length monoclonal antibodies, as well as fusion proteins containing antibody fragments (Fab, Fab', F (ab') 2, Fv), single chain (scFv) mutants, antibody moieties. , And any other modified immunoglobulin molecule containing an antigen recognition site. Furthermore, "monoclonal antibody" refers to an antibody produced by any of the embodiments including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animal methods.

0055The term "humanized antibody" refers to the form of a specific immunoglobulin chain, chimeric immunoglobulin, or fragment thereof, a non-human (eg, murine) antibody that contains a minimal non-human (eg, murine) sequence. Typically, humanized antibodies are derived from complementarity determining regions (CDRs) by residues derived from CDRs of non-human species (eg, mice, rats, rabbits, hamsters) with the desired specificity, affinity and competence. Human immunoglobulins in which residues have been replaced (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, the Fv framework region (FR) residue of human immunoglobulin has been replaced with a corresponding residue in a non-human species-derived antibody having the desired specificity, affinity and potency. Humanized antibodies can be further modified by the substitution of additional residues within the Fv framework and / or within the replaced non-human residues to refine and optimize the specificity, affinity and / or ability of the antibody. .. Generally, a humanized antibody comprises at least one variable domain containing all or substantially all of the CDR regions corresponding to non-human immunoglobulins, and typically substantially all of two or three. On the other hand, all or substantially all of the FR regions are of the human immunoglobulin consensus sequence. Humanized antibodies can also include at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to form humanized antibodies are described in US Pat. No. 5,225,539 or 5,639,641.

0056The "variable region" of an antibody, alone or in combination, refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain. The heavy and light chain variable regions each consist of four framework regions (FRs) linked by three complementarity determining regions (CDRs), also known as hypervariable regions. The CDRs in each strand are held closer together by FR, and together with the CDRs of the other strands, contribute to the formation of the antigen-binding site of the antibody. There are at least two methods for determining CDRs: (1) an approach based on sequence variability between species (ie Kabat et al., Sequences of Proteins of Immunological Interest, (5th edition, 1991, National Institutes of Health, Bethesda). There are Md.)); And (2) approaches based on crystallographic studies of antigen-antibody complexes (Al-lazikaniet al (1997) J. Molec. Biol. 273: 927-948)). Furthermore, a combination of these two approaches may be used in the art to determine CDRs.

0057The Kabat numbering system is commonly used to refer to residues within the variable domain (generally light chain residues 1-107 and heavy chain residues 1-113) (eg Kabat, etc., Sequences of Immunological Interest). . 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

0058In the case of Kabat, the numbering of amino acid positions is described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. A numbering system used for heavy or light chain variable domains of antibody compilations in (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids that correspond to the shortening or insertion into the FR or CDR of the variable domain. For example, a heavy chain variable domain has a single amino acid insertion after residue 52 of H2 (residue 52a by Kabat) and an insertion residue after heavy chain FR residue 82 (eg residues 82a, 82b by Kabat). And 82c etc.) may be included. The Kabat numbering of residues can be determined for a given antibody by alignment in the region of homology of the antibody sequence with the "standard" Kabat numbered sequence. Chothia instead points to the structural loop (Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)). The end point of Chothia's CDR-H1 loop when numbered using Kabat's numbering method varies between H32 and H34 depending on the length of the loop (because Kabat's numbering method puts insertions in H35A and H35B). Claw; 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; Will be). The AbM hypervariable region represents a compromise between Kabat's CDR and Chothia's structural loop and is used by Oxford Molecular's AbM antibody modeling software.<chemistry num="1-1"><img id="000002" he="64" wi="150" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>

0059The term "human antibody" means an antibody produced by a human or having an amino acid sequence corresponding to an antibody artificially produced using any of the techniques known in the art. This definition of a human antibody refers to an antibody comprising an intact or full-length antibody, a fragment thereof, and / or at least one human heavy chain and / or light chain polypeptide, such as a murine light chain and a human heavy chain polypeptide. Includes antibodies containing.

0060The term "chimeric antibody" refers to an antibody in which the amino acid sequence of an immunoglobulin molecule is derived from more than one species. Typically, the variable regions of both the light and heavy chains are in the variable regions of the antibody derived from one species of mammal (eg, mouse, rat, rabbit, etc.) having the desired specificity, affinity and ability. Corresponds and constant regions are homologous to sequences in antibodies derived from another (usually human) to avoid eliciting an immune response in that species.

0061The terms "epitope" or "antigen determinant" are used interchangeably herein and refer to a portion of an antigen that can be recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, the epitope can be formed from both continuous and discontinuous amino acids juxtaposed by the tertiary folding of the protein. Epitopes formed from continuous amino acids are typically retained during protein denaturation, whereas epitopes formed by tertiary folding typically disappear during protein denaturation. Epitopes typically contain at least 3 and more commonly at least 5 or 8-10 amino acids in a unique spatial conformation.

0062"Binding affinity" generally refers to the total intensity of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise stated, "binding affinity" as used herein refers to an endogenous binding affinity that reflects a 1: 1 interaction between members of a binding pair (eg, antibody and antigen). Point to. The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally tend to bind slowly and dissociate easily, whereas high-affinity antibodies generally bind rapidly and for longer. Has a tendency to remain. Various methods for measuring binding affinity are known in the art, and any of them can be used for the purposes of the present invention. Specific exemplary embodiments are as described below.

0063"Better" as used herein refers to a stronger bond between a molecule and its binding partner. "Better" as used herein refers to a stronger bond represented by a smaller number of Kd values. For example, for an antibody that has an affinity for an antigen that is "better than 0.6 nM", the affinity of the antibody for that antigen is either <0.6 nM, ie 0.59 nM, 0.58 nM, 0.57 nM, etc., or less than 0.6 nM. The value.

0064The phrase "substantially similar" or "substantially the same" as used herein will allow those skilled in the art to measure the difference between two values by that value (eg, the Kd value). Two numbers (generally one related to the antibodies of the invention) that are considered to have little or no biological and / or statistical significance within the range of the traits. , The other is related to reference / comparator antibodies) means a sufficiently high degree of similarity. The difference between the two values is less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10% as a function of the value of the reference / comparator antibody.

0065An "isolated" polypeptide, antibody, polynucleotide, vector, cell or composition is a polypeptide, antibody, polynucleotide, vector, cell or composition that is in a form not observed in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells or compositions include those that have been purified to the extent that they can no longer be in the form observed in nature. In some embodiments, the isolated antibody, polynucleotide, vector, cell, or composition is substantially pure.

0066As used herein, "substantially pure" is at least 50% pure (ie, no contaminants), at least 90% pure, at least 95% pure, at least 98% pure, at least 99% pure. Refers to a substance.

0067The term "immunoconjugate" or "conjugate" as used herein refers to a compound or derivative thereof linked to a cell binding agent (ie, an anti-FOLR1 antibody or fragment thereof), and a comprehensive formula: Defined by CLA, in the formula, C = cytotoxic substance, L = linker, and A = cell binding agent or anti-FOLR1 antibody or antibody fragment. Immunoconjugates can also be defined by a comprehensive expression in reverse order: ALC.

0068A "linker" is any chemical moiety capable of linking a drug, usually a maytansinoid, to a cell binder such as an anti-FOLR1 antibody or fragment thereof in a stable covalent manner. The linker is sensitive or substantially resistant to acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage under conditions where the compound or antibody remains active. be able to. Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid instability groups, photoinstability groups, peptidase instability groups and esterase instability groups. Linkers also include charged linkers described herein and known in the art, and hydrophilic forms thereof.

0069The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals characterized by cell population uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers are squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, and peritoneal cancer. Hepatocyte cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer colon rectal cancer, endometrial or uterine cancer, salivary adenocarcinoma, kidney Includes cancer, liver cancer, prostate cancer, genital cancer, thyroid cancer, liver cancer, and various types of head and neck cancer.

0070"Tumor" and "neoplasm" refer to any mass of tissue resulting from the growth or proliferation of either benign (non-cancerous) or malignant (cancerous) cells, including the affected area of the precancerous area.

0071The terms "cancer cells", "tumor cells" and grammatical equivalents refer to tumor or precancerous including both non-tumorogenic cells, including masses of tumor cell populations, and tumorigenic stem cells (cancer stem cells). Refers to the total population of cells derived from the affected area. As used herein, the term "tumor cells" is "non-tumor formation" when referring only to tumor cells that lack the ability to renew and differentiate from cancer stem cells so that they can be distinguished. It will be modified by the term "sex".

0072The term "subject" refers to any animal (eg, mammal) that includes, but is not limited to, humans, non-human primates, rodents, etc. that should be recipients of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein when referring to a human subject.

0073Administration "in combination" with one or more other therapeutic agents includes simultaneous (parallel) and continuous administration in any order.

0074The term "pharmaceutical formulation" refers to a preparation that is in the form of activating the biological activity of the active ingredient and does not contain additional ingredients that are unacceptably toxic to the subject to whom the formulation is administered. Such formulations can be sterilized.

0075The "effective amount" of an antibody disclosed herein is an amount sufficient to accomplish the stated purpose. The "effective amount" can be determined experimentally and in a routine manner in relation to the stated objectives.

0076The term "therapeutically effective amount" refers to the amount of antibody or other drug effective to "cure" a disease or disorder in a subject or mammal. In the case of cancer, the therapeutically effective amount of the drug reduces the number of cancer cells; reduces the size of the tumor; inhibits the infiltration of cancer cells into the surrounding organs (ie, slows to some extent, and certain practices Stop in morphology); inhibit tumor metastasis (ie, slow to some extent, and stop in certain embodiments); inhibit tumor growth to some extent; and / or one of the symptoms associated with cancer One or more can be relaxed to some extent. See the definition of "treating" herein. As long as the drug can prevent the growth of existing cancer cells, they can be cell proliferation inhibitory and / or cytotoxic. "Prophylactically effective amount" refers to an amount that is effective for a required period of time at a required dosage in order to achieve a desired preventive result. Typically, but not necessarily, the prophylactic dose is lower than the therapeutically effective dose because the prophylactic dose is used in the subject before or in the early stages of the disease.

0077The term "labeled" as used herein refers to a detectable compound or composition that is directly or indirectly conjugated to an antibody to form a "labeled" antibody. "The label can itself be detected (eg, a radioisotope label or a fluorescent label), or in the case of an enzyme label, it can catalyze a chemical modification of the detectable substrate compound or composition.

0078A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer, regardless of its 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 compounds used in "targeting therapies" and conventional chemotherapeutic agents.

0079Terms such as "treating" or "treating" or "to treat" or "to alleviate" or "to alleviate" 1) cure or slow down the symptoms of the diagnosed pathological condition or disorder. Refers to both therapeutic measures that reduce, and / or stop progression, and 2) prophylactic or preventive measures that prevent and / or slow the onset of targeted pathological conditions or disorders. That is, those in need of treatment include those who already have a disability; those who are prone to have a disability; and those who should prevent the disability. In certain embodiments, the subject is a patient: a reduction in the number or complete absence of cancer cells; a reduction in tumor size; to the interior of peripheral organs, including, for example, the spread of cancer into soft tissues and bones. Inhibition or absence of invasion of cancer cells; Inhibition or absence of tumor metastasis; Inhibition or absence of tumor growth; Relief of one or more symptoms associated with a particular cancer; Reduction of morbidity and mortality; Quality of life Improvement of tumors; reduction of tumorigenicity, frequency of tumorigenesis, or ability to form tumors; reduction of number or frequency of cancer stem cells in tumors; differentiation of tumorigenic cells into non-tumorogenic states; or any action When presenting one or more of the combinations are well "treated" with respect to the tumor according to the methods of the invention.

0080As used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length and includes DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase. Polynucleotides can include modified nucleotides such as methylated nucleotides and their analogs. If present, modification of the nucleotide structure can be done before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. The polynucleotide can be further modified after polymerization, for example by conjugation with a labeling component. Other types of modifications include, for example, "caps", substitutions with one or more naturally occurring relatives of nucleotides, internucleotide modifications, such as uncharged linkages (eg, methylphosphonate, phosphotriester, phosphoamidate, carbamate, etc.) ) And those having a charged link (eg, phosphorothioate, phosphorodithioate, etc.), such as those containing suspended moieties such as proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), inter Those with a cullator (eg aclysine, solarene, etc.), those containing a chelate-forming agent (eg, metals, radioactive metals, boron, oxidizing metals, etc.), those containing an alkylating agent, having a modified link. Includes unmodified forms of things (eg, alpha anomeric nucleic acids, etc.) and polynucleotides. Furthermore, any of the hydroxyl groups originally present in the sugar can be replaced with, for example, a phosphonate group, a phosphate group, protected with a standard protecting group, or activated to prepare another link to another nucleotide. Or can be conjugated to a solid support. OH at the 5'and 3'ends is phosphorylated or amine or charcoal It can be replaced with an organic capping group moiety of 1 to 20 elementary atoms. Other hydroxyls can also be derivatized to standard protecting groups. Polynucleotides are also analogs of the sugars of ribose or deoxyribose commonly known in the art, such as 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-. Azido-ribose, carboxyl saccharide relatives, alpha anomeric sugars, epimer sugars such as arabinose, xylose or lyxose, pyranose sugar, furanose sugar, sedhepturose, acyclic analogs and baseless nucleoside analogs such as methylriboside Can be contained. One or more phosphodiester bonds can be replaced with alternative linking groups. Such alternative linking groups have P (O) S (thioate), P (S) S (dithioate), and (O) NR phosphates.2(Amidate), P (O) R, P (O) OR', CO or CH2Including, but not limited to, embodiments of (form acetal), each R or R'in the formula independently contains an H or, optionally substituted or unsubstituted, ether (-O-) link. Alkyl (1-20C), aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl. Not all connections in the polynucleotide need be identical. The above description applies to RNA and all polynucleotides referred to herein, including DNA.

0081The term "vector" means a construct capable of delivering and expressing one or more genes or sequences in a host cell. Examples of vectors are viral vectors, naked DNA or RNA expression vectors, plasmids, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and specific vectors. Includes, but is not limited to, eukaryotic cells such as producer cells.

0082The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be straight or branched, can contain modified amino acids, and can be interrupted by non-amino acids. The term is also an amino acid that has been modified naturally or by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, eg, conjugation with a labeling component. Includes polymers. Also included are, for example, one or more analogs of amino acids (including, for example, unnatural amino acids, etc.) and other polypeptides known in the art. Since the polypeptides of the invention are based on antibodies, in certain embodiments, the polypeptides can exist as single or associated chains.

0083The terms "identity" or percent "identity" in the context of two or more nucleic acids or polypeptides are compared to give maximum equivalence without considering any conservative amino acid substitutions as part of sequence identity. Refers to two or more sequences or subsequences that have a particular percentage of nucleotide or amino acid residues that are the same or are the same when aligned (with gaps introduced as needed). Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain amino acid or nucleotide sequence alignments are known in the art. Non-limiting examples of such sequence alignment algorithms were modified by Karlin et al., 1993, Proc. Natl. Acad. Sci., 90: 5873-5877 and modified by the NBLAST and XBLAST programs (Altschul et al., 1991, Nucleic Acids Res). ., 25: 3389-3402), Karlin et al., 1990, Proc. Natl. Acad. The algorithm described in Sci., 87: 2264-2268. 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 (DNA STAR) to align sequences Another preferably available software program that can be used with. In certain embodiments, the percent identity between the two nucleotide sequences uses the GAP program in GCG software (eg, NWSgapdna.CMP matrix and gap weights 40, 50, 60, 70 or 90 and length weight 1). , 2, 3, 4, 5 and 6). In certain alternative embodiments, Needleman and Wunsch (J. Mol. Biol. The percent identity between the two amino acid sequences can be determined by using the GAP program in the GCG software package incorporating the algorithm of (48): 444-453 (1970) (eg, Blossum62 matrix or PAM250 matrix and Use gap weights 16, 14, 12, 10, 8, 6 or 4 and length weights 1, 2, 3, 4, 5). Alternatively, in certain embodiments, the percent identity between nucleotide or amino acid sequences is Myers and Miller (CABIOS,). Obtained using the algorithm of 4: 11-17 (1989)). For example, percent identity can be determined using the ALIGN program (version 2.0) and using PAM120 with a residue table, gap length penalty 12 and gap penalty 4. Appropriate parameters for maximum alignment with specific alignment software can be determined by one of ordinary skill in the art. In certain embodiments, the default parameters of the alignment software are used. In certain embodiments, the percentage identity "X" of the first amino acid sequence to the second sequence is calculated as 100x (Y / Z), where Y is the alignment of the first and second sequences. The number of amino acid residues scored as the same match (either by visual inspection or when aligned by a particular sequence alignment program), and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than the second sequence, then the percent identity of the first sequence to the second sequence will be longer than the percent identity of the second sequence to the first sequence.

0084As a non-limiting example, any particular polynucleotide has a particular percent sequence identity to the reference sequence (eg, at least 80% identical, at least 85% identical, at least 90% identical, and some. Whether at least 95%, 96%, 97%, 98%, or 99% identical in embodiments can be measured using the Bestfit program in certain embodiments (Wisconsin Sequence Analysis Package, Version 8 for Unix (Wisconsin Sequence Analysis Package, Version 8 for Unix). Registered Trademarks), GeneticsComputer Group, University Research Park, 575 Science Drive, Madison, WI 53711). Festfit finds the best segment of homology between two sequences Smithand Waterman, Advances in Applied Mathematics 2: 482 It uses the local homology algorithm of 489 (1981). When examining whether a particular sequence is, for example, 95% identical to a reference sequence according to the invention by using Bestfit and any other sequence alignment program, the parameter is that the percentage of identity is that of the reference nucleotide sequence. Set to be calculated over full length and to allow gaps in homology up to 5% of the total number of nucleotides in the reference sequence.

0085In some embodiments, the two nucleic acids or polypeptides of the invention are at least aligned when compared and aligned using a sequence comparison algorithm or for maximal equivalence as measured by visual inspection. 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, or 99% of nucleotide or amino acid residues It is said to be substantially the same to mean having the sameness. In certain embodiments, the identity spans a region of the sequence that is at least about 10, about 20, about 40-60 residue lengths, or an integer value in between, and from 60-80 residues. It is present over a long region, eg, at least about 90-100 residues, or the sequences are substantially identical over the full length of the sequence being compared, such as the codon region of a nucleotide sequence.

0086A "conservative amino acid substitution" is one in which one amino acid residue is replaced by another amino acid residue having a similar side chain. A family of amino acid residues with similar side chains is defined in the art, eg, basic side chains (eg lysine, arginine, histidine), acidic side chains (eg aspartic acid, glutamate), uncharged polar sides. Chains (eg asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (eg glycine, alanine, baalin, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta branched side chains (eg threonine, Contains valine, isoleucine) and aromatic side chains (eg tyrosine, genylalanine, tryptophan, histidine). For example, the substitution of phenylalanine for tyrosine is a conservative substitution. In certain embodiments, conservative substitutions in the sequences of the polypeptides and antibodies of the invention eliminate the binding of the polypeptide or antibody containing the amino acid sequence to the antigen to which the polypeptide or antibody binds, namely FOLR1. I won't let you. Methods for identifying conservative substitutions of nucleotides and amino acids that do not eliminate antigen binding are well known in the art (eg, Brummell et al., Biochem. 32: 1180-1 187 (1993); Kobayashi et al., Protein Eng. 12 (10): 879-884 (1999); and Burks et al., See Proc. Natl. Acad. Sci. USA 94: .412-417 (1997)).

0087As used in the present disclosure and claims, the singular markings "Aru ~" and "That ~" shall include multiple markings unless otherwise specified.

0088Whenever an embodiment is described herein with the expression "contains," another similar embodiment described in terms of "consisting of" and / or "becoming more essential" is also provided. Must be understood as a thing.

0089As used herein in phrases such as "A and / or B", the term "and / or" is any of "A and B", "A or B", "A" and "B". Intended to be included. Similarly, the term "and / or" as used in the phrase "A, B and / or C" is used in the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C "; Aoyobi B; B and C; A (single); B (single); and C (single) are each intended to be included.

0090II.FOLR1 binder The present invention provides an agent that specifically binds to human FOLR1. These agents are referred to herein as "FOLR1 binders". Full-length amino acid (aa) and nucleotide (nt) sequences for FOLR1 are known in the art and are provided herein as set forth in SEQ ID NOs: 25 and 26, respectively.

0091In certain embodiments, the FOLR1 binder is an antibody, immunoconjugate or polypeptide. In some embodiments, the FOL R1 binder is a humanized antibody. In certain embodiments, the FOLR1 binder is a humanized version of the murine Mov19 antibody (variable heavy and light chains are shown in SEQ ID NOs: 17 and 18, respectively).

0092In certain embodiments, the FOLR1 binder has the following effects: Inhibition of tumor cell growth, reduction of tumor tumorigenicity by reducing the frequency of cancer stem cells in the tumor, inhibition of tumor growth, increase of viability, trigger of tumor cell death, tumorigenic cells Has one or more of differentiation from to non-tumorogenic state, or prevention of tumor cell metastasis.

0093In certain embodiments, immunoconjugates or other agents that specifically bind to human FOLR1 trigger cell death via cytotoxic agents. For example, in certain embodiments, the antibody against the human FOLR1 antibody is conjugated to a maytansinoid that is activated in tumor cells expressing FOLR1 by protein internalization. In certain alternative embodiments, the agent or antibody is not conjugated.

0094In certain embodiments, the FOLR1 binder can inhibit tumor growth. In certain embodiments, the FOLR1 binder can inhibit tumor growth in vivo (eg, in xenograft mouse models and / or humans with cancer). In certain embodiments, the FOLR1 binder can inhibit tumor growth in humans.

0095That is, the present invention provides a humanized antibody that specifically binds to human forate receptor 1 or an antigen-binding fragment thereof, wherein the antibody comprises the following: (a) Heavy chain CDR1; RIHPYDGDTFYNQXaa containing GYFMN (SEQ ID NO: 1).1FXaa2Xaa3Heavy chain CDR2 containing (SEQ ID NO: 56) and heavy chain CDR3 containing YDGS RAMDY (SEQ ID NO: 3); and (b) Light chain CDR1 containing KASQSVSFAGTSLMH (SEQ ID NO: 7); Light containing RASNLEA (SEQ ID NO: 8). Chain CDR2; and light chain CDR3 containing QQSREYPYT (SEQ ID NO: 9); however, where Xaa1Is selected from K, Q, H and R; Xaa2Is selected from Q, H, N and R; and Xaa3Includes those selected from G, E, T, S, A and V; In certain embodiments, the antibody is a Mov19 antibody, which is said antibody comprising the heavy chain CDR2 RIHPYDGDTFYNQKFQG (SEQ ID NO: 2).

0096In certain embodiments, the present invention is a humanized antibody that specifically binds to FOLR1 containing a CDR of huMov19 with up to 4 (ie 0, 1, 2, 3 or 4) conservative amino acid substitutions per CDR. Alternatively, an antigen-binding fragment is provided. That is, in certain embodiments, the invention comprises the following: (a) heavy chain CDR1 containing GYFMN (SEQ ID NO: 1), or a variant thereof comprising a conservative amino acid substitution of 1, 2, 3 or 4; Heavy chain CDR2 containing RIHPYDGDTFYNQKFQG (SEQ ID NO: 2), or a variant thereof containing a conservative amino acid substitution of 1, 2, 3 or 4; and heavy chain CDR3 containing YDG SRAMDY (SEQ ID NO: 3), or 1, 2, 3 Or a variant thereof containing a conservative amino acid substitution of 4; and / or (b) a light chain CDR1 containing KASQSVSFAGTSLMH (SEQ ID NO: 7), or a variant thereof containing a conservative amino acid substitution of 1, 2, 3 or 4. Light chain CDR2 containing RASNLEA (SEQ ID NO: 8), or a variant thereof containing a conservative amino acid substitution of 1, 2, 3 or 4; and light chain CDR3 containing QQSREYPYT (SEQ ID NO: 9), or 1, 2, Provided are a humanized antibody that specifically binds to human forate receptor 1, or an antigen-binding fragment thereof, which comprises a variant thereof containing 3 or 4 conservative amino acid substitutions.

0097The invention also comprises the following: (a) heavy chain CDR1 containing SSYGMS (SEQ ID NO: 30); heavy chain CDR2 containing TISSGGSYTY (SEQ ID NO: 31); and / or heavy chain CDR3 containing DGEGGLYAMDY (SEQ ID NO: 32). And / or (b) light chain CDR1 containing KASDHINNWLA (SEQ ID NO: 27); light chain CDR2 containing GATSLET (SEQ ID NO: 28); and light chain CDR3 containing QQYWSTPFT (SEQ ID NO: 29); Provided is a humanized antibody (huFR1-21) that specifically binds to body 1 or an antigen-binding fragment thereof.

0098In certain embodiments, the present invention specifically binds to FOLR1 containing a CDR of huFR1-21 with up to 4 (ie 0, 1, 2, 3 or 4) conservative amino acid substitutions per CDR. An antibody or antigen-binding fragment is provided. That is, in certain embodiments, the invention comprises the following: (a) heavy chain CDR1 containing SSYGMS (SEQ ID NO: 30), or a variant thereof comprising a conservative amino acid substitution of 1, 2, 3 or 4; Heavy chain CDR2 containing TISSGGSYTY (SEQ ID NO: 31), or a variant thereof containing a conservative amino acid substitution of 1, 2, 3 or 4; and heavy chain CDR3 containing DGEGGLYAMDY (SEQ ID NO: 32), or 1, 2, 3 Or a variant thereof containing a conservative amino acid substitution of 4; and / or (b) a light chain CDR1 containing KASDHINNWLA (SEQ ID NO: 27), or a variant thereof containing a conservative amino acid substitution of 1, 2, 3 or 4. Light chain CDR2 containing GATSLET (SEQ ID NO: 28), or a variant thereof containing a conservative amino acid substitution of 1, 2, 3 or 4; and light chain CDR3 containing QQYWSTPFT (SEQ ID NO: 29), or 1, 2, Provided are a humanized antibody that specifically binds to human forate receptor 1, or an antigen-binding fragment thereof, which comprises a variant thereof containing 3 or 4 conservative amino acid substitutions.

0099In certain embodiments, the present invention specifically binds to FOLR1 containing a CDR of huFR1-48 with up to 4 (ie 0, 1, 2, 3 or 4) conservative amino acid substitutions per CDR. An antibody or antigen-binding fragment is provided. That is, in certain embodiments, the invention comprises the following: (a) heavy chain CDR1 containing TNYWMQ (SEQ ID NO: 60), or a variant thereof comprising a conservative amino acid substitution of 1, 2, 3 or 4; Heavy chain CDR2 containing AIYPGNGDSR (SEQ ID NO: 61), or a variant thereof containing a conservative amino acid substitution of 1, 2, 3 or 4; and heavy chain CDR3 containing RDGNYAAY (SEQ ID NO: 62), or 1, 2, 3 Or a variant thereof containing a conservative amino acid substitution of 4; and / or (b) a light chain CDR1 containing RASENIYSNLA (SEQ ID NO: 57), or a variant thereof containing a conservative amino acid substitution of 1, 2, 3 or 4. Light chain CDR2 containing AATNLAD (SEQ ID NO: 58), or a variant thereof containing a conservative amino acid substitution of 1, 2, 3 or 4; and light chain CDR3 containing QHFWASPYT (SEQ ID NO: 59), or 1, 2, Provided are a humanized antibody that specifically binds to human forate receptor 1, or an antigen-binding fragment thereof, which comprises a variant thereof containing 3 or 4 conservative amino acid substitutions.

0100In certain embodiments, the present invention specifically binds to FOLR1 containing a CDR of huFR1-49 with up to 4 (ie 0, 1, 2, 3 or 4) conservative amino acid substitutions per CDR. An antibody or antigen-binding fragment is provided. That is, in certain embodiments, the invention comprises the following: (a) heavy chain CDR1 comprising TNYWMY (SEQ ID NO: 66), or a variant thereof comprising a conservative amino acid substitution of 1, 2, 3 or 4; Heavy chain CDR2 containing AIYPGNSDTT (SEQ ID NO: 67), or a variant thereof containing a conservative amino acid substitution of 1, 2, 3 or 4; and heavy chain CDR3 containing RHDYGAMDY (SEQ ID NO: 68), or 1, 2, 3 Or a variant thereof containing a conservative amino acid substitution of 4; and / or (b) a light chain CDR1 containing RASENIYTNLA (SEQ ID NO: 63), or a variant thereof containing a conservative amino acid substitution of 1, 2, 3 or 4. Light chain CDR2 containing TASNLAD (SEQ ID NO: 64), or a variant thereof containing a conservative amino acid substitution of 1, 2, 3 or 4; and light chain CDR3 containing QHFWVSPYT (SEQ ID NO: 65), or 1, 2, Provided are a humanized antibody that specifically binds to human forate receptor 1, or an antigen-binding fragment thereof, which comprises a variant thereof containing 3 or 4 conservative amino acid substitutions.

0101In certain embodiments, the present invention specifically binds to FOLR1 containing a CDR of huFR1-57 with up to 4 (ie 0, 1, 2, 3 or 4) conservative amino acid substitutions per CDR. An antibody or antigen-binding fragment is provided. That is, in certain embodiments, the invention comprises the following: (a) heavy chain CDR1 containing SSFGMH (SEQ ID NO: 72), or a variant thereof comprising a conservative amino acid substitution of 1, 2, 3 or 4; Heavy chain CDR2 containing YISSGSSTIS (SEQ ID NO: 73), or a variant thereof containing a conservative amino acid substitution of 1, 2, 3 or 4; and heavy chain CDR3 containing EAYGSSMEY (SEQ ID NO: 74), or 1, 2, 3 Or a variant thereof containing a conservative amino acid substitution of 4; and / or (b) a light chain CDR1 containing RASQNINNNLH (SEQ ID NO: 69), or a variant thereof containing a conservative amino acid substitution of 1, 2, 3 or 4. Light chain CDR2 containing YVSQSVS (SEQ ID NO: 70), or a variant thereof containing a conservative amino acid substitution of 1, 2, 3 or 4; and light chain CDR3 containing QQSNSWPHYT (SEQ ID NO: 71), or 1, 2, Provided are a humanized antibody that specifically binds to human forate receptor 1, or an antigen-binding fragment thereof, which comprises a variant thereof containing 3 or 4 conservative amino acid substitutions.

0102In certain embodiments, the present invention specifically binds to FOLR1 containing a CDR of huFR1-65 with up to 4 (ie 0, 1, 2, 3 or 4) conservative amino acid substitutions per CDR. An antibody or antigen-binding fragment is provided. That is, in certain embodiments, the invention comprises the following: (a) heavy chain CDR1 containing TSYTMH (SEQ ID NO: 78), or a variant thereof comprising a conservative amino acid substitution of 1, 2, 3 or 4; Heavy chain CDR2 containing YINPISGYTN (SEQ ID NO: 79), or a variant thereof containing a conservative amino acid substitution of 1, 2, 3 or 4; and heavy chain CDR3 containing GGAYGRKPMDY (SEQ ID NO: 80), or 1, 2, 3 Or a variant thereof containing a conservative amino acid substitution of 4; and / or (b) a light chain CDR1 containing KASQNVGPNVA (SEQ ID NO: 75), or a variant thereof containing a conservative amino acid substitution of 1, 2, 3 or 4. Light chain CDR2 containing SASYRYS (SEQ ID NO: 76), or a variant thereof containing a conservative amino acid substitution of 1, 2, 3 or 4; and light chain CDR3 containing QQYNSYPYT (SEQ ID NO: 77), or 1, 2, Provided are a humanized antibody that specifically binds to human forate receptor 1, or an antigen-binding fragment thereof, which comprises a variant thereof containing 3 or 4 conservative amino acid substitutions.

0103Polypeptides comprising one of the individual light or heavy chains described herein, as well as polypeptides containing both light and heavy chains (eg, antibodies) are also provided. The polypeptides of SEQ ID NOs: 4 and 6 contain the variable domain of the heavy chain of huMov19 and the heavy chain of huMov19, respectively. The polypeptides of SEQ ID NOs: 10-13 contain variable domain light chain version 1.00, variable domain light chain version 1.60, light chain version 1.00, and light chain version 1.60 of huMov19, respectively. The polypeptides of SEQ ID NOs: 42 and 46 contain the variable domain of the heavy chain of huFR1-21 and the heavy chain of huFR1-21, respectively. The polypeptides of SEQ ID NOs: 41 and 45 contain the variable domain light chain and light chain of huFR1-21, respectively. The polypeptides of SEQ ID NOs: 97 and 113 contain the variable domain of the heavy chain of huFR1-48 and the heavy chain of huFR1-48, respectively. The polypeptides of SEQ ID NOs: 96 and 112 contain the variable domain light chain and light chain of huFR1-48, respectively. The polypeptides of SEQ ID NOs: 99 and 115 contain the variable domain of the heavy chain of huFR1-49 and the heavy chain of huFR1-49, respectively. The polypeptides of SEQ ID NOs: 98 and 114 contain the variable domain light chain and light chain of huFR1-49, respectively. The polypeptides of SEQ ID NOs: 101 and 117 contain the variable domain of the heavy chain of huFR1-57 and the heavy chain of huFR1-57, respectively. The polypeptides of SEQ ID NOs: 100 and 116 contain the variable domain light chain and light chain of huFR1-57, respectively. The polypeptides of SEQ ID NOs: 103 and 119 contain the variable domain of the heavy chain of huFR1-65 and the heavy chain of huFR1-65, respectively. The polypeptides of SEQ ID NOs: 102 and 118 contain the variable domain light chain and light chain of huFR1-65, respectively.

0104Also provided are (a) polypeptides having at least about 90% sequence identity to SEQ ID NO: 4 or 6; and / or (b) at least about 90% sequence identity to SEQ ID NOs: 10-13. It is a polypeptide containing a polypeptide having. Also provided are (a) polypeptides having at least about 90% sequence identity to SEQ ID NOs: 42 or 46; and / or (b) at least about 90% sequence identity to SEQ ID NOs: 41 and 45. It is a polypeptide containing a polypeptide having. Also provided are (a) polypeptides having at least about 90% sequence identity to SEQ ID NO: 97 or 113; and / or (b) at least about 90% sequence identity to SEQ ID NOs: 96 and 112. It is a polypeptide containing a polypeptide having. Also provided are (a) polypeptides having at least about 90% sequence identity to SEQ ID NO: 99 or 115; and / or (b) at least about 90% sequence identity to SEQ ID NOs: 98 and 114. It is a polypeptide containing a polypeptide having. Also provided are (a) polypeptides having at least about 90% sequence identity to SEQ ID NOs: 101 or 117; and / or (b) at least about 90% sequence identity to SEQ ID NOs: 100 and 116. It is a polypeptide containing a polypeptide having. Also provided are (a) polypeptides having at least about 90% sequence identity to SEQ ID NO: 103 or 119; and / or (b) at least about 90% sequence identity to SEQ ID NOs: 102 and 118. It is a polypeptide containing a polypeptide having. In certain embodiments, the polypeptide is at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least in SEQ ID NOs: 4, 6, 10-13, 41, 42, 45 or 46. Contains a polypeptide having about 99% sequence identity. That is, in certain embodiments, the polypeptide is (a) a polypeptide having at least about 95% sequence identity to SEQ ID NO: 4 or 6; And / or (b) SEQ ID NOs: 10-13 include polypeptides having at least about 95% sequence identity. In certain embodiments, the polypeptide is (a) a polypeptide having at least about 95% sequence identity to SEQ ID NO: 42 or 46; and / or (b) at least about 95% sequence to SEQ ID NOs: 41 and 45. Includes polypeptides with identity. Also provided are (a) polypeptides having at least about 95% sequence identity to SEQ ID NO: 97 or 113; and / or (b) at least about 95% sequence identity to SEQ ID NOs: 96 and 112. It is a polypeptide containing a polypeptide having. Also provided are (a) polypeptides having at least about 95% sequence identity to SEQ ID NO: 99 or 115; and / or (b) at least about 95% sequence identity to SEQ ID NOs: 98 and 114. It is a polypeptide containing a polypeptide having. Also provided are (a) polypeptides having at least about 95% sequence identity to SEQ ID NOs: 101 or 117; and / or (b) at least about 95% sequence identity to SEQ ID NOs: 100 and 116. It is a polypeptide containing a polypeptide having. Also provided are (a) polypeptides having at least about 95% sequence identity with SEQ ID NO: 103 or 119; and / or (b) at least about 95% sequence identity with SEQ ID NOs: 102 and 118. It is a polypeptide containing a polypeptide having. In certain embodiments, the polypeptide comprises (a) a polypeptide having the amino acid sequence of SEQ ID NO: 4; and / or (b) a polypeptide having the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11. In certain embodiments, the polypeptide comprises (a) a polypeptide having the amino acid sequence of SEQ ID NO: 45; and / or (b) a polypeptide having the amino acid sequence of SEQ ID NO: 46. In certain embodiments, the polypeptide is (a) a polypeptide having the amino acid sequence of SEQ ID NO: 6; And / or (b) contains a polypeptide having the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13. In certain embodiments, the polypeptide is an antibody and / or polypeptide that specifically binds to human forate receptor 1. In certain embodiments, the polypeptide is a humanized antibody that specifically binds to human forate receptor 1. For example, the present invention is an antibody or human that specifically binds to human FOLR1 comprising (a) a polypeptide having the amino acid sequence of SEQ ID NO: 4; and (b) a polypeptide having the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11. To provide a peptide antibody. In certain embodiments, the polypeptide comprising SEQ ID NO: 4 is a heavy chain variable region. In certain embodiments, the polypeptide comprising SEQ ID NO: 10 or 11 is a light chain variable region. The present invention also comprises an antibody that specifically binds to human FOL R1 comprising (a) a polypeptide having the amino acid sequence of SEQ ID NO: 6; and / or (b) a polypeptide having the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13. Alternatively, a humanized antibody is provided. The invention also comprises an antibody or humanized antibody that specifically binds to human FOL R1 comprising (a) a polypeptide having the amino acid sequence of SEQ ID NO: 45; and / or (b) a polypeptide having the amino acid sequence of SEQ ID NO: 46. I will provide a. The invention also comprises an antibody or humanized antibody that specifically binds to human FOL R1 comprising (a) a polypeptide having the amino acid sequence of SEQ ID NO: 112; and / or (b) a polypeptide having the amino acid sequence of SEQ ID NO: 113. I will provide a. The invention also comprises an antibody or humanized antibody that specifically binds to human FOL R1 comprising (a) a polypeptide having the amino acid sequence of SEQ ID NO: 114; and / or (b) a polypeptide having the amino acid sequence of SEQ ID NO: 115. I will provide a. The present invention also (a) a polypeptide having the amino acid sequence of SEQ ID NO: 116; And / or (b) an antibody or humanized antibody that specifically binds to human FOLR1 comprising a polypeptide having the amino acid sequence of SEQ ID NO: 117. The invention also comprises an antibody or humanized antibody that specifically binds to human FOL R1 comprising (a) a polypeptide having the amino acid sequence of SEQ ID NO: 118; and / or (b) a polypeptide having the amino acid sequence of SEQ ID NO: 119. I will provide a. In certain embodiments, polypeptides having a particular percentage of sequence identity to SEQ ID NOs: 4, 6, 10-13, 41, 42, 45, 46, 96-103 and 112-119 and SEQ ID NOs: The only difference from 4, 6, 10-13, 41, 42, 45, 46, 96-103 and 112-119 is the conservative amino acid substitution.

0105In certain embodiments, the FOLR1 binder comprises an anti-FOLR1 antibody selected from the group consisting of huMov19, FR-1-21, FR1-48, FR1-49, FR1-57, and FR1-65 antibodies. Essentially better or better than this.

0106In certain embodiments, the huMov19 antibody is deposited with the American Type Culture Collection (ATCC) on April 7, 2010, and is encoded by a plasmid having ATCC accession numbers PTA-10772 and PTA-10773 or 10774.

0107In certain embodiments, the FR-1-21 antibody is deposited with the ATCC on April 7, 2010 and is encoded by a plasmid bearing ATCC accession numbers PTA-10775 and PTA-10776.

0108In certain embodiments, the humanized antibody binds to FOLR1 with substantially the same affinity as the antibody chimera Mov19. The affinity or avidity of an antibody for an antigen can be determined by any suitable method well known in the art, such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA), or kinetic analysis ( For example, BIACORETMIt can be measured experimentally using analysis). Direct binding assay and competitive binding assay formats can be readily used. (For example, Berzofsky et al., "Antibody-Antigen Interactions," In Fundamental Immunology, Paul, WE, ed., Raven Press: New York, NY (1984); Kuby, JanisImmunology, WH Freeman and Company: New York, NY (1992); See the method described herein). The measured affinity of a particular antibody-antigen interaction may vary if measured under different conditions (eg, salt concentration, pH, temperature). That is, affinity and other antigen binding parameters (eg, KD or Kd, K).on, Koff) Is measured using standard solutions of antibodies and antigens, and standard buffers known in the art and such as the buffers described herein.

0109In one aspect, the binding assay can be performed using flow cytometry on cells expressing the FOLR1 antigen on the surface. For example, 1x10 per sample in 100 μL FACS buffer (RPMI-1640 medium supplemented with 2% normal goat serum).5FOLR1-positive cells such as SKOV3 were incubated with various concentrations of anti-FOLR1 antibody while incubating the cells. The cells were then pelleted, washed, and incubated with 100 μL of FITC-conjugated goat anti-mouse or goat anti-human IgG antibody (eg, available from Jackson Laboratory, 6 μg / ml in FACS buffer) for 1 hour. Cells were pelleted again, washed with FACS buffer and resuspended in 200 μL of PBS containing 1% formaldehyde. Samples were obtained using, for example, FACSCalibur flow cytometry with an HTS multiwell sampler and analyzed using CellQuest Pro (all sources: BD Biosciences, San Diego, US). For each sample, a binding curve was created by exporting the mean fluorescence intensity (MFI) for FL1 and plotting against antibody concentration on a semi-log plot. GraphPad Prism v4 (GraphPad) with a jigmoid dose response curve fitted to the binding curve and using default parameters Calculate the EC50 using a program like Software, San Diego, CA). The EC50 value can be used as a measure of the apparent dissociation constant "Kd" or "KD" for each antibody.

0110Monoclonal antibodies can be prepared using the hybridoma method, eg, those described in Kohler and Milstein (1975) Nature 256: 495. Using the hybridoma method, immunization of a mouse, hamster or other suitable host animal as described above induces lymphocyte production of an antibody that will specifically bind to the immunized antigen. Lymphocytes can also be immunized in vitro. After immunization, lymphocytes are isolated and fused with a suitable myeloma cell lineage using, for example, polyethylene glycol to form hybridomas, which are then sorted from unfused lymphocytes and myeloma cells. Monoclonal antibodies specifically directed against selected antigens determined by immunoprecipitation, immunoblotting, or by in vitro binding tests (eg, radioimmunoassay (RIA); enzyme-linked immunosorbent assay (ELISA)). The hybridomas produced are then subjected to standard methods in in vitro culture (Goding, Monoclonal Antibodies: It can grow as ascites tumors in animals in Principles and Practice, Academic Press, 1986) or in vivo. The monoclonal antibody can then be purified from the medium or ascites as described above for polyclonal antibodies.

0111Alternatively, monoclonal antibodies can also be made using recombinant DNA methods as described in US Pat. No. 4,816,567. A polynucleotide encoding a monoclonal antibody is isolated from mature B cells or hybridomas by RT-PCR or the like using an oligonucleotide primer that specifically amplifies the genes encoding the heavy and light chains of the antibody, and the sequence thereof is isolated. Determined using conventional operating methods. The isolated polynucleotides encoding heavy and light chains are then cloned into suitable expression vectors, which E. coli cells, monkey COS cells, Chinese hamsters, which do not otherwise produce immunoglobulin proteins. Monochrome antibodies are formed by host cells when transfected into host cells such as ovarian (CHO) cells, or myeloma cells. Furthermore, recombinant monoclonal antibodies of the desired species or fragments thereof are isolated from phage display libraries expressing the CDRs of the desired species 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).

0112Polynucleotides encoding monoclonal antibodies can also be modified in many different embodiments using recombinant DNA technology to form alternative antibodies. In some embodiments, eg, the constant domains of the light and heavy chains of a mouse monoclonal antibody, 1) to these regions of a human antibody, for example to form a chimeric antibody, or 2) to form a fusion antibody. Can be substituted for non-immunoglobulin polypeptides. In some embodiments, the constant region is truncated or removed to form the desired antibody fragment of the monoclonal antibody. The specificity, affinity, etc. of a monoclonal antibody can be optimized by using site-directivity of a variable region or high-density mutagenesis.

0113In some embodiments, the monoclonal antibody against human FOLR1 is a humanized antibody. In certain embodiments, therapeutic use of such antibodies reduces the antigenicity and HAMA (human anti-mouse antibody) response upon administration to a human subject.

0114Methods for manipulating, humanizing or resurfing non-human and human antibodies can also be used and are well known in the art. Antibodies that have been humanized, resurfed, or similarly engineered have one or more amino acid residues derived from sources that are non-human, such as, but not limited to, mice, rats, rabbits, non-human primates, or other mammals. Can have. These non-human amino acid residues are replaced by residues often referred to as "import" residues that are typically taken from "import" variable, stationary or other domains of known human sequences.

0115Such imported sequences may be used to reduce immunogenicity or to bind, affinity, on-rate, iflate, avidity, specificity, half-life, or any other suitable known in the art. It can be used to reduce, enhance or modulate the properties. In general, CDR residues are directly and most substantially involved in the effect on FOLR1 binding. Thus, variable and constant region non-human sequences can be replaced by human or other amino acids while maintaining some or all of the non-human or human CDR sequences.

0116The antibody can also optionally be a humanized, resurfing, engineered or human antibody engineered to retain high affinity for the antigen FOLR1 and other desirable biological properties. To achieve this goal, humanization (or human) or manipulation antibodies are performed through the process of analysis of parental sequences and various conceptual humanization and manipulation products using a three-dimensional model of parental, manipulation and humanization sequences. FOLR1 antibody and resurfacing antibody can optionally be prepared. Three-dimensional immunoglobulin models are generally available and well known in the art. A computer program can be used to illustrate and mark the estimated three-dimensional conformational structure of the selected candidate immunoglobulin sequence. Examination of these displays allows analysis of the possible role of residues in the functioning of candidate immunoglobulin sequences, namely residues that affect the ability of candidate immunoglobulins to bind to their own antigens, such as FOLR1. Become. In this way, framework (FR) residues are combined from choice, consensus and import sequences to achieve the desired antibody properties, such as increased affinity for the target antigen.

0117Humanization, resurfacing or manipulation of the antibodies of the invention can be performed in any known manner, eg, but not limited to 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. Natl. Acad. Sci.USA 89: 4285 (1992); Presta et al., J. Immunol. 151: 2623 (1993), US Pat. 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; To US96 / 18978; US91 / 09630; US91 / 05939; US94 / 01234; GB89 / 01334; GB91 / 01134; GB92 / 01755; WO90 / 14443; WO90 / 14424; WO90 / 14430; EP229246; 7,557,189; 7,538,195; and 7,342,110 It can be carried out using those described, each of which is incorporated herein by reference in its entirety, including its references.

0118In certain alternative embodiments, the antibody against FOLR1 is a human antibody. Human antibodies can be directly prepared using various techniques known in the art. Immortalized human B lymphocytes isolated from immunized individuals that produce antibodies that are immunized in vitro or directed against the target antigen can be formed (eg, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R). Liss, p. 77 (1985); see Boemer et al., 1991, J. Immunol., 147 (1): 86-95; and US Pat. No. 5,750,373). Furthermore, human antibodies include, for example, Vaughan et al., 1996, Nat. Biotech., 14: 309-314, Sheets et al., 1998, Proc. Nat'l. Acad. Sci., 95: 6157-6162, Hoogenboom and Winter, 1991. , J. Mol. Biol., 227: 381, and Marks, et al., 1991, J. As described in Mol.Biol., 222: 581, a phage library can be selected from its phage library expressing human antibodies. Techniques for the formation and use of antibody phage libraries are also US Pat. Nos. 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; It is also described in .Bio., Doi: 10.1016 / j.jmb. 2007.12.018 (each of which is incorporated herein by reference in its entirety). Affinity maturation and chain shuffling methods (Marks et al., 1992, Bio / Technology 10: 779-783; incorporated herein by reference in their entirety) are known in the art and are high affinity human antibodies. Can be used to form.

0119Humanized antibodies can also be made in transgenic mice containing the human immunoglobulin locus, which is capable of producing a complete repertoire of human antibodies by immunization in the absence of endogenous immunoglobulin production. This approach is described in US Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.

0120The present invention also includes bispecific antibodies that specifically recognize human forate receptor 1. Bispecific antibodies are antibodies that can specifically recognize and bind to at least two different epitopes. Different epitopes are in the same molecule (eg, the same human forate receptor 1), or, for example, the antibody is human forate receptor 1, and, for example, 1) T cell receptor (eg CD3) or Fc receptor (eg CD64, CD32 or CD16). It can be on a different molecule, as in both cases where it specifically recognizes and binds to an effector molecule on a leukocyte, such as), or 2) a cytotoxic agent detailed below.

0121The exemplified bispecific antibodies can bind to two different epitopes, at least one of which occurs in the polypeptides of the invention. Alternatively, the anti-antigenic arm of the immunoglobulin molecule can be combined with an arm that binds to a triggering molecule on leukocytes, such as a T cell receptor molecule (eg, CD2, CD3, CD28 or B7) or an Fc receptor for IgG. The cell defense mechanism can be concentrated on cells expressing a specific antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells expressing a particular antigen. These antibodies carry an antigen-binding arm and an arm that binds to cytotoxic agents or various radioactive chelate-forming agents such as EOTUBE, DPTA, DOTA or TETA. Methods for producing 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 US Pat. No. 5,731,168). Antibodies more expensive than divalent are also intended. For example, trispecific antibodies can be prepared (Tutt et al., J. Immunol. 147: 60 (1991)). That is, in certain embodiments, the antibody against FOLR1 is multispecific.

0122In certain embodiments, antibody fragments are provided, for example, to increase tumor penetration. Various methods are known for the production of antibody fragments. Traditionally, these fragments are induced 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, et al. 229: 81). In certain embodiments, antibody fragments are produced recombinantly. Fab, Fv and scFv antibody fragments are all expressed in E. coli or other host cells and can be secreted from them, which allows for the mass production of these fragments. Such antibody fragments can also be isolated from the antibody phage libraries discussed above. The antibody fragment can also be a linear antibody, eg, as described in US Pat. No. 5,641,870, and can be unispecific or bispecific. Other techniques for the production of antibody fragments are known to those of skill in the art.

0123According to the present invention, a method can be adapted for the production of a single chain antibody specific for human forate receptor 1 (see US Pat. No. 4,946,778). Furthermore, a monoclonal Fab fragment having the desired specificity for human forate receptor 1 by adapting the method for the construction of a Fab expression library (Huse, et al., Science 246: 1275-1281 (1989)). Allows for rapid and effective identification of derivatives, fragments, analogs or homologues. Antibody fragments can be produced by techniques in the art, eg, but not limited to (a) F (ab') 2 fragments produced by pepsin digestion of antibody molecules; (b) Reducing disulfide bridges of F (ab') 2 fragments. Fab fragments formed by, (c) Fab fragments formed by treatment of antibody molecules with papain and reducing agents, and (d) Fv fragments are included, but not limited to.

0124Especially in the case of antibody fragments, it may be more desirable to modify the antibody to increase its serum half-life. This can be done, for example, by incorporating salvage receptor binding epitopes into the antibody fragment by mutation of the appropriate region in the antibody fragment, or by incorporating the epitope into a peptide tag, which is then either terminal or central. Can be achieved by fusing to (eg, by DNA or peptide synthesis).

0125Heteroconjugated antibodies are also included within the scope of the invention. Heteroconjugate antibodies consist of two covalently linked antibodies. Such antibodies have been proposed, for example, for targeting immune cells to unwanted cells (US Pat. No. 4,676,980). It is also intended that it is possible to prepare antibodies in vitro using known methods in synthetic protein chemistry, such as those using cross-linking agents. For example, an immunotoxin can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolates and methyl-4-mercaptobutylimidase.

0126For the purposes of the present invention, it is not surprising that the modified antibody can contain any type of variable region that results in antibody association with the human FOLR1 polypeptide. In this regard, the variable region can include or derive from any type of mammal that can induce a humoral response and generate immunoglobulins against the desired tumor association antigen. .. That is, the variable region of the modified antibody can be, for example, of human, murine, non-human primate (eg, cynomolgus monkey, macaque, etc.) or wolf origin. In some embodiments, both the variable and constant regions of the modified immunoglobulin are human. In other embodiments, the variable region of a compatible antibody (usually derived from a non-human source) can be manipulated or specially modified to improve binding properties or reduce the immunogenicity of the molecule. In this regard, the variable regions useful in the present invention can be modified through inclusion of humanized or otherwise imported amino acid sequences.

0127In certain embodiments, the variable domains in both heavy and light chains are modified by at least partial replacement of one or more CDRs, and optionally by partial framework region replacement and sequence changes. .. CDRs can be derived from antibodies of the same class as or even subclasses of the antibody from which the framework region is derived, but from antibodies of different classes and, in certain embodiments, from different species. Is also intended. It may not always be necessary to replace all of the CDRs with a complete CDR from the donor's variable region in order to transfer the antigen binding capacity of one variable domain to another. Rather, in some cases it may only be necessary to transfer the residues required to maintain the activity of the antigen binding site. According to the description given in U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762, this is either by performing routine experiments or by trial and error to obtain functional antibodies with reduced immunogenicity. By conducting the above test, it can be sufficiently performed within the ability of those skilled in the art.

0128Despite the modification of the variable region, as is known by those skilled in the art, the modified antibody of the present invention has enhanced tumorigenesis when compared to an antibody of approximately the same immunogenicity containing native or unmodified constant regions. Antibodies in which at least one or more parts of the constant region domain have been deleted or otherwise modified (eg, complete) to provide the desired biochemical properties, such as retained or reduced serum half-life. Long antibody or immunoreactive fragment thereof) will be included. In some embodiments, the constant region of the modified antibody will include the human constant region. Modifications to constant regions compatible with the present invention include additions, deletions or substitutions of one or more amino acids in one or more domains. That is, the modified antibody disclosed herein comprises a modification or modification to one or more of the three heavy chain constant domains (CH1, CH2 or CH3) and / or to the light chain constant domain (CL). Can be done. In some embodiments, modified constant regions are intended in which one or more domains are partially or totally deleted. In some embodiments, the modified antibody will comprise a domain-deleted construct or variant in which the entire CH2 domain has been removed (ΔCH2 construct). In some embodiments, the omitted constant region domain is replaced by a short amino acid spacer (eg, 10 residues) that provides some of the molecular flexibility typically conferred by the non-existent constant region. Become.

0129In addition to their arrangement, constant regions are also known in the art to mediate several effector functions. For example, binding of the C1 component of complement to an antibody activates the complement system. Complement activation is important in opsonization and lysis of cellular pathogens. Complement activation also stimulates the inflammatory response and may be involved in autoimmune hypersensitivity. Furthermore, the antibody binds to the cell via the Fc region, and the Fc receptor site is on the antibody Fc region that binds to the Fc receptor (FcR) on the cell. There are many Fc receptors specific for different classes of antibodies, including IgG (gamma receptor), IgE (eta receptor), IgA (alpha receptor) and IgM (Miu receptor). Binding of antibodies to Fc receptors on the cell surface is a number of important and diverse biological responses, such as enclosing and disrupting antibody-coated particles, immune complex clearance, and killer cell lysis of antibody-coated target cells (antibodies). Triggers the regulation of dependent cell-mediated cytotoxicity (called ADCC), release of inflammatory mediators, placenta metastasis and immunoglobulin production.

0130In certain embodiments, the FOLR1-binding antibody provides modified effector function, which in turn affects the biological profile of the antibody administered. For example, deletion or inactivation of the constant region domain (via point mutation or other means) reduces Fc receptor binding of the modified antibody in the circulation, thereby enhancing tumor localization. Can be done. In another example, modification of the constant region consistent with the present invention may relax complement fixation and thereby reduce serum half-life and non-specific association of conjugated cytotoxicity. Yet another modification of the constant region can be used to eliminate disulfide linkages or oligosaccharide moieties that result in enhanced localization due to increased antigen specificity or antibody flexibility. Similarly, modifications of the constant region according to the present invention can be readily made using well-known biochemical or molecular manipulation techniques well within the knowledge of those skilled in the art.

0131In certain embodiments, the antibody FOLR1 binder 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 cellular cytotoxicity (CDC) activity. In certain embodiments, the antibody does not bind to Fc receptors and / or complement factors. In certain embodiments, the antibody has no effector function.

0132In certain embodiments, the modified antibody will be engineered to fuse the CH3 domain directly to the hinge region of each modified antibody. In another construct, it may be desirable to provide a peptide spacer between the hinge region and the modified CH2 and / or CH3 domains. For example, the construct which is compatible is CH2 domain has been deleted, and the remaining CH3 domain (Fix can be expressed as linked to the hinge region decoration or unmodified) is 5-20 amino acid spacer .. Such spacers can be added, for example, to ensure that the regulatory elements of the stationary domain remain available or that the hinge region remains flexible. However, it should be noted that amino acid spacers have been found to be immunogenic in some cases and elicit an unwanted immune response to the construct. Thus, in certain embodiments, any spacer added to the construct will be relatively non-immunogenic to maintain the desired biochemical quality of the modified antibody, or will be omitted altogether. Will be.

0133In addition to the deletion of the entire constant region domain, of course, the antibodies of the invention may be provided by partial deletion or substitution of several or even a single amino acid. For example, mutation of a single amino acid within a selected area of the CH2 domain may be sufficient to substantially reduce Fc binding, thereby enhancing tumor localization. Similarly, it may be desirable to simply delete one or more parts of the constant region domain that control the effector function to be modified (eg, complement C1Q binding). Such partial deletion of the constant region may improve the selected properties of the antibody (serum half-life) while leaving other desired functions associated with the constant region domain of interest intact. it can. Furthermore, as mentioned above, the constant regions of the disclosed antibodies can be modified via mutations or substitutions of one or more amino acids that enhance the profile of the resulting construct. In this regard, the modified antibody arrangement and immunogenicity profile may be substantially maintained while disrupting the activity conferred by the conserved binding site (eg, Fc binding). Certain embodiments add one or more amino acids to the constant region to enhance desired properties such as reduced or increased effector function, or to allow more cytotoxic or carbohydrate binding. Can be included. In such embodiments, it may be desirable to insert or replicate specific sequences derived from selected constant region domains.

0134The present invention further includes variants and equivalents that are substantially homologous to the chimeric, humanized and human antibodies or antibody fragments thereof described herein. These can include, for example, conservative substitution mutations, i.e. substitutions with one or more similar amino acids. For example, conservative substitutions are by another within the same general class of one amino acid, eg by another acidic amino acid of one acidic amino acid. , Refers to the substitution of one basic amino acid with another basic amino acid, or one neutral amino acid with another neutral amino acid. The intent of conservative amino acid substitutions is well known in the art.

0135The polypeptide of the invention can be a recombinant polypeptide, a native polypeptide, or a synthetic polypeptide comprising an antibody against human FOLR1 or a fragment thereof. As is known in the art, some amino acid sequences of the present invention can be varied without significantly affecting the structure or function of the protein. That is, the present invention further includes variants of the polypeptide that exhibit or include a region thereof of an antibody or fragment thereof against a human forlate receptor protein. Such mutants include deletions, insertions, inversions, repetitions and type substitutions.

0136Polypeptides and analogs can be further modified to contain additional chemical moieties that are not normally part of the protein. These derivatized moieties can improve protein solubility, biological half-life, or absorption. The portion can also reduce or eliminate any unwanted side effects of the protein or the like. An overview of these parts can be found in REMINGTON'S PHARMACEUTICAL SCIENCES, 20th, Mack Publishing Co., Easton, PA (2000).

0137The isolated polypeptides described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthesis methods to constructing DNA sequences that encode isolated polypeptide sequences and express these sequences in a suitable transformed host. In some embodiments, the DNA sequence is constructed using recombinant techniques by isolating or synthesizing the DNA sequence encoding the wild-type protein of interest. In some cases, the functional analog can be obtained by mutagenizing the sequence by site-directed mutagenesis. See, for example, Zoeller et al., Proc. Nat'l. Acad. Sci. USA 81: 5662-5066 (1984) and US Pat. No. 4,588,585.

0138In some embodiments, the DNA sequence encoding the polypeptide of interest is constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and by selecting codons that are favorable to the host cell from which the recombinant polypeptide of interest will be produced. Standard methods can be applied to synthesize isolated polynucleotide sequences that encode the isolated polypeptide of interest. For example, a back-translated gene can be constructed using the complete amino acid sequence. Furthermore, DNA oligomers containing nucleotide sequences encoding for a particular isolated polypeptide can be synthesized. For example, several small oligonucleotides encoding for a portion of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically contain 5'or 3'overhangs for complementary assembly.

0139After assembly (by synthesis, site-directed mutagenesis or other methods), the polynucleotide sequence encoding the particular isolated polypeptide of interest is inserted into an expression vector and in the desired host. It will operably link to an expression control sequence suitable for protein expression. Proper assembly can be confirmed by nucleotide sequencing, restriction mapping, and expression of the biologically active polypeptide in a suitable host. As is well known in the art, in order to obtain high expression levels of a transfected gene in a host, the gene acts on a transcriptional and translational expression control sequence that is functional in the selected expression host. Must be connected as much as possible.

0140In certain embodiments, recombinant expression vectors are used to amplify and express DNA encoding an antibody against human FOL R1 or a fragment thereof. A recombinant expression vector is a synthetic or cDNA-induced DNA fragment that encodes a polypeptide chain of an anti-FOLR1 antibody or fragment thereof operably linked to an appropriate transcriptional or translational regulatory element derived from a mammalian, microbial, viral or insect gene. It is a replicable DNA construct having. Transcriptional units are generally (1) genetic elements or elements that have a regulatory role in gene expression, such as transcriptional promoters or enhancers, (2) structures or coding sequences that are transcribed into mRNA and translated into proteins, and ( 3) Includes an assembly of appropriate transcription and translation initiation and termination sequences detailed below. Such regulatory elements can include operator sequences to control transcription. The ability to replicate in a host normally conferred by an origin of replication, as well as select genes that facilitate recognition of transformants, are also incorporated. DNA regions are operably linked when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operably linked to DNA for a polypeptide if it is expressed as a precursor that participates in the secretion of the polypeptide; the promoter it transcribes the sequence. When regulated, it is operably linked to the coding sequence; or the ribosome binding site is operably linked to the coding sequence if it is positioned to allow translation. Structural elements intended for use in the Kobo expression system include leader sequences that allow the extracellular secretion of translated proteins by the host cell. Alternatively, if the recombinant protein is expressed in the absence of a leader or transport sequence, it can include an N-terminal methionine residue. This residue is optionally cleaved from the expressed recombinant protein to give the final product.

0141The choice of expression control sequence and expression vector will depend on the choice of host. A wide variety of expression host / vector combinations can be used. Useful expression vectors for eukaryotic hosts include, for example, vectors containing expression control sequences derived from SV40, bovine papillomavirus, adenovirus and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids such as Escherichia coli-derived plasmids such as pCR1, pBR322, pMB9 and their derivatives, broad host range plasmids such as M13 and filament-like single strands. Includes DNA phage

0142Suitable host cells for the expression of FOLR1-binding polypeptides or antibodies (or FOLR1 proteins for use as antigens) are of prokaryotes, corn, insects and higher eukaryotes under the control of the appropriate promoter. Includes cells. Prokaryotes include Gram-negative or Gram-positive organisms such as E. coli or Bacillus. Higher eukaryotic cells include established cell lines of mammalian origin as described below. Cell-free translation systems are also used. Appropriate cloning and expression vectors using bacterial, mold, corn, and mammalian cell hosts have been described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, NY, 1985), the relevant disclosure of which is by reference. Incorporated herein. Other information on methods of protein production, including antibody production, is described, for example, in U.S. Patent Application Publication 2008/0187954, U.S. Patents 6,413,746 and 6,660,501 and International Patent Publication WO04009823, each of which is hereby referred to in its entirety. Incorporated into the book.

0143Various mammalian and insect cell culture systems are also conveniently used to express recombinant proteins. Expression of recombinant proteins in mammalian cells can be accomplished because such proteins are generally correctly folded, properly modified, and fully functional. Examples of suitable mammalian host cell lines are HEK-293 and HEK-293T, the COS-7 lineage of monkey kidney cells described by Gluzman (Cell 23: 175, 1981), and other cell lines such as L cells. , C127,3T3, Chinese Hamster Ovary (CHO), HeLa and BHK cell lines. Mammalian expression vectors require non-transcriptional elements such as replication origins, suitable promoters and enhancers linked to the gene to be expressed, and 5'or 3'flanking non-transcriptional sequences, and 5'or 3'untranslated sequences, eg. Ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and transcription termination sequences. The baculovirus system for the production of non-homologous proteins in insect cells is Luccow and Summers, Considered in Bio / Technology 6:47 (1988).

0144The protein produced by the transformed host can be purified according to any suitable method. Such standard methods include chromatography (eg ion exchange, affinity and sizing column chromatography), centrifugation, solubility differences, or any other standard protein purification technique. Binding of affinity tags such as hexahistidine, maltose binding domain, influenza capsule sequence and glutathione-S-transferase to the protein allows for easy purification by passing over an appropriate affinity column. The isolated protein can also be physically characterized using techniques such as proteolysis, nuclear magnetic resonance and X-ray crystallography.

0145For example, the supernatant from a system that secretes recombinant protein into the medium can first be concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. After the concentration step, the concentrate can be applied to a suitable purification matrix. Alternatively, an anion exchange resin, such as a matrix or substrate having a suspended diethylaminoethyl (DEAE) group, can be used. The matrix can be acrylamide, agarose, dextran, cellulose or other types commonly used in protein purification. Alternatively, a cation exchange emperor can be used. Suitable cation exchangers include various insoluble matrices containing sulfopropyl or carboxymethyl groups. Finally, the FOLR1 binder is further purified using one or more Reverse Phase High Performance Liquid Chromatography (RP-HPLC) steps using a hydrophobic RP-HPLC medium, such as silica gel with suspended methyl or other aliphatic groups. can do. Homogeneous recombinant proteins can also be obtained using some or all of the purification steps described above in various combinations.

0146Recombinant proteins produced in bacterial cultures can be isolated, for example, by initial extraction from cell pellet, followed by one or more concentration, salting out, aqueous ion exchange or size exclusion chromatography steps. High performance liquid chromatography (HPLC) can be used for the final purification step. The microbial cells used in the expression of the recombinant protein can be destroyed by any convenient method, such as freezing and thawing cycling, sonication, mechanical destruction or the use of cytolytic agents.

0147Methods known in the art for purifying antibodies and other proteins also include, for example, those described in US Patent Application Publications 2008/0312425, 2008/0177048 and 2009/0187005, each of which. Is incorporated herein by reference in its entirety.

0148In certain embodiments, the FOLR1 binder is a polypeptide that is not an antibody. Various methods for identifying and producing non-antibody polypeptides that bind to protein targets with high affinity are known in the art. 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). Each is incorporated herein by reference in its entirety. In certain embodiments, phage display techniques have been used to identify / produce FOLR1-binding polypeptides. In certain embodiments, the polypeptide comprises a type of protein scaffold selected from the group consisting of protein A, lipocalin, fibronectin domain, ankyrin consensus repeat domain and thioredoxin.

0149In some embodiments, the agent is a non-protein molecule. In certain embodiments, the agent is a small molecule. Combinatorial chemistry libraries and techniques useful in identifying non-protein FOLR1 binders are known in the art. 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) can be referred to, each of which is incorporated herein by reference in its entirety. In certain other embodiments, the agent is a carbohydrate, glycosaminoglycan, glycoprotein, or proteoglycan.

0150In certain embodiments, the agent is a nucleic acid aptamer. Aptamers are polynucleotide molecules that are selected based on their ability to bind to another molecule (eg, from a random or mutagenetic pool). In some embodiments, the aptamer comprises a DNA polynucleotide. In most particular embodiments, the aptamer comprises an RNA polynucleotide. In certain embodiments, the aptamer comprises one or more modified nucleic acid residues. Methods of making and screening nucleic acid aptamers for binding to proteins are well known in the art. For example, US patent 5,270,163, US patent 5,683,867, US patent 5,763,595, US patent 6,344,321, US patent 7,368,236, US patent 5,582,981, US patent 5,756,291, US patent 5,840,867, US patent 7,312,325, US patent 7,329,742, International patent publication WO02 / 077262, International patent Publication WO 03/070984, US Patent Application Publication 2005/0239134, US Patent Application Publication 2005/0124565 and US Patent Application Publication 2008/0227735, each of which is incorporated herein by reference in its entirety.

0151III. Immunoconjugate The present invention also includes anti-FOLR1 antibodies, antibody fragments, functional equivalents, improved antibodies and aspects thereof disclosed herein linked or conjugated to a cytotoxic agent (drug) or prodrug. It relates to a gate (also referred to herein as an immunoconjugate). That is, in certain embodiments, the present invention provides an immunoconjugate comprising a humanized antibody that specifically binds to human forate receptor 1 or an antigen-binding fragment thereof, wherein the antibody is described below: (a) GYFMN ( Heavy chain CDR1 containing SEQ ID NO: 1); RIHPYDGDTFYNQXaa1FXaa2Xaa3Heavy chain CDR2 containing (SEQ ID NO: 56) and heavy chain CDR3 containing YDGS RAMDY (SEQ ID NO: 3); and (b) Light chain CDR1 containing KASQSVSFAGTSLMH (SEQ ID NO: 7); Light containing RASNLEA (SEQ ID NO: 8). Chain CDR2; and light chain CDR3 containing QQSREYPYT (SEQ ID NO: 9); however, where Xaa1Is selected from K, Q, H and R; Xaa2Is selected from Q, H, N and R; and Xaa3Includes those selected from G, E, T, S, A and V; In certain embodiments, the antibody is a huMov19 antibody, which is said antibody comprising the heavy chain CDR2 RIHPYDGDTFYNQKFQG (SEQ ID NO: 2). In other embodiments, the antibody is FR1-21, and (a) heavy chain CDR1 containing SSYGMS (SEQ ID NO: 30); heavy chain CDR2 containing TISSGGSYTY (SEQ ID NO: 31); and / or DGEGGL YAMDY (SEQ ID NO: 32). ) Containing heavy chain CDR3; and (b) light chain CDR1 containing KASDHINNWLA (SEQ ID NO: 27); light chain CDR2 containing GATSLET (SEQ ID NO: 28); and (b) light chain CDR3 containing QQYWSTPFT (SEQ ID NO: 29). In other embodiments, the antibody is FR1-48 and (a) heavy chain CDR1 containing TNYWMQ (SEQ ID NO: 60); heavy chain CDR2 containing AIYPGNGDSR (SEQ ID NO: 61); and / or RDGNYAAY (SEQ ID NO: 62). ) Containing heavy chain CDR3; and / or (b) light chain CDR1 comprising RASENIYSNLA (SEQ ID NO: 57); light chain CDR2 containing AATNLAD (SEQ ID NO: 58); and light chain CDR3 containing QHFWASPYT (SEQ ID NO: 59). ;including. In other embodiments, the antibody is FR1-49, and (a) heavy chain CDR1 containing TNYWMY (SEQ ID NO: 66); heavy chain CDR2 containing AIYPGNSDTT (SEQ ID NO: 67); and / or RHDYGAMDY (SEQ ID NO: 68). ) And / or (b) light chain CDR1 containing RASENIYTNLA (SEQ ID NO: 63); light chain CDR2 containing TASNLAD (SEQ ID NO: 64); and light chain CDR3 containing QHFWVSPYT (SEQ ID NO: 65). ;including. In other embodiments, the antibody is FR1-57, and (a) heavy chain CDR1 containing SSFGMH (SEQ ID NO: 72); heavy chain CDR2 containing YISSGSSTIS (SEQ ID NO: 73); and / or EAYGSSMEY (SEQ ID NO: 74). ) Including heavy chain CDR3; And / or (b) light chain CDR1 containing RASQNINNNLH (SEQ ID NO: 69); light chain CDR2 containing YVSQSVS (SEQ ID NO: 70); and light chain CDR3 containing QQSNSWPHYT (SEQ ID NO: 71); In other embodiments, the antibody is FR1-65 and (a) heavy chain CDR1 containing TSYTMH (SEQ ID NO: 78); heavy chain CDR2 containing YINPISGYTN (SEQ ID NO: 79); and / or GGAYGRKPMDY (SEQ ID NO: 80). ) And / or (b) light chain CDR1 containing KASQNVGPNVA (SEQ ID NO: 75); light chain CDR2 containing SASYRYS (SEQ ID NO: 76); and light chain CDR3 containing QQYNSYPYT (SEQ ID NO: 77). ;including.

0152Suitable drugs or prodrugs are known in the art. In certain embodiments, the drug or prodrug is a cytotoxic agent. The cytotoxic agent used in the cytotoxic conjugate of the present invention is any compound that results in cell death, induces cell death, or, in some embodiments, reduces cell viability. And can, for example, mitancinoids and mitancinoid analogs, benzodiazepines, taxoids, CC-1065 and CC-1065 analogs, duocalmycin and duocalmycin analogs, enesin, such as calikeamycin, drastatin and drastatin. Includes analogs such as auristatin, tomymycin derivatives, leptomycin derivatives, methotrexate, cisplatin, carboplatin, daunorubicin, doxorubicin, vincristine, vinblastine, melfaran, mitomycin C, chlorambusyl and morpholinodoxorubicin. In certain embodiments, the cytotoxic agent is a maytansinoid and a maytansinoid analog.

0153Such conjugates can be prepared by using a linking group to link the drug or prodrug to the antibody or functional equivalent. Suitable linking groups are well known in the art and include, for example, disulfide groups, thioether groups, acid instability groups, photoinstability groups, peptidase instability groups and esterase instability groups.

0154The drug or prodrug can be linked to the anti-FOLR1 antibody or fragment thereof, for example via a disulfide bond. The linker molecule or cross-linking agent contains a reactive chemical group capable of reacting with the anti-FOLR1 antibody or fragment thereof. In certain embodiments, the reactive chemical groups for the reaction with the cell binder are N-succinimidyl ester and N-sulfosuccinimidyl ester. Furthermore, the linker molecule contains a reactive chemical group, in certain embodiments a dithiopyridyl group that can react with a drug to form a disulfide bond. In certain embodiments, the linker molecule is such as-succinimidyl 3- (2-pyridyldithio) propionate (SPDP) (see, eg, Carlsson et al., Biochem.J., 173: 723-737 (1978)), N-succinimidyl. 4- (2-Pyridyldithio) butanoate (SPDB) (eg, US Pat. No. 4,563, 304), N-Succinimidyl 4- (2-pyridyldithio) 2-sulfobutanoate (sulfo-SPDB) (see US Patent Publication 20090274713), N-succinimidyl 4- (2-pyridyldithio) pentanoate (SPP) ( For example, CAS Registry Number 341498-08-6), including 2-iminothiolane or acetylsuccinic anhydride. For example, an antibody or cell binding agent can be modified with a cross-linking reagent, and an antibody or cell binding agent containing a free or protected thiol group thus derived is then a disulfide or thiol-containing maytansinoid. A conjugate is produced by reacting with a noid. The conjugate can be purified by chromatography, such as, but not limited to, HPLC, size exclusion, adsorption, ion exchange and affinity capture, dialysis or tangential flow filtration. In certain embodiments, the anti-FOLR1 antibody is linked to the cytotoxic agent via an SPDB or sulfo-SPDB linker. In certain embodiments, the huMov19 antibody is linked to the cytotoxic agent via an SPDB or sulfo-SPDB linker.

0155In another aspect of the invention, the anti-FOLR1 antibody is linked to a cytotoxic agent via a disulfide bond and polyethylene glycol when enhancing the titer, solubility or efficacy of the immunoconjugate. Such a cleavable hydrophilic linker is described in WO 2009/0134976. An additional advantage of this linker design is the desired high monomer ratio and minimal antibody drug conjugate aggregation. Particularly intended in this aspect are cell binder conjugates and polyethylene glycol spacers with a narrow range of drug loading of 2-8 ((CH).2CH2O)n = 1-14The drug linked via a disulfide bond (-SS-) carrying) exhibits relatively high strong biological activity against cancer cells, and has high conjugate state and high monomer ratio and minimal. It is described as having the desired biochemical property of protein aggregation.

0156Particularly intended in this aspect are anti-FOLR1 antibody drug conjugates of formula (I) or conjugates of formula (I'): A- [Xl-(-CH2-CH2O-)n-YC]m (I) [CY-(-CH2-CH2O-)n-Xl]m-A (I') And During the ceremony: A indicates an anti-FOLR1 antibody or fragment; C indicates a cytotoxic drug; X indicates an aliphatic, aromatic or heterocyclic unit bound to a cell binder via a thioether bond, an amide bond, a carbamate bond, or an ether bond; Y represents an aliphatic, aromatic or heterocyclic unit bound to the drug via a disulfide bond; l is 0 or 1; m is an integer from 2 to 8; and n is an integer from 1 to 24. In some embodiments, m is an integer of 2-6. In some embodiments, m is an integer of 3-5.

0157Furthermore, in certain embodiments, n is an integer of 2-8. Alternatively, the drug can first be modified to introduce a reactive ester suitable for reaction with the cell binder, eg, as disclosed in US Pat. Nos. 6,441,163 and 7,368,565. Reactions of these drugs with cell-binding agents containing activated linker moieties provide another way to generate cell-binding agent drug conjugates. Maytansinoids can also be linked to anti-FOLR1 antibodies or fragments using, for example, the PEG linking group described in US Pat. No. 6,716,821. These PEG-cleaveable linking groups are soluble in both water and non-aqueous solvents and can be used to link one or more cytotoxic agents to the cell binding agent. Illustrated PEG linking groups include a functional sulfidyl or disulfide group at one end and a heterobifunctional PEG linker that reacts with cytotoxic and cell binding agents at both ends of the linker via an active ester at the other end. Include. Again, as a general example of the synthesis of cytotoxic conjugates using PEG linking groups, US Pat. No. 6,716, 821 can be referenced, which is incorporated herein by reference in its entirety. The initiation of synthesis is the reaction of a cytotoxic agent carrying a reactive PEG moiety with one or more cell binders, thereby allowing the terminal active ester of each reactive PEG moiety by the amino acid residues of the cell binder. Substitution occurs, which results in a cytotoxic conjugate containing one or more covalently bound cytotoxic agents to the cell-binding agent via the PEG linking group. Alternatively, a reactive disulfide moiety (eg, pyridyl disulfide) can be introduced by modifying the cell binder with a bifunctional PEG crosslinker, which is then conjugated with a thiol-containing maytansinoid. Can be obtained. Alternatively, the thiol moiety can be introduced by modifying the cell bond with a bifunctional PEG crosslinker, which is then treated with a reactive disulfide-containing maytansinoid (eg, pyridyl disulfide). Can obtain a conjugate.

0158Antibody-maytansinoid conjugates with uncleavable linkages can also be prepared. Such cross-linking agents have been reported in the art (Thermo Scientific Pierce Crosslinking Technical). See Handbook and US Patent Publication 2005/0169933), and N-succinimidyl 4- (maleimidemethyl) cyclohexanecarboxylate (SMCC), N-succinimidyl 4- (N-maleimidemethyl) -cyclohexane-1-carboxy- (6-). Amidocaproate), ie the "long chain" analog of SMCC (LC-SMCC), κ-maleimideundecanoic acid N-succinimidyl ester (KMUA), β-maleimide propanoic acid N-succinimidyl ester (BMPS) , Γ-Maleimidebutyric acid N-succinimidyl ester (GMBS), ε-maleimide caproic acid N-hydroxysuccinimide ester (EMCS), m-maleimidebenzoyl-N-hydroxysuccinimide ester (MBS), N- (α-maleimideacetoxy) )-Succiniimide ester (AMAS), succinimidyl-6- (β-maleimidepropionamide) hexanoate (SMPH), N-succinimidyl 4- (p-maleimidephenyl) -butyrate (SMPB), and N- (p-maleimidephenyl) Isocyanate (PMPI), N-succinimidyl-4- (iodoacetyl) -aminobenzoate (SIAB), N-succinimidyl iodoacetate (SIA), N-succinimidylbromoacetate (SBA), and N-succinimidyl Includes, but is not limited to, 3- (bromoacetamide) propionate (SBAP). In certain embodiments, the antibody is succinimidyl 4- (N-maleimidemethyl) cyclohexane-1-carboxylate (SMCC), sulfo- as described in the literature to introduce 1-10 reactive groups. Modified with cross-linking agents such as SMCC, maleimide benzoyl-N-hydroxysuccinimide ester (MBS), sulfo-MBS or succinimidyl-iodoacetate (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 a thiol-containing maytansinoid 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 antibody is treated with a thiol-containing maytansinoid (1-2 molar equivalence / maleimide group) and the antibody-maytansinoid conjugate is gel filtered through a Sephadex G25 column, chromatographed on a ceramic hydroxyapatite column. Purify by chromatography, dialysis or tangential flow filtration or a combination of these methods. Typically, an average of 1-10 maytansinoids are linked per antibody. One method is to introduce a maleimide group by modifying the antibody with succinimidyl 4- (N-maleimidemethyl) -cyclohexane-1-carboxylate (SMCC) and then react the modified antibody with a thiol-containing maytansinoid. A thioether-linked conjugate is obtained by allowing the antibody to form a thioether-linked conjugate. Again, conjugates with 1-10 drug molecules per antibody molecule are produced. Maytansinoid conjugates of antibodies, antibody fragments, protein hormones, protein growth factors and other proteins are prepared in the same embodiment. 690-697 (1979)). The modified antibody is then reacted with a thiol-containing maytansinoid 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 antibody is treated with a thiol-containing maytansinoid (1-2 molar equivalence / maleimide group) and the antibody-maytansinoid conjugate is gel filtered through a Sephadex G25 column, chromatographed on a ceramic hydroxyapatite column. Purify by chromatography, dialysis or tangential flow filtration or a combination of these methods. Typically, an average of 1-10 maytansinoids are linked per antibody. One method is to introduce a maleimide group by modifying the antibody with succinimidyl 4- (N-maleimidemethyl) -cyclohexane-1-carboxylate (SMCC) and then react the modified antibody with a thiol-containing maytansinoid. A thioether-linked conjugate is obtained by allowing the antibody to form a thioether-linked conjugate. Again, conjugates with 1-10 drug molecules per antibody molecule are produced. Maytansinoid conjugates of antibodies, antibody fragments, protein hormones, protein growth factors and other proteins are prepared in the same embodiment. 690-697 (1979)). The modified antibody is then reacted with a thiol-containing maytansinoid 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 antibody is treated with a thiol-containing maytansinoid (1-2 molar equivalence / maleimide group) and the antibody-maytansinoid conjugate is gel filtered through a Sephadex G25 column, chromatographed on a ceramic hydroxyapatite column. Purify by chromatography, dialysis or tangential flow filtration or a combination of these methods. Typically, an average of 1-10 maytansinoids are linked per antibody. One method is to introduce a maleimide group by modifying the antibody with succinimidyl 4- (N-maleimidemethyl) -cyclohexane-1-carboxylate (SMCC) and then react the modified antibody with a thiol-containing maytansinoid. A thioether-linked conjugate is obtained by allowing the antibody to form a thioether-linked conjugate. Again, conjugates with 1-10 drug molecules per antibody molecule are produced. Maytansinoid conjugates of antibodies, antibody fragments, protein hormones, protein growth factors and other proteins are prepared in the same embodiment. (1979)). The modified antibody is then reacted with a thiol-containing maytansinoid 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 antibody is treated with a thiol-containing maytansinoid (1-2 molar equivalence / maleimide group) and the antibody-maytansinoid conjugate is gel filtered through a Sephadex G25 column, chromatographed on a ceramic hydroxyapatite column. Purify by chromatography, dialysis or tangential flow filtration or a combination of these methods. Typically, an average of 1-10 maytansinoids are linked per antibody. One method is to introduce a maleimide group by modifying the antibody with succinimidyl 4- (N-maleimidemethyl) -cyclohexane-1-carboxylate (SMCC) and then react the modified antibody with a thiol-containing maytansinoid. By letting it form a thioether-linked conjugate. Again, conjugates with 1-10 drug molecules per antibody molecule are produced. Maytansinoid conjugates of antibodies, antibody fragments, protein hormones, protein growth factors and other proteins are prepared in the same embodiment. (1979)). The modified antibody is then reacted with a thiol-containing maytansinoid 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 antibody is treated with a thiol-containing maytansinoid (1-2 molar equivalence / maleimide group) and the antibody-maytansinoid conjugate is gel filtered through a Sephadex G25 column, chromatographed on a ceramic hydroxyapatite column. Purify by chromatography, dialysis or tangential flow filtration or a combination of these methods. Typically, an average of 1-10 maytansinoids are linked per antibody. One method is to introduce a maleimide group by modifying the antibody with succinimidyl 4- (N-maleimidemethyl) -cyclohexane-1-carboxylate (SMCC) and then react the modified antibody with a thiol-containing maytansinoid. A thioether-linked conjugate is obtained by allowing the antibody to form a thioether-linked conjugate. Again, conjugates with 1-10 drug molecules per antibody molecule are produced. Antibodies, antibody fragments, protein hormones, protein growth factors and other protein mitansi

0159In another aspect of the invention, the FOLR1 antibody (eg, huMov19, FR1-21, FR1-48, FR1-49, FR1-57 or FR1-65) to the drug via a non-cleavable bond with PEG spacers in between ) Are concatenated. Suitable cross-linking reagents containing hydrophilic PEG chains that form a linker between the drug and the anti-FOLR1 antibody or fragment are well known or commercially available in the art (eg Quanta Biodesign, Powell, Ohio). .. Suitable PEG-containing crosslinkers can also be synthesized from commercially available PEGs themselves using standard synthetic chemical techniques known in the art. By reacting the drug with a bifunctional PEG-containing crosslinker by the methods detailed in US Patent Application Publication 20090274713 and WO 2009/0134976,l-(-CH2-CH2-O-)n-YpIt can be conjugated by making a compound of -D and then reacting it with a cell binder. Alternatively, a thiol-reactive group (eg maleimide or haloacetamide) is introduced by modifying the cell bond with a bifunctional PEG crosslinker and then treated with a thiol-containing mytancinoid to form a conjugate. Can be done. Alternatively, the thiol moiety is introduced by modifying the cell junction with a bifunctional PEG crosslinker, which is then treated with a thiol-reactive maytansinoid (eg, a maleimide or haloacetamide-carrying maytansinoid). It can be made into a conjugate by processing.

0160Therefore, another aspect of the present invention is the anti-FOLR1 antibody drug conjugate of formula (II) or formula (II'): A- [Xl-(-CH2-CH2-O-)n-Yp-C]m (II) [CYp-(-CH2-CH2-O-)n-Xl]m-A (II') In the formula, A represents an anti-FOLR1 antibody or fragment; C indicates drug; X indicates an aliphatic, aromatic or heterocyclic unit bound to a cell binder via a thioether bond, an amide bond, a carbamate bond, or an ether bond; Y is an aliphatic, aromatic or heterocyclic bond attached to a drug via a covalent bond selected from the group consisting of thioether bonds, amide bonds, carbamate bonds, ether bonds, amine bonds, carbon-carbon bonds and hydrazone bonds. Indicates the unit; l 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 certain embodiments, m is an integer from 2 to 8; and n is an integer from 1 to 24. In certain embodiments, m is an integer of 2-6. In certain embodiments, n is an integer of 2-8.

0161In certain embodiments, m is an integer of 3-5. In certain embodiments, the antibody is huMov19. 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 linkers are N-succinimidyl esters or N-sulfosuccinimidyl esters for reaction with anti-FOLR1 antibodies or fragments thereof, as well as maleimide-or haloacetyl systems for reaction with compounds. Includes a linker with a portion. The PEG spacer can be incorporated into any of the cross-linking agents known in the art by the methods described herein.

0162Many of the linkers disclosed herein are detailed in US Patent Publications 20050169933 and 20090274713, and WO2009 / 0134976; their contents are incorporated herein by reference in their entirety.

0163In the present invention, about 2 to about 8 drug molecules (drug loading), such as maytansinoids, are linked to an anti-FOLR1 antibody or fragment thereof, and the conjugate anti-tumor adoption is linked to the same cell binding agent. It includes aspects that are far more effective than drug loading, where the number of drugs taken is low or high. As used herein, "drug loading" refers to the number of drug molecules (eg, maytansinoids) that can bind to a cell binder (eg, an anti-FOLR1 antibody or fragment thereof). In one aspect, the average number of drug molecules that can bind to a cell binder is about 2 to about 8 (eg, 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 pieces). In certain embodiments, the drug is N2’-Deacetyl-N2’-(3-Mercapto-1-oxopropyl)-maitansine (DM1) or N2’-Deacetyl-N2’-(4-Mercapto-4-methyl-1-oxopentyl) Maitansine (DM4). That is, in certain embodiments, the antibody huMov19 is conjugated to DM1 or DM4. In another embodiment, antibody FR-1-21 is conjugated to DM1 or DM4. In another embodiment, antibody FR-1-48 is conjugated to DM1 or DM4. In another embodiment, antibody FR-1-49 is conjugated to DM1 or DM4. In another embodiment, antibody FR-1-57 is conjugated to DM1 or DM4. In another embodiment, antibody FR-1-65 is conjugated to DM1 or DM4.

0164That is, in one aspect, the immunoconjugate contains one maytansinoid per antibody. In another aspect, immunoconjugates contain two maytansinoids per antibody. In another aspect, immunoconjugates contain 3 maytansinoids per antibody. In another aspect, immunoconjugates contain 4 maytansinoids per antibody. In another aspect, immunoconjugates contain 5 maytansinoids per antibody. In another aspect, immunoconjugates contain 6 maytansinoids per antibody. In another aspect, immunoconjugates contain 7 maytansinoids per antibody. In another aspect, immunoconjugates contain 8 maytansinoids per antibody.

0165In one aspect, immunoconjugates contain from about 1 to about 8 maytansinoids per antibody. In another aspect, immunoconjugates contain about 2 to about 7 maytansinoids per antibody. In another aspect, immunoconjugates contain about 2 to about 6 maytansinoids per antibody. In another aspect, immunoconjugates contain from about 2 to about 5 maytansinoids per antibody. In another aspect, immunoconjugates contain about 3 to about 5 maytansinoids per antibody. In another aspect, immunoconjugates contain about 3 to about 4 maytansinoids per antibody.

0166In one feature, compositions containing immunoconjugates average about 2 to about 8 bound drug molecules (eg, maytansinoids) per antibody (eg, 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 pieces). In one aspect, compositions containing immunoconjugates have an average of about 1 to about 8 drug molecules (eg, maytansinoids) per antibody. In one aspect, compositions containing immunoconjugates have an average of about 2 to about 7 drug molecules (eg, maytansinoids) per antibody. In one aspect, compositions containing immunoconjugates have an average of about 2 to about 6 drug molecules (eg, maytansinoids) per antibody. In one aspect, compositions containing immunoconjugates have an average of about 2 to about 5 drug molecules (eg, maytansinoids) per antibody. In one aspect, compositions containing immunoconjugates have an average of about 3 to about 5 drug molecules (eg, maytansinoids) per antibody. In one aspect, compositions containing immunoconjugates have an average of about 3 to about 4 drug molecules (eg, maytansinoids) per antibody. In one aspect, compositions containing immunoconjugates have an average of about 3.5 to about 4 drug molecules (eg, maytansinoids) per antibody.

0167In one aspect, compositions containing immunoconjugates average about 2 ± 0.5, about 2.5 ± 0.5, about 3 ± 0.5, about 3.5 ± 0.5 of bound drug molecules (eg, maytansinoids) per antibody. Approximately 4 ± 0.5 pcs, Approximately 4.5 ± 0.5 pcs, Approximately 5 ± 0.5 pcs, Approximately 5.5 ± 0.5 pcs, Approximately 6 ± 0.5 pcs, Approximately 6.5 ± 0.5 pcs, Approximately 7 ± 0.5 pcs, Approximately 7.5 ± 0.5 pcs Or have about 8 ± 0.5 pieces. In one aspect, compositions containing immunoconjugates have an average of about 3.5 ± 0.5 drug molecules (eg, maytansinoids) per antibody.

0168The anti-FOLR1 antibody or fragment thereof can be modified by reacting a bifunctional cross-linking reagent with the anti-FOLR1 antibody or fragment thereof, thereby causing covalent attachment of the linker molecule to the anti-FOLR1 antibody or fragment thereof. As used herein, a "bifunctional cross-linking reagent" is any chemical moiety that covalently binds a cell binder to a drug, such as the drugs described herein. Alternatively, a portion of the connecting portion is provided by the drug. In this regard, the drug comprises a linking moiety that is a portion of the larger linker molecule used to link the cell binder to the drug. For example, to form the maytansinoid DM1, the side chain of the C-3 hydroxyl group of maitansine is modified to have a free sulfidyl group (SH). This thiolated form of maitansine can react with modified cell binders to form conjugates. Thus, refurbishment is assembled from two components, one given by the cross-linking reagent and the other by the side chain derived from DM1.

0169The drug molecule can be linked to the antibody molecule via an intermediate carrier molecule such as serum albumin.

0170As used herein, the phrase "linked to a cell binder" or "linked to an anti-FOLR1 antibody or fragment" is at least one bound to a cell binder anti-FOLR1 antibody or fragment via a suitable linking group. Refers to a conjugate containing two drug derivatives or a precursor thereof. In certain embodiments, one linking group is SMCC.

0171In certain embodiments, the cytotoxic agents useful in the present invention are maytansinoids and maytansinoid analogs. Examples of suitable maytansinoids include esters of maytansinol and maytansinol analogs. Includes any drug that inhibits microtubule formation and is highly toxic to mammalian cells, such as mitansinol and mitansinol analogs.

0172Examples of suitable mitansinol esters include those with a modified aromatic ring and those with modifications at other positions. Such suitable maytansinoids are US Pat. Nos. 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; And 7,473,796.

0173In certain embodiments, the immunoconjugates of the invention are cytotoxic agents, officially named N.2’-Deacetyl-N2’-(3-Mercapto-1-oxopropyl)-Maytansine, a thiol-containing maytansinoid (DM1), is used. DM1 is represented by the following structural formula (III).<chemistry num="1-2"><img id="000003" he="50" wi="79" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>

0174In another embodiment, the conjugates of the invention, as cytotoxic agents, are thiol-containing maytansinoids N.2’-Deacetyl-N2’-(4-Methyl-4-mercapto-1-oxopropyl) -maitansine (eg DM4) is used. DM4 is represented by the following structural formula (IV).<chemistry num="2"><img id="000004" he="55" wi="90" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>

0175Another maytansinoid containing a side chain containing a steric hindrance thiol bond is N represented by the following structural formula (V).2’-Deacetyl-N2’-(4-Mercapto-1-oxopentyl) -maitansine (referred to as DM3).<chemistry num="3"><img id="000005" he="48" wi="78" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>

0176Each of the maytansinoids taught in US Pat. Nos. 5,208,020 and 7,276,497 can also be used in the conjugates of the present invention. In this regard, all disclosures of 5,208,020 and 7,276,697 are incorporated herein by reference.

0177Many positions of maytansinoids serve as positions for chemically connecting the connecting parts. For example, the C-3 position having a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with hydroxyl, and the C-20 position having a hydroxy group are all expected to be useful. In certain embodiments, the C-3 position is utilized. In certain embodiments, the C-3 position of maytansinol is utilized.

0178The structural diagram of a specific conjugate is shown below.<chemistry num="4"><img id="000006" he="64" wi="146" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry><chemistry num="5"><img id="000007" he="60" wi="142" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry><chemistry num="6"><img id="000008" he="69" wi="129" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry><chemistry num="7"><img id="000009" he="61" wi="131" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry><chemistry num="8"><img id="000010" he="69" wi="115" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry><chemistry num="9"><img id="000011" he="63" wi="129" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry><chemistry num="10"><img id="000012" he="64" wi="136" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry><chemistry num="11"><img id="000013" he="64" wi="139" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>In certain embodiments, the antibody is huMov19. In another embodiment, the antibody is FR1-21.

0179Several descriptions for producing such antibody maytansinoid conjugates are described in US Pat. Nos. 6,333,410, 6,441,163, 6,716,821 and 7,368,565, each of which is incorporated herein by reference in its entirety.

0180In general, a solution of the antibody in aqueous buffer can be incubated with a molar excess of maytansinoid having a disulfide moiety carrying a reactive group. The reaction mixture can be quenched by adding excess amine (eg ethanolamine, taurine, etc.). The maytansinoid-antibody conjugate can then be purified by gel filtration. The number of maytansinoid molecules bound per antibody molecule can be determined by spectroscopically measuring the absorbance ratio at 252 nm and 280 nm. An average of 1 to 10 maytansinoid molecules / antibodies are used, and in certain embodiments an average of 2 to 5 is used. The average number of maytansinoid molecules / antibodies can be, for example, 1-10, 2-5, 3-4, 3.5-4 or about 3.5. In one aspect, the average number of maytansinoid molecules is about 3.5 ± 0.5. In one aspect, the average number of maytansinoid molecules is about 3.5-4.

0181The ability of antibody conjugates with maytansinoid drugs to suppress the growth of various unwanted cell lines can be assessed in vitro. For example, cell lines such as the human KB cell lineage can be readily used to assess the cytotoxicity of these compounds. Cells to be evaluated are exposed to the compound for 4-5 days, and cell viability can be measured directly in the assay by known methods. Next IC50The value can be calculated from the assay results.

0182For example, the benzodiazepine compounds described in US Patent Application 2010/0203007 (eg, indolinobenzodiazepines or oxazolidinobenzodiazepines), their derivatives, and their intermediates are also used to prepare anti-FOLR1 antibody fragments or conjugates. It's okay.

0183Useful benzodiazepines have the following formulas (XIV), (XV) and (XVI):<chemistry num="12"><img id="000014" he="35" wi="132" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry><chemistry num="13"><img id="000015" he="29" wi="123" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry><chemistry num="14"><img id="000016" he="29" wi="106" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>[Double line between N and C in the formula--Indicates a single bond or a double bond, but if it is a double bond X is absent, Y is H, and if it is a single bond X is H or An amino-protected moiety that converts a compound into a prodrug; Y is -OR, ester indicated by -OCOR', carbonate indicated by -OCOOR', carbamate indicated by -OCONR'R ", hydroxylamine indicated by NR'R", amide indicated by -NRCOR', NRCOP. Peptides designated by, where P is an amino acid or a peptide containing 2 to 20 amino acid units, a thioester represented by SR', a sulfoxide represented by SOR', and -SO.2Sulfone indicated by -R', -SO3Sulfite, indicated by -OSO3Selected from bisulfite, halogen, cyano, azide, or thiol indicated by, where R, R'and R'are the same or different, H, substituted or unsubstituted with 1-10 carbon atoms. Linear, branched or cyclic alkyl, alkenyl or alkynyl, polyethylene glycol units (-OCH)2CH2) N, where n is an integer from 1 to 2000, an aryl having 6 to 10 carbon atoms, a heterocycle having 3 to 10 carbon atoms, where the substituent is OR7, NR8R9, NO2, NRCOR', SR10Selected from, sulfoxides indicated by SOR', -SO2Sulfone indicated by R', -SO3Sulfite, indicated by -OSO3Bisle Fight, SO2Sulfonamide, cyano, azide, -COR indicated by NRR'11, OCOR11Or OCONR11R12, But here R7, R8, R9, R10, R11And R12The definition of is as shown above, and in some cases R "is OH; W is C = O, C = S, CH2, BH, SO or SO2Is; R1, R2, R3, R4, R1', R2', R3'And R4'Is H, substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl, polyethylene glycol units (-OCH) each independently having 1 to 10 carbon atoms.2CH2) N, where n is an integer from 1 to 2000, or halogen, guanidinium [-NH (C = NH) NH2], OR7, NR8R9, NO2, NRCOR', SR10, Sulfoxide, indicated by SOR', -SO2Sulfone, Sulfite-SO, indicated by R'3, Bisle Fight-OSO3, SO2Sulfonamide, cyano, azide, -COR indicated by NRR'11, OCOR11Or OCONR11R12Selected from Substituents Selected from, where R7, R8, R9, R10, R11And R12Are independently H, substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl, polyethylene glycol units (-OCH) each having 1 to 10 carbon atoms.2CH2) N, where n is an integer from 1 to 2000, selected from aryls with 6 to 10 carbon atoms, heterocycles with 3 to 10 carbon atoms, and in some cases R10Is SR13Or COR13And here R13Is a linear, branched or cyclic alkyl, alkenyl or alkynyl, polyethylene glycol unit (-OCH) with 1 to 10 carbon atoms.2CH2) N, where n is an integer from 1 to 2000, selected from aryls with 6 to 10 carbon atoms, heterocycles with 3 to 10 carbon atoms, and in some cases R11Is OR14And here R14Has the same definition as R, and in some cases R1, R2, R3, R4, R1', R2', R3', Or R4Any one of the'is a linking group that allows ligation to the cell binding agent via a covalent bond, or polypyrrolo, polyindyl, polyimidazolyl, polypyrrolomidazolyl, polypyrroloin drill or polyimidazolone. Selected from drill units that optionally carry linking groups that allow ligation to cell binding agents; Z is (CH2)nHowever, n is 1, 2 or 3, CR15R16, NR17, O or S, where R15, R16And R17Are independently H, linear, branched or cyclic alkyl, polyethylene glycol units (-OCH) with 1-10 carbon atoms.2CH2) N, where n is chosen from integers from 1 to 2000; R6Is OR, SR or NRR', where R and R'have the same definition as above; X'is CH2, NR, CO, BH, SO or SO2Selected from, where R has the same definition as above; Y'is O, CH2, NR or S, where R has the same definition as above; Z'is CH2Or (CH2)nWhere n is 2, 3 or 4, but X', Y'and Z'are all CH at the same time.2Is never; A and A'are the same or different, and O, -CRR'O, S, -CRR'S, -NR15Or CRR'NHR15Selected from, where R and R'have the same definition as above, and R15Has the same definition as above for R; D and D'are the same or different, and are linear, branched or cyclic alkyl, alkenyl or alkynyl with 1-10 carbon atoms independently, optionally halogen, OR.7, NR8R9, NO2, NRCOR', SR10, Sulfoxide, indicated by SOR', -SO2Sulfone, Sulfite-SO, indicated by R'3, Bisle Fight-OSO3, SO2Sulfonamide, cyano, azide, -COR indicated by NRR'11, OCOR11Or OCONR11R12Replaced by any one of, R7, R8, R9, R10, R11And R12The definition of is as above, polyethylene glycol unit (-OCH)2CH2)n, But where n is an integer from 1 to 2000, selected from; L is an optionally substituted heterocycle with any phenyl group or 3-10 carbon atoms, where the substituent is a linking group that allows ligation to the cell binding agent via a covalent bond. Alternatively, a linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms, optionally halogen, OR.7, NR8R9, NO2, NRCOR', SR10, Sulfoxide, indicated by SOR', -SO2Sulfone, Sulfite-SO, indicated by R'3, Bisle Fight-OSO3, SO2Sulfonamide, cyano, azide, -COR indicated by NRR'11, OCOR11Or OCONR11R12Replaced by any one of, R7, R8, R9, R10, R11And R12The definition of is as above, polyethylene glycol unit (-OCH)2CH2)n, But where n is an integer from 1 to 2000, selected from; in some cases L itself is a linking group that allows ligation to the cell binding agent via covalent bonds] compounds, or theirs. Includes pharmaceutically acceptable solvates, salts, hydrates, or hydrous salts, their optical isomers, racemic mixtures, diastereomers, enantiomers or polymorphic crystal structures of these compounds, wherein 2 Although the compound of the metric optionally carries a linking group that allows ligation to the cell binding agent; however, the compound has more than one linking group that allows ligation of the cell binding agent via a covalent bond. I don't have it.

0184Double line between N and C on one side--Indicates a single bond or a double bond, but if it is a double bond X is absent, Y is H, and if it is a single bond X is H or An amino-protected moiety that converts a compound into a prodrug; Y is -OR, NR'R , Sulfite-SO3Or Bisle Fight-OSO3Selected from, where R is H, a linear, branched or cyclic alkyl, alkenyl or alkynyl, polyethylene glycol unit with 1-10 carbon atoms (-OCH).2CH2)nWhere n is an integer from 1 to 2000, selected from aryls with 6 to 10 carbon atoms, heterocycles with 3 to 10 carbon atoms; W is C = O, CH2Or SO2Is; R1, R2, R3, R4, R1', R2', R3'And R4'Is H, NO independently2Or selected from linking groups that allow ligation to the cell binder via covalent bonds; R6Is OR18And here R18Has the same definition as R; Z is (CH2)nHowever, n is 1, 2 or 3, CR15R16, NR17, O or S, where R15, R16And R17Are independently H, linear, branched or cyclic alkyl, polyethylene glycol units (-OCH) with 1-10 carbon atoms.2CH2) N, where n is chosen from integers from 1 to 2000; X'is CH2Or selected from C = O; Y'is O, NR or S, where R has the same definition as above; Z'is CH2Or (CH2)2Is; A and A'are O respectively; D and D'are selected from straight, branched or cyclic alkyl, alkenyl or alkynyl, which are the same or different and independently have 1-10 carbon atoms; L is an optionally substituted heterocycle with any phenyl group or 3-10 carbon atoms, where the substituent is a linking group that allows ligation to the cell binding agent via a covalent bond. Alternatively, a linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms, optionally halogen, OR.7, NR8R9, NO2, NRCOR', SR10, Sulfoxide, indicated by SOR', -SO2Sulfone, Sulfite-SO, indicated by R'3, Bisle Fight-OSO3, SO2Sulfonamide, cyano, azide, -COR indicated by NRR'11, OCOR11Or OCONR11R12Polyethylene glycol unit (-OCH) substituted with any one of2CH2)n, Where n is selected from those integers from 1 to 2000; in some cases L itself is a linking group that allows ligation to the cell binding agent via covalent bonds; or their pharmaceuticals. The above are acceptable solvates, salts, hydrates, or hydrous salts, their optical isomers, racemic mixtures, diastereomers, enantiomers or polymorphic crystal structures of these compounds.

0185In another feature, the compound has the following formula (XVII):<chemistry num="15"><img id="000017" he="39" wi="139" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>[Double line between N and C in the formula--Indicates a single bond or a double bond, but if it is a double bond X is absent, Y is H, and if it is a single bond X is H or An amino-protected moiety that converts a compound into a prodrug, and Y is OH, an ether represented by -OR, sulfite-SO.3, Or Bisle Fight-OSO3Selected from, where R is selected from linear, branched or cyclic alkyl, alkenyl or alkynyl with 1-10 carbon atoms; One of R2 and R3 is a linking group that allows ligation to a cell binder via a covalent bond, and the other is H; One of L', L "or L'" is a linking group that allows ligation to a cell binder, the remainder is H; L'can be a linking group, and G is CH or Is N]. Other examples are described in US patent application 61 / 150,201, the entire contents of which are incorporated herein by reference. That is, in certain embodiments, the antibody huMov19 is conjugated to a benzodiazepine having the structure shown in XIX-XXII. In another embodiment, antibody FR-1-21 is conjugated to a benzodiazepine having the structure shown in XIX-XXII above.

0186IV. Polynucleotide In certain embodiments, the present invention includes polynucleotides that specifically bind to FOLR1 or that include a polynucleotide encoding a fragment of such a polypeptide. For example, the present invention provides a polynucleotide comprising a nucleic acid sequence that encodes an antibody against human FOLR1 or encodes a fragment of such an antibody. The polynucleotides of the invention can be in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and if it is single-stranded, it can be a coding strand or a non-coding (antisense) strand. it can.

0187In certain embodiments, the polynucleotide has been isolated. In certain embodiments, the polynucleotide is substantially pure.

0188The present invention provides a polynucleotide comprising a polynucleotide encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 4, 10, 11, 41, 42 and 88-103. Also provided are sequences of at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% in SEQ ID NOs: 4, 10, 11, 41, 42 and 88-103. A polynucleotide encoding a polypeptide having identity.

0189Polynucleotide SEQ ID NOs: 5, 14 and 15 contain coding sequences for huMov19 variable domain heavy chain, variable domain light chain version 1.00 and variable domain light chain version 1.60, respectively.

0190The present invention also provides polynucleotides comprising sequences selected from the group consisting of SEQ ID NOs: 5, 14, 15, 37, 38, 43, 44, 47, 48 and 120-127. Also provided are SEQ ID NOs: 5, 14, 15, 37, 38, 43, 44, 47, 48 and 120-127 at least about 95%, at least about 96%, at least about 97%, at least about 98%. , Or a polynucleotide having at least about 99% sequence identity. That is, in certain embodiments, the polynucleotide is (a) a polynucleotide having at least about 95% sequence identity to SEQ ID NO: 5; and / or (b) at least about 95% sequence to SEQ ID NO: 14 or 15. Contains polynucleotides with identity. In certain embodiments, the polynucleotide comprises (a) a polynucleotide having the amino acid sequence of SEQ ID NO: 5; and / or (b) a polynucleotide having the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

0191In certain embodiments, the polynucleotide comprises a coding sequence for a mature polypeptide fused to the polynucleotide in the same reading frame, eg, supporting expression and secretion of the polypeptide from a host cell (eg, a polypeptide from a cell). Leader sequence that acts as a secretory sequence to control the transport of). A polypeptide having a leader sequence is a preprotein and can have a leader sequence that is cleaved by a host cell to form a mature form of the polypeptide. Polynucleotides can also be encoded for mature proteins plus proproteins, which are additional 5'amino acid residues. A mature protein with a pro sequence is a proprotein and is an inactive form of the protein. When the pro sequence is cleaved, the active mature protein remains.

0192In certain embodiments, the polynucleotide comprises, for example, a coding sequence for a mature polypeptide fused in the same reading frame with a marker sequence that allows purification of the encoded polypeptide. For example, the marker sequence can be a hemagglutinin tag provided by the pQE-9 vector that allows purification of the mature polypeptide fused to the marker in the case of a bacterial host, or a mammalian host (eg COS-7 cells). ) Can be a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein.

0193The present invention further relates to variants of the lower polynucleotides encoding fragments, analogs and derivatives, for example.

0194Polynucleotide variants can contain modifications in the coding and / or non-coding regions. In some embodiments, the polynucleotide variant contains a modification that results in a silent substitution, loading, or deletion, but does not alter the properties or activity of the encoded polypeptide. In some embodiments, nucleotide variants are generated by silent substitution by degeneracy of the genetic code. Polynucleotide variants are produced for a variety of reasons, for example to optimize codon expression for a particular host (changing codons in human mRNA to those preferred by bacterial hosts such as E. coli). You can also do it.

0195Vectors and cells containing the polynucleotides described herein are also provided.

0196V. Usage and pharmaceutical composition The FOLR1 binders of the invention (including antibodies, immunoconjugates and polypeptides) are useful in a variety of applications including, but not limited to, therapeutic methods such as the treatment of cancer. In certain embodiments, the agent is useful for inhibiting tumor growth, inducing differentiation, reducing tumor volume, and / or reducing tumorigenicity of tumors. The method of use may be in vitro, ex vivo, or in vivo. In certain embodiments, the FOLR1 binder or antibody or immunoconjugate or polypeptide is an antagonist of human FOLR1 to which it binds.

0197In one aspect, the anti-FOLR1 antibodies and immunoconjugates of the present invention are useful for detecting the presence of FOLR1 in biological samples. The term "detect", as used herein, includes quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues. In certain embodiments, such tissues are normal and / or cancerous tissues that express FOLR1 at higher levels relative to other tissues, such as B cells and / or B cell-related tissues. Include. In certain embodiments, overexpression of FOL R1 detects the presence of ovarian cancer, lung cancer, brain cancer, breast cancer, uterine cancer, kidney cancer or pancreatic cancer.

0198In one aspect, the invention provides a method of detecting the presence of FOLR1 in a biological sample, in certain embodiments, the method is under conditions that allow binding of an anti-FOLR1 antibody to FOLR1. It involves contacting the anti-FOLR1 antibody with a biological sample and detecting whether a complex is formed between the anti-FOLR1 antibody and FOLR1.

0199In one aspect, the invention provides a method of diagnosing disorders associated with increased expression of FOLR1. In certain embodiments, the method is to contact the test cell with the anti-FOLR1 antibody; by detecting the binding of the anti-FOLR1 antibody to FOLR1, the level of expression of FOLR1 by the test cell (quantitatively or qualitatively). ) Measure; and the level of FOLR1 expression by the test cell by the control cell (eg, normal cells of the same tissue origin as the test cell, or cells expressing FOLR1 at levels comparable to such normal cells). Higher levels of FOLR1 expression by test cells when compared to control cells, including comparing to levels of FOLR1 expression, indicate the presence of disorders associated with increased expression of FOLR1. In certain embodiments, test cells are obtained from individuals suspected of having a disorder associated with increased expression of FOLR1. In certain embodiments, the disorder is a cell proliferation disorder, such as cancer or tumor.

0200In certain embodiments, methods of diagnosis or detection, such as those described above, are membrane preparations obtained from cells that are expressed on the surface of the cell or that are expressing FOLR1 on their surface. Includes detecting the binding of anti-FOLR1 antibody to FOLR1 in. In certain embodiments, the method involves contacting the cell with the anti-FOLR1 antibody under conditions that allow binding of the anti-FOLR1 antibody to FOLR1, and combining between the anti-FOLR1 antibody and FOLR1 on the cell surface. Includes detecting if a body is formed. An exemplary assay for detecting the binding of an anti-FOLR1 antibody to FOLR1 expressed on the cell surface is the "FACS" assay.

0201Binding of anti-FOLR1 antibody to FOLR1 can be detected using a specific alternative method. Such methods include well-known antigen binding tests in the art, such as Western blots, radioimmunoassays, ELISAs (enzyme-linked immunosorbent assay). Includes, but is not limited to, "sandwich" immunoassays, immunoprecipitation assays, fluorescent immunoassays, protein A immunoassays and immunohistochemistry (IHC).

0202In certain embodiments, the anti-FOLR1 antibody is labeled. Labels are labels or moieties that are directly detected (eg, fluorescence, chromophore, electron density, chemical luminescent, and radioactive labels) and enzymes or moieties that are indirectly detected, such as through enzymatic reactions or molecular interactions. Includes, but is not limited to, ligand-like parts.

0203In certain embodiments, the anti-FOLR1 antibody is immobilized on an insoluble matrix. Immobilization means isolating the anti-FOLR1 antibody from any FOLR1 that remains free in solution. This has traditionally been anti-FOLR1 prior to assay operation, such as by adsorption on a water-insoluble matrix or surface (Bennich et al., US Pat. No. 3,720,760) or by covalent coupling (eg using glutaraldehyde cross-linking). It is achieved either by insolubilizing the antibody or, for example, by insolubilizing the anti-FOLR1 antibody after the formation of a complex between the anti-FOLR1 antibody and FOLR1 by immunoprecipitation.

0204Any of the above embodiments of diagnosis or detection may be performed in place of or in addition to the anti-FOLR1 antibody, using the immunoconjugates of the present invention.

0205In certain embodiments, the disease treated with a FOLR1 binder or antagonist (eg, huMov19 antibody or immunoconjugate) is cancer. In certain embodiments, the cancer is to whom a FOLR1 binder (eg, an antibody) binds. It is characterized by a tumor that expresses a forate receptor 1.

0206The present invention provides a method of treating cancer, comprising administering to a subject (eg, a subject in need of treatment) a therapeutically effective amount of a FOLR1 binder. In certain embodiments, the cancers are colonic rectal cancer, pancreatic cancer, lung cancer, ovarian cancer, liver cancer, breast cancer, brain cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, bladder cancer, nerves. It is selected from the group consisting of glioblastoma and cancer of the head and neck. In certain embodiments, the cancer is ovarian cancer. In certain embodiments, the cancer is lung cancer. In certain embodiments, the subject is a human.

0207The present invention further provides methods for inhibiting tumor growth using the antibodies or other agents described herein. In certain embodiments, the method of inhibiting tumor growth involves contacting cells with a FOLR1 binder (eg, an antibody) in vitro. For example, an immortal cell line or cancer cell expressing FOLR1 is cultured in a medium, and an antibody or other agent is added thereto to inhibit tumor growth. In some embodiments, tumor cells are isolated from a patient sample, such as a biopsy tissue, pleural effusion or blood sample, cultured in medium and added with a FOL R1 binding agent to inhibit tumor growth.

0208In some embodiments, the method of inhibiting tumor growth involves contacting a tumor or tumor cell with a FOLR1 binder (eg, an antibody) in vivo. In certain embodiments, contacting the tumor or tumor cells with the FOLR1 binder is performed in an animal model. Tumor growth can be inhibited, for example, by administering a FOLR1 binder to a xenograft that expresses one or more FOLR1 grown in immunocompromised mice (eg, NOD / SCID mice). In some embodiments, cancer stem cells are isolated from a patient sample, such as biopsy tissue, pleural effusion or blood sample, injected into immunocompromised mice, and then administered with a FOLR1 binding agent to inhibit tumor cell growth. To do. In some embodiments, the FOLR1 binder prevents tumor growth by administration at the same time as or immediately after the introduction of tumorigenic cells into the animal. In some embodiments, the FOL R1 binder is administered as a therapeutic agent after the tumorigenic cells have grown to a certain size.

0209In certain embodiments, the method of inhibiting tumor growth comprises administering to the subject a therapeutically effective amount of a FOLR1 binder. In certain embodiments, the subject is a human. In certain embodiments, the subject has or has had a tumor removed.

0210In certain embodiments, the tumor expresses a forate receptor to which a FOLR1 binder or antibody binds. In certain embodiments, the tumor overexpresses human FOLR1.

0211In certain embodiments, the tumors are brain cancer, colorectal tumor, pancreatic tumor, lung tumor, ovarian tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, gastrointestinal tumor, melanoma, cervical tumor, bladder tumor. , Glioblastoma, and head and neck tumors. In certain embodiments, the tumor is an ovarian tumor.

0212Furthermore, the present invention provides a method of reducing the tumorigenicity of a tumor in a subject, including administering to the subject a therapeutically effective amount of a FOLR1 binder. In certain embodiments, the tumor comprises cancer stem cells. In certain embodiments, the incidence of cancer stem cells in tumors is reduced by administration of the agent.

0213That is, in certain embodiments, the present invention provides a method of treating cancer with a huMov19 antibody and an immunoconjugate. In certain embodiments, the huMov19 immunoconjugate is huMov19-SPDB-DM4; huMov19-sulfo-SPP-DM1; huMov19-SPP-DM1; or huMov19-PEG4-Mal-DM4.

0214The present invention further provides a method of culture of tumorigenic cells to non-tumorogenic cells, including contacting tumorigenic cells with a FOLR1 binding agent (eg, having a tumor containing tumorigenic cells or By administering a FOLR1 binding agent to subjects who have had such tumors removed). In certain embodiments, the tumorigenic cells are ovarian tumor cells.

0215The present invention also provides a method of reducing the activation of myofibroblasts in an effective amount of a FOL R1 binder, polypeptide or antibody, including contacting the support of a solid tumor.

0216The present invention further provides a pharmaceutical composition comprising one or more of the FOLR1 binders described herein. In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable vehicle. These pharmaceutical compositions can be used to inhibit tumor growth and treat cancer in human patients.

0217In certain embodiments, the pharmaceutical product is prepared for storage and use by mixing a pharmaceutically acceptable vehicle (eg, carrier, excipient) with the purified antibody or agent of the invention. , The Science and Practice of Pharmacy 20th Edition Mack Publishing, 2000). Suitable pharmaceutically acceptable vehiclesNon-toxic buffers such as phosphates, citrates and other organic acids; salts such as sodium chloride; antioxidants such as ascorbic acid and methionine; preservatives (eg octadecyldimethylbenzylammonium chloride; hexamethonium chloride; Benzalconium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (eg about 10) Less than amino acid residues); proteins such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates such as monosaccharides, disaccharides , Glucose, mannose, dextrin; chelate-forming agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and nonionic surface activity. Agents such as, but not limited to, TWEEN or polyethylene glycol (PEG).

0218The pharmaceutical compositions of the present invention can be administered in any number of ways for either topical or systemic administration. Administration is topical (eg, transmucosal including transvaginal and anal delivery), such as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; lungs (eg powder or aerosol inhalation or Absorption, eg by nebulizer; intratracheal, intranasal, epidermal and transdermal); oral; or non-enteral, eg intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial (eg intracranial) Can be intrathecal or intraventricular) administration.

0219The immunoconjugate antibody of the present invention can be combined with a second compound having anti-cancer properties in a pharmaceutical complex or a dosage regimen as a combination therapy. The second compound of the pharmaceutical complex or dosage regimen preferably has complementary activity on the ADC of the complex so that they do not adversely affect each other. Pharmaceutical compositions comprising a FOLR1 binder and a second anti-cancer agent are also provided.

0220For the treatment of disease, the appropriate dose of the antibody or agent of the invention is the type of disease to be treated, the severity and course of the disease, the responsiveness of the disease, whether the antibody or agent is for therapeutic or prophylactic purposes. It will be administered, or it will depend on the previous treatment, the patient's medical history, etc., all at the discretion of the doctor in charge. Antibodies or agents span a single treatment or a series of treatments that last from days to months, or until cure is successful or disease reduction (eg, reduction in tumor size) is achieved. Can be administered. The optimal dosing regimen can be calculated from the measurement of drug accumulation in the patient's body and will vary depending on the relative titer of the individual antibody or agent. The dosing physician can easily determine the optimal dose, dosing regimen and repeat rate. In certain embodiments, the dose is 0.01 μg-100 mg / kg body weight and can be administered daily, weekly, monthly or at least once annually. In certain embodiments, the antibody or other FOLR1 binder is administered once every two weeks or once every three weeks. In certain embodiments, the dose of antibody or other FOLR1 binder is from about 0.1 mg to about 20 mg / kg body weight. The doctor in charge can estimate the repeat rate of dosing based on the measured values of the residence time and concentration of the drug in the body fluid or tissue.

0221Combined therapies can produce "synergistic effects" and have been found to be "synergistic", i.e., when the effects achieved when the active ingredients are used together are when the compounds are used separately. Greater than the sum of the resulting effects. The synergistic effect is whether the active ingredients are (1) co-formulated and co-administered or delivered in a complex unit dosage form; (2) alternately or in parallel as separate formulations; or (3) some This can be achieved with other dosage forms. When delivered in alternating therapy, synergies can be achieved if the compounds are administered or delivered sequentially, eg, by different injections in separate syringes. Generally, during alternate therapy, the effective doses of each active ingredient are administered sequentially, i.e. consecutively, but in the case of combination therapy, the effective doses of two or more active ingredients are administered together.

0222Kit containing VI.FOLR1 binder The present invention provides a kit that includes an antibody, immunoconjugate or other agent described herein and can be used to carry out the methods described herein. In certain embodiments, the kit comprises at least one purified antibody against human forate receptor 1 in one or more containers. In some embodiments, the kit is required to perform a detection assay that includes all of the controls, instructions for performing the assay, and any required software for analysis and display of the results. Or it contains all of the sufficient ingredients. Those skilled in the art know that the disclosed antibodies, immunoconjugates and other agents of the present invention can be readily incorporated into one of the well-known and established kit formats in the art.

0223Further provided is a kit containing a FOLR1-binding agent (eg, FOLR1-binding antibody) and a second anti-cancer agent. In certain embodiments, the second anti-cancer agent is a chemotherapeutic agent (eg gemcitabine or irinotecan).

0224Embodiments of the invention can be further clarified by reference to the following non-limiting examples, which describe in detail the preparation of the specific antibodies of the present disclosure and the methods for using the antibodies of the present disclosure. .. It is known to those skilled in the art that many changes to both materials and methods can be made without departing from the scope of this disclosure.

The examples and embodiments described herein are for illustration purposes only, and various modifications or alterations based therein will be suggested to those skilled in the art and are within the spirit and scope of the present application. It must be understood that it should be. (Example 1) Chimerization of the murine monoclonal antibody Mov19 The variable region amino acid sequence of Mov19 was obtained from the NCBI database (Axon CAA68253 for light chain (SEQ ID NO: 24) and CAA68252 for heavy chain (SEQ ID NO: 23)) and then codon-optimized to Blue Heron Biotechnology. Was synthesized by. The light chain variable region was cloned into the EcoRI and BsiWI sites in the pAbKZeo plasmid, and the heavy chain variable region was cloned into the HindIII and Apa1 sites in the pAbG1Neo plasmid. (Example 2) Humanized Mov19 antibodies of the murine monoclonal antibodies Mov19 and FR1-21 have been previously reported by framework resurfacing methods (Roguska M. et al., Proc. Natl. Acad. Sci.USA 1994 Feb; 91: 969-973) and Humanized according to (Roguska et al., Protein Eng. 9 (10): 895-904 (1996)). If desired, mean solvent accessibility for each variable region framework residue was calculated using the closely related solved antibody structure in the PDB database, and positions above 30% mean accessibility were surfaced. Marked as a residue (Pedersen JT et al., J. Mol. Biol. 1994; 235: 959-973). Human surface replacement sequences were selected by aligning the surface position of the murine antibody sequence to the corresponding position of the human antibody germ cell lineage sequence in the Kabat database (Johnson, G. and Wu, TT). (2001) Nucleic Acids Research, 29: 205-206). The most homologous human light chain variable region surface (IMGT locus IGKV2D-30 * 01 for clone DPK19, Mov19, and IMGT locus IGKV1 / OR2-0 * 01 for FR1-21) and the most homologous human heavy chain variable Replace the murine Mov19 framework surface position by selecting the region surface (IMGT locus IGHV1-69 * 08 for clones 8M27, Mov18, and IMGT locus IGHV5-51 * 02 for FR1-21 for FR1-21). , 6CDR (Table 1) remained unmodified. The surface positions and residues of murine and human Mov19 and FR1-21 are shown in FIGS. 1A to 1D.<tables num="1-1"><img id="000018" he="232" wi="134" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></tables><tables num="1-2"><img id="000019" he="48" wi="134" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></tables> None of the residue changes pose the problem of affecting the interaction of the CDRs of Mov19 or FR1-21 with their target epitopes on forate receptor 1, so surface reversion mutations of any antibody. No consideration was given to the humanized sequence. However, the resurfed Mov19 sequence introduced a consensus N-linked glycosylation site in light chain N74 (light chain version 1.00), thus creating a second humanized light chain version to eliminate this site. Examination of Kabat's human light chain sequence database revealed that threonine was the most common residue observed at position 74 of the light chain, and thus humanized Mov19 light chain version 1.60 to have threonine at position 74. Was built. Since position 74 is not a surface residue, this residue substitution does not affect humanization by resurfing. The alignment of the variable region sequences of murine and humanized Mov19 and FR1-21 is shown in FIG. Codon-optimized variable region sequences of humanized Mov19 and FR1-21, Blue Heron Synthesized by Biotechnology. The sequences are flanked by the appropriate constant sequences in the single-stranded mammalian expression plasmid and restriction enzyme sites to facilitate in-frame cloning. The light chain variable region was cloned into the EcoRI and BsiWI sites in the pAbKZeo plasmid. The resulting plasmid DNA encoding the huMov19 light chain was deposited with ATCC as ATCC Accession Nos. PTA-10773 and PTA-10774, and the resulting plasmid DNA encoding the FR1-21 light chain was deposited as ATCC Accession No. PTA-10776. Deposited. The heavy chain variable region was cloned into the HindIII and Apa1 sites of the pAbG1 Neo plasmid. The resulting plasmid DNA encoding the huMov19 heavy chain was deposited with ATCC under ATCC Accession No. PTA-10772, and the resulting plasmid DNA encoding the FR1-21 heavy chain was deposited under ATCC Accession No. PTA-10775. Next, huMov19 was prepared by transfecting these plasmids as described in Example 3. Methods described herein by pairing a plasmid encoding any huMov19 strain (ie, deposited as ATCC Accession Nos. PTA-10773 and PTA-10774) with a plasmid encoding the huMov19 heavy chain. According to, and as is well known in the art, the huMov19 antibody can be made. (Example 3) Recombinant antibody expression Suspended HEK-293T cells with chimeric and humanized antibody constructs using standard calcium phosphate manipulation methods (BD Biosciences, CalPhos Milk Transfection Kit, Catalog No. 631312) or using modified PEI methods. In 293T cells (Durocher Y, Perret S, Kamen A High-level and high-throughputrecombinant protein production by transient transfection of suspension-growinghuman 293-EBNA1 cells. Nucleic Acids Res. 2002 Jan 15; 30 (2): E9) Transiently prepared in a spinner flask. PEI transient transfection has been previously reported, except that this time HEK-293T cells were grown in Freestyle 298 (Invitrogen) and the culture volume remained undiluted after the addition of the PEI-DNA complex. It was carried out as per (Durocher, Y. et al., Nucleic Acids Res. 30 (2): E9 (2002)). Both attachment and transient transfection of the suspension were incubated for 1 week, and then the clarified supernatant was purified by Protein A column followed by CM column ion exchange chromatography as described below. As shown in FIG. 3, the expression of huMov19 was at least 10-fold higher than that of chimeric Mov19 in the transfected cells. (Example 4) Antibody purification Antibodies were purified from clarified cell culture supernatants using standard methods such as protein A or G chromatography (HiTrap protein A or GHP, 1 mL, Amersham Biosciences). If desired, the supernatant was prepared for chromatography by adding 1/10 volume of 1M Tris / HCl buffer, pH 8.0. The pH controlled supernatant was filtered through a 0.22 μm membrane filter and loaded onto a column equilibrated with binding buffer (PBS, pH 7.3). The column was washed with binding buffer until a stable baseline with no absorbance at 280 nm was obtained. The antibody was eluted with 0.15 M NaCl-containing 0.1 M acetate buffer, pH 2.8, using a flow rate of 0.5 mL / min. A fraction of about 0.25 mL was taken and neutralized by adding 1/10 volume of 1M Tris / HCl, pH 8.0. The peak fraction was dialyzed against 1xPBS twice overnight and sterilized by filtration through a 0.2 μm membrane filter. Purified antibody is A280It was quantified by the absorbance of. The protein A purified fraction was further purified by ion exchange chromatography (IEX) by carboxymethyl (CM) chromatography. If desired, samples from Protein A purification were buffered in binding buffer (10 mM calcium phosphate, 10 mM sodium chloride, pH 7.5) and filtered through a 0.22 μm filter. The prepared samples were then loaded onto CM First Flow Resins (GE Lifesciences) equilibrated with starting buffer at a flow rate of 120 cm / hr. The column size was chosen to have sufficient capacity to bind all antibodies in the sample. The column was then washed with binding buffer until a stable baseline with no absorbance at 280 nm was obtained. Antibodies were eluted by initiating a gradient of 10 mM to 500 mM sodium chloride in 20 column volumes (CV). Fractions with UV readings above 50 mAu of the tumor peak were collected. Purity (percentage of monomeric and soluble high molecular weight agglutinates) is available on the Agilent HPLC 1100 System (Agilent, Santa). Evaluation was performed by size exclusion chromatography (SEC) on TSKgel G3000 SWXL, 7.8x300 mm using SWXL guard column, 6.0x40 mm (Tosoh Bioscience, Montgomeryville, PA) using Clala, CA). Fractions of the desired purity (> 95%) were pooled, buffer exchanged with PBS (pH 7.4) using the TFF system, and sterilized by filtration through a 0.2 μm membrane filter. Purified antibodies were further tested by the SEC for their purity and IgG concentrations were determined by absorbance measurements at 280 nm using an extinction factor of 1.47. Dilution was performed as needed. Alternatively, ceramic hydroxyapatite (CHT) can also be used for final purification of both murine and humanized antibodies with good selectivity. Type II CHT resin with a particle size of 40 μm (BioRad Laboratories) was also applied to the final purification of the antibody using a protocol similar to that described for IEX chromatography. The starting buffer for CHT was 20 mM sodium phosphate, pH 7.0, and the antibody was eluted with a gradient of 20-160 mM sodium phosphate over 20 CV. (Example 5) Development of anti-FOLR1 antibody for mice Two different immunization / screening series were used. The first series resulted in the formation of FR1-21 clones, and the second series resulted in the formation of FR1-48, FR1-49, FR1-57 and FR1-65 clones. In the first series, the mouse is about 5x106Subcutaneous immunization was performed with FOLR1-expressing KB cells (American Tissue Culture Collection, ATCC CCL-17). In the second series, mice were immunized with 300-19 cells expressing human FOLR1 on their surface. To generate these cells, the human FOL R1 amino acid sequence was obtained from the NCBI website (Axon NP_057937), then codon-optimized, synthesized by Blue Heron Biotechnology, and flanked by EcoRI and XbaI restriction sites. Facilitated cloning into the pSRa mammalian expression vector. Pre-B cell lineage derived from 300-19 cells, ie Balb / c mice (Reth et al., Nature, 317: By transfecting 353-355 (1985)) with a pSRa-FolR1 expression plasmid, huFOLR1 was stably expressed at high levels on the cell surface. Standard immunization protocols known in the art, such as those used in ImmunoGen, Inc, were applied to both series. Immunized mice were boosted with antigen 3 days prior to sacrifice for hybridoma formation. A single cell suspension in RPMI-1640 medium was obtained by grinding the spleen of mice according to standard animal protocols, eg, between two sterile frosted microscope slides. Spleen cells are centrifuged, pelleted, washed and fused with murine mieroma, eg P3X63Ag8.653 cells (Kearney et al., J Immunol, 123: 1548-1550 (1979)) using polyethylene glycol-1500 (Roche783641). I let you. Fusion cells were resuspended in RPMI-1640 selective medium containing hypoxanthine-aminopterin-thymidine (HAT) (Sigma H-0262), and 37 ° C 5% CO2Selected for growth in a 96-well flat bottom culture plate (Corning-Costar 3596, 0.2 mL cell suspension per well) below. After incubation for 5 days, 0.1 mL of culture supernatant was removed from each well and replaced with 0.1 mL of RPMI-1640 medium containing a hypoxanthine-thymidine (HT) supplement (Sigma H-0137). 37 ° C 5% CO until hybridoma clones can be used for antibody screening2Incubation was continued below. Other techniques for immunization and hybridoma production, such as Langone et al. (Edited "Immunochemical Techniques, Part I", Methods in Enzymology, Academic Press, volume 121, Florida) and Harlow et al., ("Antibodies: A Laboratory Manual"; Cold The one described in Spring Harbor Laboratory Press, New York (1988)) can also be used.<tables num="1-3"><img id="000020" he="228" wi="150" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></tables> (Example 6) Hybridoma screening and selection FOLR1-300-19 cells and KB cells transfected with human FOLR1 were used for the first and second series of screens, respectively. Hybridoma culture supernatant by flow cytometry for the secretion of mouse monoclonal antibodies that bind to FOLR1-positive cells, such as FOLR1-expressing 300-19 or KB cells, but not to FOLR1-negative cells, such as non-transfected 300-19 cells. Was screened. FOLR1-positive cells or non-transfected 300-19 cells (1x10 per sample) in 0.1 mL of hybridoma supernatant in 0.1 mL FACS buffer (RPMI-1640 medium supplemented with 2% normal goat serum)5Incubated for 3 hours with any of the). The cells were then centrifuged, pelleted, washed and incubated with 0.1 mL of PE-conjugated goat anti-mouse IgG antibody (eg, available from Jackson Laboratory, 6 μg / ml in FACS buffer) for 1 hour. Cells were centrifuged, pelleted again, washed with FACS buffer and resuspended in 0.2 mL of PBS containing 1% formaldehyde. Cell-associative fluorescence was measured using FACSCalibur flow cytometry or FACS array flow cytometry with an HTS multiwell sampler, and CellQuest Pro (all sources are BD). Analysis was performed using Biosciences, San Diego, US). Hybridoma clones with positive findings were subcloned by limiting dilution. One subclone was selected from each hybridoma that showed the same reactivity to FOLR1 as the parent cell by flow cytometry and submitted for subsequent analysis. Stable subclones were cultured and the isotypes of each secreted anti-FOLR1 antibody were identified using a commercially available isotyping reagent (Roche 1493027). The murine antibody was purified from protein A from the hybridoma medium clarified as described above. These antibodies were referred to as FR-1 antibodies. (Example 7) Purification of murine monoclonal antibody Antibodies were purified from hybridoma subclone supernatants using standard methods such as protein A or G chromatography (HiTrap protein A or GHP, 1 mL, Amersham Biosciences). If desired, the supernatant was prepared for chromatography by adding 1/10 volume of 1M Tris / HCl buffer, pH 8.0. The pH controlled supernatant was filtered through a 0.22 μm membrane filter and loaded onto a column equilibrated with binding buffer (PBS, pH 7.3). The column was washed with binding buffer until a stable baseline with no absorbance at 280 nm was obtained. The antibody was eluted with 0.15 M NaCl-containing 0.1 M acetate buffer, pH 2.8, using a flow rate of 0.5 mL / min. A fraction of about 0.25 mL was taken and neutralized by adding 1/10 volume of 1M Tris / HCl, pH 8.0. The peak fraction was dialyzed against 1xPBS twice overnight and sterilized by filtration through a 0.2 μm membrane filter. The purified antibody was quantified by the absorbance of A280. (Example 8) Binding characterization by flow cytometry The binding specificity was tested by flow cytometry using the purified antibody. Figure 4 shows a FACS histogram showing no binding of anti-FOLR1 to FOLR1 expression 300-19 cells and no binding to parent 300-19 cells. FOLR1-expressing 300-19 cells or non-transfected 300-19 cells (cells 1x10 per sample) in 0.1 mL FACS buffer (RPMI-1640 medium supplemented with 2% normal goat serum)5Each antibody was incubated with any of the) for 3 hours. The cells were then pelleted, washed and incubated with 0.1 mL of FITC-conjugated goat anti-mouse IgG antibody (eg, available from Jackson Laboratory, 6 μg / ml in FACS buffer) for 1 hour. The cells were pelleted again, washed with FACS buffer and resuspended in 200 μL of PBS containing 1% formaldehyde. Samples were obtained using FACSCalibur flow cytometry or FACS array flow cytometry with an HTS multiwell sampler and analyzed using CellQuest Pro (all sources from BD Biosciences, San Diego, US). The FACS histogram of the anti-FOLR1 antibody showed a shift in fluorescence, but not the parent 300-19 cells. No significant fluorescence shift was detected when any of the cell lines were incubated with FITC-conjugated goat anti-human IgG antibody alone. (Example 9) Cloning and sequencing of VL and VH regions of muFR1-21 5x10 using R Easy kit (QIAgen) according to the manufacturer's protocol6Whole cell RNA was prepared from hybridoma cells. Then, cDNA was synthesized from total RNA using the SuperScript II cDNA synthesis kit (Invitrogen). Procedures for the first round of denatured PCR reactions on cDNA derived from hybridoma cells are Wang et al., ((2000) J Immunol Methods. Jan 13; 233 (1-2): 167-77) and Co et al., Etc. ((1992) J Immunol. Feb 15; 148 (4): 1149-54) was based on the method described. The VH sequence was amplified by PCR using the following denatured primers: EcoMH1CTTCCGGAATTCSARGTNMAGCTGSAGSAGTC (SEQ ID NO: 50), EcoMH2CTTCCGGAATTCSARGTNMAGCTGSAGSAGTCWGG (SEQ ID NO: 51) and BamIgG1GGAGGATCCATAGACAGATGGGGGTGTCGTTTTGGC (SEQ ID NO: 52). The VL sequence was amplified by PCR using the following denatured primers: SacIMKGGAGCTCGAYATTGTGMTSACMCARWCTMCA (SEQ ID NO: 53) and HindKLTATAGAGCTCAAGCTTGGATGGTGGGAAGATGGATACAGTTGGTGC (SEQ ID NO: 54). (Mixed bases are as follows: N = G + A + T + C, S = G + C, Y = C + T, M = A + C, R = A + G, W = A + T Defined). The PCR reaction mixture was then run on a 1% low melting point agarose gel, the 300-400 bp band was excised, purified using a ZymoDNA minicolumn, and sent to Agencourt Biosciences for sequencing. Variable region cDNA was formed from both directions by using the respective 5'and 3'PCR primers as sequencing primers. The amino acid sequences of the VH and VL regions were obtained by interpreting the results of DNA sequencing using Vector NTI software. NCBI's IgBlast site (http://www.ncbi.nlm.nih.gov/igblast/) was used to discover artificial factors for 5'end primer sequencing in preliminary variable region cDNA sequences. We searched for the sequence of the murine germ cell lineage from which it was derived. The full-length murine antibody sequence predicted by combining the clarified variable region sequence with the NCBI reference sequence for the constant region of a particular antibody was then constructed. The predicted molecular weights of the rat Fr1-21 light and heavy chains were then calculated and compared to the mass measured by liquid chromatography / mass spectroscopic analysis (LC / MS). The murine FR1-21 heavy chain matched the measured mass, while the light chain required follow-up sequencing work to examine the 5'end sequence. The CD37-1LClead1 PCR primer (ttttgaattcgccaccatgaagtttccttctcaacttct) was designed to anneal to the germline ligation leader sequence of the murine antibody so that this new PCR reaction would provide a complete variable region cDNA that was not modified by the primer. PCR reaction, band purification, and sequencing were performed as described above, and the new complete sequence encoded a light chain that matched the molecular weight of the light chain of Fr1-21 as measured by LC / MS. (Example 10) Expression of reference antibody Morphotech anti-FOLR1 antibody, MorAb-003 (Farletuzumab), amino acid sequence was obtained from the list of World Health Organization (WHO) International Nonproprietary Names for Pharmaceutical Substances (INN), codon-optimized and synthesized by Blue Heron Biotechnology. The light chain variable region sequences are flanked by EcoRI and BsiWI restriction enzyme sites and the heavy chain variable region sequences are HindIII and Apa1 restriction enzymes for cloning into their respective constant sequences and inframes in single chain mammalian expression plasmids. It is flanked by the part. Cloning, expression and purification were performed as described for humanized Mov19 and Fr1-21 above. (Example 11) ADCC activity of huMov19 Lactate dehydrogenase (LDH) has been used to measure antibody-dependent cellular cytotoxicity (ADCC) in tumor cells using newly isolated human natural killer (NK) cells as effector cells (eg). Shields, J. Biol. Chem., 276 (9): 6591-6604 (2001)). Human blood from normal donors (Research Blood Components, Inc., Brighton,) using a modified protocol for NK Isolation Kit II (Miltenyi Biotech, 130-091-152). First isolated from MA). Blood was diluted 2-fold with 1xPBS. 25 mL of diluted blood was carefully layered on 25 mL Ficoll Paque in a 50 mL triangular tube and centrifuged at 400 G for 45 minutes at RT. Peripheral blood mononuclear cells (PBMC) were collected from the interface, transferred to a new 50 mL triangular test tube, and washed once with 1xPBS. PBMCs were resuspended in 2 mL of NK isolation buffer (1xPBS, 0.5% BSA, 2 mM EDTA), then 500 μL of biotin antibody cocktail was added to the cell suspension. The biotin antibody cocktail contains a biotinylated antibody that binds to lymphocytes other than NK cells and results in negative selection of NK cells. The mixture was incubated for 10 minutes at 4 ° C, then 1.5 mL of NK isolation buffer and 1 mL of anti-biotin microbeads were added. The cell-antibody mixture was incubated at 4 ° C for an additional 15 minutes. The cells were then washed once with 50 mL of NK isolation buffer and resuspended in 3 mL of NK isolation buffer. Next, the MACSLC column is turned into an automatic MACS separator (Miltenyi). It was mounted on Biotech) and pre-washed with 3 mL of NK isolation buffer. The cell suspension was automatically applied to the column, washed, and the eluted fraction with unlabeled NK cells was collected in a new 50 mL volume triangular test tube. Play the resulting NK cells overnight in 30 mL of complete RPMI medium (RPMI-1640 with 5% fetal bovine serum, 1% penicillin-streptomycin, 1 mM HEPES, 1 mM sodium pyruvate, 1% 100XMEM non-essential amino acid solution). Ting. Subsequent assays and all dilutions were performed in RPMI medium (RPMI-1640 medium supplemented with 20 mM HEPES, pH 7.4, 0.1% BSA and 1% penicillin-streptomycin). Antibodies of various concentrations in RHBP medium were dispensed in duplicate at 50 μL / well in a round-bottomed 96-well plate. Target cells in RHBP medium 106It was resuspended at cells / mL and added at 100 μL / well to each well containing antibody dilution. Plates containing target cells and antibody dilutions were incubated at 37 ° C for 30 minutes. Next, NK cells were added to the wells containing the target cells at 50 μL / well. A typical ratio was 1 target cell to 3-4 NK cells. At least the following controls: NK cells alone, target cells alone (spontaneous LDH release), target cells and NK cells (antibody-independent LDH release), target cells and 10% Triton X-100 (maximum LDH release), respectively. Set for the experiment. Cytolysis was allowed by incubating the mixture at 3 ° C for 4 hours. The plate was centrifuged at 1200 rpm for 10 minutes and 100 μL of supernatant was carefully transferred to a new flat bottom 96-well plate. The LDH reaction mixture (100 μL / well) obtained from the Cytotoxic T Cell Detection Kit (Roche 1644793) was added to each well and incubated for 5-30 minutes at room temperature. The optical density of the sample was measured at 490 nm (OD)490). The percent-specific lysis of each sample was determined using the following formula: percent-specific lysis = (sample value-spontaneous release) / (maximum release-spontaneous release) * 100. Incubation with huMov19 results in good ADCC activity on IGROV-1 cells in the presence of human NK effector cells. ADCC activity against IGROV-1 was compared for huMov19, huFR-1-21, Mor003 and chTK1 (isotype control) (Fig. 6). Administration of 0.9 ng / mL huMov19 resulted in approximately 30% IGROV-1 cytolysis, similar to the activity observed with other anti-FOLR1 antibodies. ADCC activity of huMov19 is 0.20 ng / mL EC50HuFR-1-21 has 0.11 ng / mL EC50Mor003 was 0.16 ng / mL, and chTK1 showed no activity on IGROV-1 cells. (Example 12) Preparation of anti-FOLR1 immunoconjugate Preparation of huMov19v1.6-sulfo-SPDB-DM4 The illustrated 2-sulfo-SPDB linker was dissolved in DMA. The huMov19v1.6 antibody was incubated at pH 7.5 at 25 ° C. for about 2 hours at 8 mg / ml with a 12-fold molar excess of 2-sulfo-SPDB linker. 50 mM potassium phosphate buffer containing 50 mM NaCl, 2 mM EDTA, SEPHADEX equilibrated at pH 6.5TMThe reaction mixture was purified using a G25F column. The maytansinoid DM4 was dissolved in dimethylacetamide (DMA, final concentration 5%) and a 1.7-fold molar excess compared to the linker was added dropwise to the sulfo-SPDB modified antibody. The reaction mixture was adjusted to pH 7.5 with 1 mHEPES buffer. After incubating overnight at room temperature, the conjugated antibody was parallelized with 10 mM histidine, 250 mM glycine, 1% sucrose, pH 5.5, SEPHADEX.TMPurified by chromatography on G25F. The number of DM4 molecules linked per antibody molecule was determined using the previously reported extinction coefficients for antibodies and maytansinoids (Widdison, WC, et al., J Med Chem, 49: 4392-4408 (2006)). .. The percentage of all free maytansinoid species was determined as described above. 3.5-5 DM4 molecule conjugates were obtained per huMov19v1.6 antibody, with <1% present as unconjugated maytansinoids.Preparation of huMov19v1.6-SPP-DM1 An exemplary N-succinimidyl 4- (2-pyridylthio) pentanoate (SPP) linker was dissolved in ethanol. Incubate huMov19v1.6 antibody at 8 mg / ml in 50 mM potassium phosphate buffer (pH 6.5) containing 50 mM NaCl, 2 mM EDTA, and 5% ethanol for approximately 2 hours at room temperature with a 6.5-6 fold molar excess of SPP linker. did. SPP-modified antibody was diluted 2-fold in PBS, pH 6.5 and modified with a 1.5-fold molar excess of maytansinoid DM1 by addition of a concentrated solution of DM1 in dimethylacetamide (DMA) (15-30 mM). It was. After adjusting the DMA concentration to 5% and incubating overnight at room temperature, the conjugated antibody was equilibrated with 10 mM, 250 mM glycine, 1% sucrose pH 5.5, SEPHADEX.TMPurified by chromatography on G25F. The number of DM1 molecules linked per antibody molecule was determined using the previously reported extinction coefficients for antibody and DM1 (Liu et al., Proc. Natl. Acad. Sci. USA, 93, 8618-8623 (1996)). The percentage of free maytansinoids present after the conjugate reaction is 20-50 μg of the conjugate injected onto a HiSepTM column equilibrated in 25% acetonitrile in 100 mM ammonium acetate buffer, pH 7.0. Then, it was determined by eluting into acetonitrile. Peak areas of total free maytansinoid molecular species (identified by gradient elution and elution time comparisons with known standards) were measured using an absorbance detector set at a wavelength of 252 nm, and bound maytansinoids (identified by gradient elution and elution time comparisons with known standards). The percentage of total free maytansinoid molecular species was calculated by comparing with the peak area associated with (eluting into conjugate peaks) in the flow-through fraction of the column. Conjugates with 3.5-4 DM1 molecules per huMov19v1.6 were obtained, with <1% present as unconjugated maytansinoids.Preparation of huMov19v1.6-SPDB-DM4 An exemplary N-succinimidyl 4- (2-pyridylthio) butanoate (SPDB) linker was dissolved in ethanol. Incubate huMov19v1.6 antibody at 8 mg / ml in 50 mM potassium phosphate buffer (pH 6.5) containing 50 mM NaCl, 2 mM EDTA, and 3% ethanol for approximately 2 hours at room temperature with a 5.5-5 fold molar excess of SPDB linker. did. SPDB-decorated antibody was diluted 2-fold in PBS, pH 6.5 and modified with a 1.5-fold molar excess of maytansinoid DM4 by addition of a concentrated solution of DM4 (15-30 mM) in dimethylacetamide (DMA). It was. After incubating overnight at room temperature, the conjugated antibody was equilibrated with 10 mM histidine, 250 mM glycine and 1% sucrose pH 5.5 for SEPHADEX.TMPurified by chromatography on G25F. The number of DM4 molecules linked per antibody molecule was determined using the previously reported extinction coefficients for antibodies and maytansinoids (Widdison, WC, et al., J Med Chem, 49: 4392-4408 (2006)). ). The percentage of total free maytansinoid species was determined as described above. Conjugates with 3.5-4 DM4 molecules per huMov19v1.6 antibody were obtained, with <1% present as unconjugated maytansinoids.Preparation of huMov19v1.0-sulfo-mal-DM4 NHS-3-sulfo-mal linker and DM4 were dissolved separately in DMA.The linker and DM4 thiol were mixed in a solution of DMA containing 40% 200 mM succinate buffer, 2 mM EDTA, pH 5.0 to a molar ratio of DM4 to linker 1.6: 1 and a final concentration of DM4 of 10 mM. The mixture was reacted at 25 ° C for 2 hours. Acid in solution of huMov19 v1.0 antibody in phosphate buffer (pH 7.5) under final conjugation conditions of 4 mg / ml antibody, 90% phosphate buffer / 10% DMA pH 7.5 (v / v). The reaction mixture was added without purification so that a considerable amount of excess of 9.6 mol of linker was added. After incubating overnight at room temperature, the conjugation mixture was purified by chromatography on SEPHADEX G25 equilibrated in PBS pH 7.5. HuMov19v1.0-sulfo-mal-DM4 was then dialyzed against a buffer containing 9.55 mM phosphate, 139.6 mM NaCl, pH 6.5. The number of DM4 molecules linked per antibody molecule was determined using the previously reported extinction coefficients for antibodies and maytansinoids (Widdison, WC, et al., J Med Chem, et al. 49: 4392-4408 (2006)). The percentage of total free maytansinoid species was determined as described above. Conjugates with 3.5-4 DM4 molecules per huMov19v1.0 antibody were obtained, with <1% present as unconjugated maytansinoids.Preparation of huMov19v1.0-SMCC-DM1 NHS-sulfo-SMCC linker and DM1 were dissolved separately in DMA.The linker and DM1 thiol were mixed in a solution of DMA containing 40% 200 mM succinate buffer, 2 mM EDTA, pH 5.0 to a molar ratio of DM1 to the linker of 1.2: 1 and a final concentration of DM1 of 3.75 mM. .. The mixture was reacted at 20 ° C for 75 minutes. HuMov19 v1.0 antibody in phosphate buffer (pH 7.5) under final conjugation conditions of 4 mg / ml antibody, 88% 50 mM calcium phosphate, 50 mM NaCl, 2 mM EDTA, pH 7.5 / 12% DMA pH 7.5 (v / v) The reaction mixture was added to the solution of the antibody without purification so that a considerable amount of excess linker 6.4 mol was added to the antibody. After incubation at 20 ° C for 2 hours, the conjugation mixture was purified by chromatography on SEPHA DEX G25 equilibrated in PBS pH 7.5. Next, huMov19v1.0-SMCC-DM1 was dialyzed against a buffer containing 250 mM glycine and 10 mM histidine pH 5.5. The number of DM1 molecules linked per antibody molecule was determined using the previously reported extinction coefficients for antibodies and maytansinoids (Widdison, WC, et al., J Med Chem, 49: 4392-4408 (2006)). The percentage of total free maytansinoid species was determined as described above. Conjugates with 3.5-4 DM1 molecules per huMov19v1.0 antibody were obtained, with <2.8% present as unconjugated maytansinoids.Preparation of huMov19v1.0-PEG4-mal-DM1 A one-step reagent of NHS-PEG4-mal-DM1 was dissolved in DMA. HuMov 19v1.0 antibody was incubated overnight at 25 ° C. in 5 mg / ml with a 5.7-fold molar excess of NHS-PEG4-mal-DM1 in 50 mM KPi, 50 mM NaCl, 2 mM EDTA, pH 7.5 and 10% DMA by volume. The reaction mixture was purified on a SEPHADE G25 column parallelized to PBS pH 7.5. huMov19v1.0-PEG4-mal-DM1 was dialyzed against a buffer containing 250 mM glycine and 10 mM histidine pH 5.5. The number of DM1 molecules linked per antibody molecule was determined using the previously reported extinction coefficients for antibodies and maytansinoids (Widdison, WC, et al., J Med Chem, et al. 49: 4392-4408 (2006)). The percentage of total free maytansinoid species was determined as described above. Conjugates with 3.5-4 DM1 molecules per huMov19v1.0 antibody were obtained, with <1.1% present as unconjugated maytansinoids. (Example 13) Binding affinity of antibody and conjugate The binding affinities of the anti-FOLR1 antibody and its SPDB-DM4, PEG4Mal-DM4, SMCC-DM1 or anti-FOLR1-sulfo-SPDB-DM4 conjugates were assayed by flow cytometry. FOLR1-expressing SKOV3 cells were incubated with varying concentrations of anti-FOLR1 antibody and conjugates thereof and treated as described above for flow cytometric analysis. Data analysis was performed using CellQuest Pro (BD Biosciences, San Diego, US), and for each sample, the mean fluorescence intensity (MFI) for FL1 was exported and plotted against antibody concentration on a semi-logarithmic plot. Dose response curves were created by non-linear regression and the values for the apparent equilibrium dissociation constant (Kd) of the test sample for binding to SKOV3 cells were determined by GraphPad's Prism v4 (GraphPad Software, San). Calculated using Diego, CA) and shown in Figure 5. The results indicate that conjugation to each DM1 or DM4 via any of the linkers used did not significantly alter the affinity of any of the antibodies (eg, huMov19). (Example 14) In vitro cytotoxic assay The ability of the exemplified muFR1-9, muFR1-13, muFR1-22, muFR1-23, huFR1-23, muFR1-21 and huFR1-21 conjugates to inhibit cell growth, such as Kovtun YV, (Cancer Res 66: 3214) -Measured using an in vitro cytotoxic assay by the method described in 3221 (2006)). Complete RPMI medium (RPMI-1640, 10% fetal bovine serum, 2 mM glutamine, 1% gentamicin, all reagents from Invitrogen) 1000 cells per 100 μL well in 96 well plates with various concentrations of PEG4-mal in FOLR1-expressing KB cells -DM4 conjugate was added. Antibodies and conjugates were diluted in complete R PMI medium by 3-fold serial dilution and 100 μL was added per well. The final concentration was typically 3x10-8M to 4.6x10-12M. Control wells containing cells and medium but no conjugate and wells containing only medium were included in each assay plate. 5% CO2The plates were incubated at 37 ° C for 4-6 days in a moist atmosphere. WST-8 reagent, 10% v / v (Dojindo Molecular Technologies, Gaithersburg, MD, US) was then added to the wells and the plates were incubated at 37 ° C for 2-6 hours. WST-8 is reduced by dehydrogenase in living cells to give the largest orange formazan product that is soluble in tissue culture. The formazan product produced is directly proportional to the number of living cells. 450nm (A) in a multi-well plate reader450) And 650nm (A650) Was measured to analyze the plate. First, the background of cell opacity (A)650) A650Subtracted from. Next obtained A *450The survival rate of cells was measured using. Background A *450Absorbance is the value of the medium and wells with only WST-8. Survival rate is calculated as follows: Percent survivability = 100x (A *)450Administration sample-A *450Background) / (A *450Unadministered sample-A *450Background). Survival values were plotted against antibody or conjugate concentrations in a semi-log plot for each dose. Next, IC50s were obtained from these data using GraphPad's Prism v4 (GraphPad Software, San Diego, CA), and are shown in Fig. 5. According to the results shown in FIG. 5, all conjugates are similarly active in their cytotoxic titers against FOLR1-expressing KB cells. To further confirm the specificity of anti-FOLR1 maytansinoid conjugates to FOLR1, their activity was evaluated in the presence of excess non-conjugated antibody to KB cells. Addition of an excessive amount of competing non-conjugated antibody to the conjugate suppressed their cytotoxicity, as shown in FIG. These data indicate that the conjugate kills KB cells in an antigen-dependent manner. According to additional data, huMov19-SPDB-DM4 induces cell cycle arrest during the G2 / M phase of KB cells in an in vitro assay. (Example 15) In vivo efficacy of huMov19-PEG4 Mal-DM4 and huMov19-SPDB-DM4 conjugates in comparison to similar non-targeting conjugates in KB xenograft modelsFOL R1 targeting cleavable conjugates huMov19-SPDB-DM4 and non-targeting huC242-PEG4Mal in comparison to non-targeting huC242-SPDB-DM4 using an established xenograft model of KB cells subcutaneously transplanted into SCID mice. -The uncut conjugate huMov19-PEG4Mal-DM4 in comparison with DM4 was tested. Mice were randomly assigned to receive groups by body weight and either single (SPDB conjugate) on day 3 after cell inoculation, or 3 times, 5 and on days 3, 10, and 17 after weekly cell inoculation. It was administered using a 10 mg / kg conjugate. The median tumor volumes of the various different dosing groups are plotted in FIG. Administration of either huMov19-SPDB-DM4 or huMov19-PEG4Mal-DM4 resulted in a reduction in median tumor volume compared to PBS controls, whereas administration of any of the corresponding non-targeting conjugates had a significant effect. Did not bring. (Example 16) In vivo efficacy of anti-FOLR1-PEG4Mal-DM4 conjugate in KB xenograft modelUsing an established xenograft model of KB cells subcutaneously transplanted into SCID mice, the exemplified anti-FOLR1 antibodies, huMov19, muFR-1-9, muFR-1-13, muFR-1-22, muFR- PEG4 Mal-DM4 conjugates of 1-23 and huFR-1-21 were tested. Mice were randomly assigned by body weight and administered 10 mg / kg of any of the conjugates listed above, or PBS alone, once 3 days after cell inoculation. huMov19-PEG4Mal-DM4 is associated with the PEG4Mal-DM4 conjugate of muFR-1-9, muFR-1-13, muFR-1-22, muFR-1-23 and huFR-1-21 in its cytotoxic titer in vitro. It turned out to be similar. huMov19-PEG4Mal-DM4 and FR-1-21-PEG4Mal-DM4 were significantly higher in vivo than any of the other conjugates, resulting in a more pronounced phenomenon of median tumor volume (Figs. 9 and 10). ). Titers were also found to be dose-dependent (Fig. 11), and linker selection played a role as well (Figs. 12 and 13). (Example 17) In vitro efficacy of anti-FOLR1-sulfo-SPDB-DM4 conjugate in xenograft model Three ovarian serous adenocarcinoma xenografts: anti-FOLR1huMov19-sulfo-SPDB-DM4 conjugates were tested in OVCAR-3, IGROV-1 and OV-90. Each of these xenograft tumors showed comparable FOLR1 expression levels to patient tumors when measured using carved immunohistochemical (IHC) staining on formalin-fixed paraffin-embedded sections. ing. Mice with established subcutaneous xenograft tumors (approximately 100 mm)3) In contrast, huMov19-SPP-DM1 conjugate was administered by a single intravenous injection at 1.2, 2.5 and 5.0 mg / kg (based on antibody concentration; Figures 14-16 show the concentration of maytansinoid conjugate at μg / kg). Shows). Conjugates were active in all three models evaluated. For OVCAR-3 xenografts, the minimum effective dose (MED) was 1.2 mg / kg (Fig. 14). Higher 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 dose groups, respectively. Conjugate administration resulted in potent antitumor activity in both the IGRO V-1 and OB-90 xenograft models, with a single injection of 2.5 mg / kg MED (FIGS. 15 and 16). These data show the potent antitumor activity of huMov19-sulfo-SPDB-DM4 against ovarian xenograft tumors with FOLR1 expression levels comparable to patient tumors. (Example 18) Linker's effect on immunoconjugate efficacy The anti-FOLR1 antibody huMov19 was ligated to DM1 or DM4 via the disulfide-containing cleavable linker SPP, SPDB, or sulfo-SPDB, or via the non-cleavable linker SMCC. The in vitro cytotoxic activity of these conjugates on KB, IGROV-1 and JEG-3 cell lines was investigated. KB (cervical cancer) cells had> 2,000,000 antibody binding sites per cell, according to FACS analysis. IGROV-1 (ovarian cancer) cells had 260,000 antibody binding sites per cell, and JEG-3 (choriocarcinoma) cells had 40,000 antibody binding sites per cell. The results of in vitro cytotoxicity are summarized in Table 2 below. Cleavable conjugates showed significantly higher in vitro activity compared to SMCC conjugates.<tables num="2"><img id="000021" he="57" wi="143" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></tables> In vivo activity of conjugates in FOLR1-positive KB and OVCAR-3-tumor models was also tested. According to the results shown in FIG. 17, cleavable SPDB-DM4 and sulfo-SPDB-DM4 conjugates were more potent in vivo than non-cleavable SMCC-DM1 conjugates. Furthermore, among the cleavable conjugates, the SPP-DM1 conjugate was less active in both xenograft models than either the SPDB-DM4 or sulfo-SPDB-DM4 conjugate (Fig. 18). The latter two conjugates were similarly active against KB tumors, and the sulfo-SPDB-DM4 conjugate was more active against the OVCAR-3 model. The data obtained using the OVCAR-3 model are summarized in Table 3 below.<tables num="3"><img id="000022" he="66" wi="150" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></tables> According to these data, immunoconjugates containing a cleavable linker show elevated efficacy both in vitro and in vivo, and anti-FOLR1 immunoconjugates containing sulfo-SPDB are highly active in tumor models. is there. (Example 19) In vitro and in vivo efficacy of huFR1 antibody SMCC-DM1 conjugate Anti-FOLR1 huFR1-48, huFR1-49, huFR1-57, and huFR1-65 were conjugated to SMCC linker and DM1 and their effects on KB cells were analyzed in vivo using the xenograft model as described above. Each of the antibodies showed similar efficacy in the KB cell model, with huFR1-48, huFR1-49, huFR1-57, and huFR1-65 immunoconjugates producing variable but significant in vivo efficacy at doses of 200 μg / kg. In the case of a xenograft model system (Table 4 and FIG. 19).<tables num="4"><img id="000023" he="71" wi="135" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></tables> All publications, patents, patent applications, internet sites, and accession number / database sequences (including both polynucleotide and polypeptide sequences) cited herein are the respective publications, patents, patents. The entire application, Internet site, and accession number / database sequence is incorporated herein by reference in its entirety for all purposes, to the extent that it is incorporated herein by reference in a specific and individual manner.<maths num="1"><img id="000024" he="218" wi="158" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></maths><maths num="2"><img id="000025" he="216" wi="158" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></maths><maths num="3"><img id="000026" he="217" wi="158" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></maths><maths num="4"><img id="000027" he="220" wi="158" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></maths><maths num="5"><img id="000028" he="219" wi="158" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></maths><maths num="6"><img id="000029" he="215" wi="158" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></maths><maths num="7"><img id="000030" he="238" wi="120" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></maths><maths num="8"><img id="000031" he="239" wi="100" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></maths><maths num="9"><img id="000032" he="211" wi="158" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></maths><maths num="10"><img id="000033" he="215" wi="158" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></maths><maths num="11"><img id="000034" he="216" wi="158" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></maths><maths num="12"><img id="000035" he="218" wi="158" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></maths><maths num="13"><img id="000036" he="220" wi="158" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></maths><maths num="14"><img id="000037" he="123" wi="158" file="JP5778700B2_D0001.tif" img-format="tif" img-content="drawing" /></maths>

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JP2008500025A	Cites	Japan
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JP2010503386A	Cites	Japan
Singh, R. et al.,Antibody-cytotoxic Agent Conjugates: Preparation and Characterization,Methods in Molecular Biology (Therapeutic Antibodies Methods and Protocols),Humana Press,2009年,vol. 525,pp. 445-467	Non-patent	–

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2013-05-30	Written request for application examinationJAPANESE INTERMEDIATE CODE: A621A621	A621