Bispecific antibodies and uses thereof

文档序号：496576 发布日期：2022-01-07 浏览：2次中文

阅读说明：本技术 双特异性抗体及其用途 (Bispecific antibodies and uses thereof ) 是由刘跃蔡文燕史家栋于 2018-08-01 设计创作，主要内容包括：本公开涉及双特异性抗体或其抗原结合片段,其中所述双特异性抗体或其抗原结合片段以不同的结合亲和力特异性结合两种不同抗原。(The present disclosure relates to bispecific antibodies or antigen-binding fragments thereof, wherein the bispecific antibodies or antigen-binding fragments thereof specifically bind two different antigens with different binding affinities.)

1. A bispecific antibody or antigen-binding fragment thereof comprising:

a first heavy chain variable region which is a heavy chain variable region,

a second heavy chain variable region which is different from the first heavy chain variable region,

a first light chain variable region, and

a second light chain variable region which is different from the light chain variable region,

wherein the first heavy chain variable region and the first light chain variable region associate with each other to form a first antigen binding region that specifically binds to a first antigen, and

the second heavy chain variable region and the second light chain variable region associate with each other forming a second antigen-binding region that specifically binds a second antigen, wherein the binding affinity of the first antigen-binding region when bound to the first antigen is at least 10-fold greater than the binding affinity of the second antigen-binding region when bound to the second antigen, wherein the first light chain variable region and the second light chain variable region are the same.

2. The bispecific antibody or antigen-binding fragment thereof of claim 1, wherein the first antigen-binding region is greater than 10⁸M^-1Specifically binds to the first antigen.

3. The bispecific antibody or antigen-binding fragment thereof of claim 1, wherein the binding affinity of the first antigen-binding region when bound to the first antigen is at least 100-fold greater than the binding affinity of the second antigen-binding region when bound to the second antigen.

4. The bispecific antibody or antigen-binding fragment thereof of claim 1, wherein the bispecific antibody or antigen-binding fragment thereof comprises a first heavy chain comprising the first heavy chain variable region and a second heavy chain comprising the second heavy chain variable region, wherein the first and second heavy chains are associated with each other by way of a knob-and-hole structure.

5. The bispecific antibody or antigen-binding fragment thereof of claim 1, wherein the first antigen is a cancer-specific antigen and the second antigen is CD 3.

6. The bispecific antibody or antigen-binding fragment thereof of claim 1, wherein the first antigen is CD20 and the second antigen is CD 3.

7. The bispecific antibody or antigen-binding fragment thereof of claim 1, wherein the first antigen is a cancer-specific antigen and the second antigen is a cancer-associated antigen.

8. The bispecific antibody or antigen-binding fragment thereof of claim 1, wherein the first antigen is PD-L1 and the second antigen is CD 55.

9. The bispecific antibody or antigen-binding fragment thereof of claim 1, comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein

The first polypeptide comprises a sequence identical to SEQ ID NO: 34. 35 or 36 at least 90% identical;

the second polypeptide comprises a sequence identical to SEQ ID NO: 37. 38 or 39 amino acid sequences that are at least 90% identical;

the third polypeptide comprises a sequence identical to SEQ ID NO: an amino acid sequence that is at least 90% identical; and is

The fourth polypeptide comprises a sequence identical to SEQ ID NO: 40 amino acid sequence that is at least 90% identical.

10. The bispecific antibody or antigen-binding fragment thereof of claim 1, comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein

The first polypeptide comprises a sequence identical to SEQ ID NO: 65 amino acid sequences that are at least 90% identical;

the second polypeptide comprises a sequence identical to SEQ ID NO: 66 amino acid sequences that are at least 90% identical;

the third polypeptide comprises a sequence identical to SEQ ID NO: 67 or 68 is at least 90% identical; and is

The fourth polypeptide comprises a sequence identical to SEQ ID NO: 67 or 68 is at least 90% identical.

11. A method of making a bispecific antibody or antigen-binding fragment thereof, the method comprising:

(a) selecting a first antigen and a second antigen, and identifying a first antibody or antigen-binding fragment thereof that binds to the first antigen and a second antibody or antigen-binding fragment thereof that binds to the second antigen, wherein the first antibody or antigen-binding fragment thereof comprises a first heavy chain variable region (VHa) and a first light chain variable region (VLa), and the second antibody or antigen-binding fragment thereof comprises a second heavy chain variable region (VHb) and a second light chain variable region (VLb);

(b) determining the amino acid sequences of VHa, VLa, VHb and VLb;

(c) aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is greater than 80%;

(d) designing a common light chain variable region (VLc), wherein when the VLc is associated with VHa, the VLc retains affinity for the first antigen;

(e) redesigning the VHa and VHb sequences, thereby obtaining VHa 'and VHb', to increase the difference in biochemical or biophysical characteristics between a first protein and a second protein, wherein the first protein comprises two polypeptides each comprising VHa 'and two polypeptides each comprising VLc, and the second protein comprises two polypeptides each comprising VHb' and two polypeptides each comprising VLc; and is

(f) Producing a bispecific antibody or antigen-binding fragment thereof having two light chain variable regions and two heavy chain variable regions, wherein the two light chain variable regions each comprise a VLc and the two heavy chain variable regions comprise a VHa 'and a VHb', respectively.

12. The method of claim 11, wherein in step (d) the binding affinity of the VLc-VHb to the second antigen may be reduced.

13. The method of claim 11, wherein the method further comprises:

(g) a buffer system was developed to purify the bispecific antibody or antigen-binding fragment thereof.

14. An antibody or antigen-binding fragment thereof that binds CD3, comprising:

a light chain variable region (VL) comprising CDRs 1, 2 and 3, wherein the VL CDR1 region comprises an amino acid sequence at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence at least 80% identical to a selected VL CDR3 amino acid sequence;

wherein the selected VH CDR1, 2,3 amino acid sequences are set forth in SEQ ID NO: 22-24, and the selected VL CDR1, 2,3 amino acid sequences are set forth in SEQ ID NOs: 28-30.

15. The antibody or antigen-binding fragment thereof of claim 14, wherein the heavy chain variable region comprises an amino acid sequence identical to SEQ ID NO:2 and the light chain variable region comprises a sequence at least 90% identical to SEQ ID NO:3 sequences that are at least 90% identical.

16. An antibody or antigen-binding fragment thereof that binds PD-L1, comprising:

wherein the selected VH CDR1, 2, and 3 amino acid sequences and the selected VL CDR1, 2, and 3 amino acid sequences are one of:

(1) the selected VH CDR1, 2 and 3 amino acid sequences are respectively shown in SEQ ID NO: 41-43, and the selected VL CDR1, 2,3 amino acid sequences are set forth in SEQ ID NOs: 53-55;

(2) the selected VH CDR1, 2 and 3 amino acid sequences are respectively shown in SEQ ID NO: 41-43, and the selected VL CDR1, 2,3 amino acid sequences are set forth in SEQ ID NOs: 59-61.

17. The antibody or antigen-binding fragment thereof of claim 16, wherein the heavy chain variable region comprises an amino acid sequence identical to SEQ ID NO:4 or SEQ ID NO 12 and the light chain variable region comprises a sequence at least 90% identical to SEQ ID NO:6 or SEQ ID NO: 7 sequences at least 90% identical.

18. A cell comprising one or more nucleic acids encoding the bispecific antibody or antigen-binding fragment thereof of claim 1.

19. A method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising the bispecific antibody or antigen-binding fragment thereof of claim 1.

20. The method of claim 19, wherein the subject has a solid tumor, melanoma, pancreatic cancer, a hematologic malignancy, non-hodgkin's lymphoma, or chronic lymphocytic leukemia.

21. A bispecific antibody or antigen-binding fragment thereof that binds CD20 and CD3, comprising:

a first polypeptide comprising a first heavy chain variable region (VH1) comprising Complementarity Determining Regions (CDRs) 1, 2 and 3, wherein the VH1CDR 1 region comprises an amino acid sequence at least 80% identical to SEQ ID NO:16, the VH1CDR2 region comprises an amino acid sequence at least 80% identical to SEQ ID NO:17 and the VH1CDR 3 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 18;

a second polypeptide comprising a second heavy chain variable region (VH2) comprising CDRs 1, 2 and 3, wherein the VH2 CDR1 region comprises an amino acid sequence at least 80% identical to SEQ ID NO:22, the VH2 CDR2 region comprises an amino acid sequence at least 80% identical to SEQ ID NO:23 and the VH2 CDR3 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 24;

a third polypeptide comprising a first light chain variable region (VL1) comprising CDRs 1, 2 and 3, wherein the VL 1CDR 1 region comprises an amino acid sequence at least 80% identical to SEQ ID NO:28, the VL 1CDR2 region comprises an amino acid sequence at least 80% identical to SEQ ID NO:29 and the VL 1CDR 3 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 30; and

a fourth polypeptide comprising a second light chain variable region (VL2) comprising CDRs 1, 2 and 3, wherein the VL2 CDR1 region comprises an amino acid sequence at least 80% identical to SEQ ID NO:28, the VL2 CDR2 region comprises an amino acid sequence at least 80% identical to SEQ ID NO:29 and the VL2 CDR3 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 30.

22. A nucleic acid encoding an immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH), wherein the VH comprises a sequence at least 97% identical to SEQ ID NO:2, and wherein the VH binds CD3 when paired with a light chain variable region (VL).

23. A bispecific antibody or antigen-binding fragment thereof that binds PD-L1 and CD55, comprising:

a first polypeptide comprising a first heavy chain variable region (VH1) comprising Complementarity Determining Regions (CDRs) 1, 2 and 3, wherein the VH1CDR 1 region comprises an amino acid sequence at least 80% identical to SEQ ID NO:41, the VH1CDR2 region comprises an amino acid sequence at least 80% identical to SEQ ID NO:42 and the VH1CDR 3 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 43;

a second polypeptide comprising a second heavy chain variable region (VH2) comprising CDRs 1, 2 and 3, wherein the VH2 CDR1 region comprises an amino acid sequence at least 80% identical to SEQ ID NO:47, the VH2 CDR2 region comprises an amino acid sequence at least 80% identical to SEQ ID NO:48 and the VH2 CDR3 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 49;

a third polypeptide comprising a first light chain variable region (VL1) comprising CDRs 1, 2 and 3, wherein the VL 1CDR 1 region comprises an amino acid sequence at least 80% identical to SEQ ID NO:53, the VL 1CDR2 region comprises an amino acid sequence at least 80% identical to SEQ ID NO:54 and the VL 1CDR 3 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 55; and

a fourth polypeptide comprising a second light chain variable region (VL2) comprising CDRs 1, 2 and 3, wherein the VL2 CDR1 region comprises an amino acid sequence at least 80% identical to SEQ ID NO:53, the VL2 CDR2 region comprises an amino acid sequence at least 80% identical to SEQ ID NO:54 and the VL2 CDR3 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 55.

24. A bispecific antibody or antigen-binding fragment thereof that binds PD-L1 and CD55, comprising:

a first polypeptide comprising a first heavy chain variable region (VH1) comprising Complementarity Determining Regions (CDRs) 1, 2 and 3, wherein the VH1CDR 1 region comprises an amino acid sequence at least 80% identical to SEQ ID NO:41, the VH1CDR2 region comprises an amino acid sequence at least 80% identical to SEQ ID NO:42 and the VH1CDR 3 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 43;

a second polypeptide comprising a second heavy chain variable region (VH2) comprising CDRs 1, 2 and 3, wherein the VH2 CDR1 region comprises an amino acid sequence at least 80% identical to SEQ ID NO:47, the VH2 CDR2 region comprises an amino acid sequence at least 80% identical to SEQ ID NO:48 and the VH2 CDR3 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 49;

a third polypeptide comprising a first light chain variable region (VL1) comprising CDRs 1, 2 and 3, wherein the VL 1CDR 1 region comprises an amino acid sequence at least 80% identical to SEQ ID NO:59, the VL 1CDR2 region comprises an amino acid sequence at least 80% identical to SEQ ID NO:60 and the VL 1CDR 3 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 61; and

a fourth polypeptide comprising a second light chain variable region (VL2) comprising CDRs 1, 2 and 3, wherein the VL2 CDR1 region comprises an amino acid sequence at least 80% identical to SEQ ID NO:59, the VL2 CDR2 region comprises an amino acid sequence at least 80% identical to SEQ ID NO:60 and the VL2 CDR3 region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 61.

25. Use of a composition comprising the bispecific antibody or antigen-binding fragment thereof of claim 1 for the preparation of a medicament for treating a subject having cancer.

26. The use of claim 25, wherein the cancer is a solid tumor, melanoma, pancreatic cancer, a hematologic malignancy, non-hodgkin's lymphoma, lymphoma or chronic lymphocytic leukemia.

Technical Field

The present disclosure relates to bispecific antibodies or antigen-binding fragments thereof.

Background

Bispecific antibodies are artificial proteins that can bind two different types of antigens or two different epitopes simultaneously. This dual specificity opens up a wide range of applications, including redirecting T cells to tumor cells, blocking two different signaling pathways simultaneously, dual targeting of different disease mediators and drug (payload) delivery to the targeted site. The approval of cetuximab (catumaxomab) (anti-EpCAM and anti-CD 3) and bonatumomab (anti-CD 19 and anti-CD 3) has become an important milestone for the development of bispecific antibodies.

Since bispecific antibodies have various applications, there is a continuing need to develop various therapeutic agents based on bispecific antibodies.

Disclosure of Invention

The present disclosure relates to unbalanced bispecific antibodies or antigen-binding fragments, wherein the bispecific antibodies or antigen-binding fragments specifically bind two different antigens with different binding affinities.

In some aspects, the present disclosure relates to a bispecific antibody or antigen-binding fragment comprising: a first heavy chain variable region, a second heavy chain variable region, a first light chain variable region, and a second light chain variable region, wherein the first heavy chain variable region and the first light chain variable region associate with each other forming a first antigen binding region that is greater than 10⁷M^-1、10⁸M^-1、10⁹M^-1、10¹⁰M^-1、10¹¹M^-1Or 10¹²M^-1And the second heavy chain variable region and the second light chain variable region associate with each other to form a second antigen-binding region that binds specifically to a first antigen with a binding affinity of less than 10⁹M^-1、10⁸M^-1、10⁷M^-1、10⁶M^-1、10⁵M^-1Or 10⁴M^-1Specifically binds to the second antigen.

In some embodiments, the second antigen binding region is greater than 10⁷M^-1、10⁶M^-1、10⁵M^-1Or 10⁴M^-1Specifically binds to the second antigen.

In some embodiments, the binding affinity of the first antigen-binding region when bound to the first antigen is at least 100-fold, 1000-fold, or 10000-fold greater than the binding affinity of the second antigen-binding region when bound to the second antigen.

In some embodiments, the first light chain variable region and the second light chain variable region are at least 90%, 95%, 99%, or 100% identical.

In some aspects, the present disclosure relates to a bispecific antibody or antigen-binding fragment comprising: a first arm comprising a first heavy chain variable region and a first light chain variable region, a second arm comprising a second heavy chain variable region and a second light chain variable region; wherein the first arm is greater than 10⁷M^-1、10⁸M^-1、10⁹M^-1、10¹⁰M^-1、10¹¹M^-1Or 10¹²M^-1Specifically binds to a first antigen with a binding affinity of less than 10, and the second arm⁹M^-1、10⁸M^-1、10⁷M^-1、10⁶M^-1、10⁵M^-1Or 10⁴M^-1Specifically binds to the second antigen.

In some embodiments, the second arm is greater than 10⁷M^-1、10⁶M^-1、10⁵M^-1Or 10⁴M^-1Specifically binds to the second antigen.

In some embodiments, the binding affinity of the first arm when bound to the first antigen is at least 100-fold, 1000-fold, or 10000-fold greater than the binding affinity of the second arm when bound to the second antigen.

In some embodiments, the first light chain variable region and the second light chain variable region are at least 90%, 95%, 99%, or 100% identical.

In some aspects, the present disclosure relates to a bispecific antibody or antigen-binding fragment comprising: a first heavy chain comprising a first heavy chain variable region, a second heavy chain comprising a second heavy chain variable region, a first light chain comprising a first light chain variable region, and a second light chain comprising a second light chain variable region, wherein the first heavy chain variable region and the first light chain variable region associate with each other to form a first antigen-binding region that is greater than 10⁷M^-1、10⁸M^-1、10⁹M^-1、10¹⁰M^-1、10¹¹M^-1Or 10¹²M^-1And the second heavy chain variable region and the second light chain variable region associate with each other to form a second antigen-binding region that binds specifically to a first antigen with a binding affinity of less than 10⁹M^-1、10⁸M^-1、10⁷M^-1、10⁶M^-1、10⁵M^-1Or 10⁴M^-1Specifically binds to the second antigen.

In some embodiments, the second antigen binding region is greater than 10⁷M^-1、10⁶M^-1、10⁵M^-1Or 10⁴M^-1Specifically binds to the second antigen.

In some embodiments, the first light chain and the second light chain are at least 90%, 95%, 99%, or 100% identical.

In some embodiments, the first heavy chain and the second chain are associated with each other by means of a knob and hole structure.

In some embodiments, the first antigen is a cancer specific antigen and the second antigen is CD 3.

In some embodiments, the first antigen is CD20 and the second antigen is CD 3.

In some embodiments, the first heavy chain variable region comprises a heavy chain variable region identical to SEQ ID NO:1, and a second heavy chain variable region comprising a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:2, and the first and second light chain variable regions comprise a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:3 sequences that are at least 80%, 85%, 90% or 95% identical.

In some embodiments, the first antigen is a cancer-specific antigen and the second antigen is a cancer-associated antigen.

In some embodiments, the first antigen is PD-L1 and the second antigen is CD 55.

In some embodiments, the first heavy chain variable region comprises a heavy chain variable region identical to SEQ ID NO:4, and a second heavy chain variable region comprising a sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO:5, 85%, 90% or 95% identical, and the first and second light chain variable regions comprise a sequence identical to SEQ ID NO:6 or SEQ ID NO: 7 sequences that are at least 80%, 85%, 90% or 95% identical.

In some aspects, the present disclosure relates to a method of making a bispecific antibody or antigen-binding fragment, the method comprising: selecting a first antigen and a second antigen, and identifying a first antibody or antigen-binding fragment thereof that binds to the first antigen and a second antibody or antigen-binding fragment thereof that binds to the second antigen, wherein the first antibody or antigen-binding fragment thereof comprises a first heavy chain variable region (VHa) and a first light chain variable region (VLa), and the second antibody or antigen-binding fragment thereof comprises a second heavy chain variable region (VHb) and a second light chain variable region (VLb); determining the amino acid sequences of VHa, VLa, VHb and VLb; aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is greater than 80%; designing a common light chain variable region (VLc), wherein when the VLc is associated with VHa, the VLc retains affinity for the first antigen; redesigning the VHa and VHb sequences, thereby obtaining VHa 'and VHb', to increase the difference in biochemical or biophysical characteristics between a first protein and a second protein, wherein the first protein comprises two polypeptides each comprising VHa 'and two polypeptides each comprising VLc, and the second protein comprises two polypeptides each comprising VHb' and two polypeptides each comprising VLc; producing a bispecific antibody or antigen-binding fragment thereof having two light chain variable regions and two heavy chain variable regions, wherein the two light chain variable regions each comprise a VLc and the two heavy chain variable regions comprise a VHa 'and a VHb', respectively.

In some embodiments, in step (d), the binding affinity of the VLc-VHb to the second antigen may be decreased.

In some embodiments, the method further comprises developing a buffer system to purify the bispecific antibody or antigen-binding fragment thereof.

In some aspects, the present disclosure relates to a method of making a bispecific antibody or antigen-binding fragment, the method comprising: selecting a first antigen and a second antigen, and identifying a first antibody or antigen-binding fragment thereof that binds to the first antigen and a second antibody or antigen-binding fragment thereof that binds to the second antigen, wherein the first antibody or antigen-binding fragment thereof comprises a first heavy chain variable region (VHa) and a first light chain variable region (VLa), and the second antibody or antigen-binding fragment thereof comprises a second heavy chain variable region (VHb) and a second light chain variable region (VLb); determining the amino acid sequences of VHa, VLa and VLb; aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is less than 80%; replacing all light chain variable regions in the phage display antibody library with VLa and panning against the second antigen to obtain a third heavy chain variable region (VHc); redesigning the VHa and VHc sequences, thereby obtaining VHa 'and VHc', to increase the difference in biochemical or biophysical characteristics between a first protein and a second protein, wherein the first protein comprises two polypeptides each comprising VHa 'and two polypeptides each comprising VLa, and the second protein comprises two polypeptides each comprising VHc' and two polypeptides each comprising VLa; producing a bispecific antibody or antigen-binding fragment thereof having two light chain variable regions and two heavy chain variable regions, wherein the two light chain variable regions each comprise VLa and the two heavy chain variable regions comprise VHa 'and VHc', respectively.

In some embodiments, the method further comprises developing a buffer system to purify the bispecific antibody or antigen-binding fragment thereof.

In some aspects, the present disclosure relates to a method of making a bispecific antibody or antigen-binding fragment, the method comprising: selecting a first antigen and a second antigen, and identifying a first antibody or antigen-binding fragment thereof that binds to the first antigen and a second antibody or antigen-binding fragment thereof that binds to the second antigen, wherein the first antibody or antigen-binding fragment thereof comprises a first heavy chain variable region (VHa) and a first light chain variable region (VLa), and the second antibody or antigen-binding fragment thereof comprises a second heavy chain variable region (VHb) and a second light chain variable region (VLb); determining the amino acid sequences of VHa, VLa, VHb and VLb; aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is less than 80%; replacing all light chain variable regions in a phage display antibody library with a plurality of light chain variable regions, wherein the light chain variable regions are at least 80%, 85%, 90%, 95%, or 99% identical to VLa or VLb; panning against the second antigen; selecting a common light chain variable region (VLc) and third heavy chain variable region (VHc), wherein VHa-VLc binds the first antigen with a desired affinity and VHc-VLc binds the second antigen with a desired affinity; redesigning the VHa and VHc sequences, thereby obtaining VHa 'and VHc', to increase the difference in biochemical or biophysical characteristics between a first protein and a second protein, wherein the first protein comprises two polypeptides each comprising VHa 'and two polypeptides each comprising VLc, and the second protein comprises two polypeptides each comprising VHc' and two polypeptides each comprising VLc; producing a bispecific antibody or antigen-binding fragment thereof having two light chain variable regions and two heavy chain variable regions, wherein the two light chain variable regions each comprise a VLc and the two heavy chain variable regions comprise VHa 'and VHc', respectively.

In some embodiments, in step (d), the plurality of light chain variable regions are produced by error-prone PCR.

In some embodiments, the method further comprises developing a buffer system to purify the bispecific antibody or antigen-binding fragment thereof.

In one aspect, the disclosure provides methods of making bispecific antibodies or antigen-binding fragments thereof. The method involves one or more of the following steps:

(a) a first antigen and a second antigen are selected, and a first antibody or antigen-binding fragment thereof that binds to the first antigen and a second antibody or antigen-binding fragment thereof that binds to the second antigen are identified. In some embodiments, the first antibody or antigen-binding fragment thereof comprises a first heavy chain variable region (VHa) and a first light chain variable region (VLa), and the second antibody or antigen-binding fragment thereof comprises a second heavy chain variable region (VHb) and a second light chain variable region (VLb);

(b) determining the amino acid sequences of VHa, VLa, VHb and VLb;

(c) aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is less than 80%;

(d) replacing all light chain variable regions in the phage display antibody library with a plurality of light chain variable regions. In some embodiments, the light chain variable region is at least 80%, 85%, 90%, 95%, or 99% identical to VLa or VLb;

(e) panning against the second antigen;

(f) a common light chain variable region (VLc) and a third heavy chain variable region (VHc) are selected. In some embodiments, VHc-VLc binds with the second antigen with a desired affinity;

(g) determining that the homology between VLa and VLc is greater than 80%;

(h) a common light chain variable region (VLd) was designed. In some embodiments, the VLd retains affinity for the first antigen when the VLd is associated with VHa, and the VLd has a desired affinity for the second antigen when the VLd is associated with VHc;

(i) optionally, redesigning the VHa and VHc sequences, thereby obtaining VHa 'and VHc', to increase the difference in biochemical or biophysical characteristics between a first protein and a second protein, wherein the first protein comprises two polypeptides each comprising VHa 'and two polypeptides each comprising VLd, and the second protein comprises two polypeptides each comprising VHc' and two polypeptides each comprising VLd; and is

(j) Optionally, a bispecific antibody or antigen-binding fragment thereof is produced having two light chain variable regions and two heavy chain variable regions. In some embodiments, the two light chain variable regions each comprise a VLd and the two heavy chain variable regions comprise VHa 'and VHc', respectively.

In one aspect, the disclosure provides methods of making bispecific antibodies or antigen-binding fragments thereof. The method involves one or more of the following steps:

(b) determining the amino acid sequences of VHa, VLa, VHb and VLb;

(c) aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is greater than 80%;

(d) a common light chain variable region (VLc) was designed. In some embodiments, when the VLc is associated with VHa, the VLc retains affinity for the first antigen; and is

(e) Optionally, a bispecific antibody or antigen-binding fragment thereof is produced having two light chain variable regions and two heavy chain variable regions. In some embodiments, the two light chain variable regions each comprise a VLc and the two heavy chain variable regions comprise VHa and VHb, respectively.

In one aspect, the disclosure also provides methods of making bispecific antibodies or antigen-binding fragments thereof. The method involves one or more of the following steps:

(b) determining the amino acid sequences of VHa, VLa and VLb;

(c) aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is less than 80%;

(d) replacing all light chain variable regions in a phage display antibody library with said VLa and panning against said second antigen to obtain a third light chain variable region (VHc); and is

(e) Optionally, a bispecific antibody or antigen-binding fragment thereof is produced having two light chain variable regions and two heavy chain variable regions. In some embodiments, the two light chain variable regions each comprise VLa and the two heavy chain variable regions comprise VHa and VHc, respectively.

In one aspect, the disclosure further provides methods of making bispecific antibodies or antigen-binding fragments thereof. The method involves one or more of the following steps:

(b) determining the amino acid sequence of VHa, VLa, VHb and/or VLb;

(c) aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is less than 80%;

(e) panning against the first and/or second antigen;

(f) a common light chain variable region (VLc) and a third heavy chain variable region (VHc) are selected. In some embodiments, VHa-VLc binds the first antigen with a desired affinity and VHc-VLc binds the second antigen with a desired affinity; and is

(g) Optionally, a bispecific antibody or antigen-binding fragment thereof is produced having two light chain variable regions and two heavy chain variable regions. In some embodiments, the two light chain variable regions each comprise a VLc and the two heavy chain variable regions comprise VHa and VHc, respectively.

In another aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to CD3, comprising: a heavy chain variable region (VH) comprising Complementarity Determining Regions (CDRs) 1, 2 and 3, wherein the VH CDR1 region comprises an amino acid sequence at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence at least 80% identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2 and 3, wherein the VL CDR1 region comprises an amino acid sequence at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence at least 80% identical to a selected VL CDR3 amino acid sequence; wherein the selected VH CDR1, 2, and 3 amino acid sequences and the selected VL CDR1, 2, and 3 amino acid sequences are one of: the selected VH CDR1, 2 and 3 amino acid sequences are respectively shown in SEQ ID NO: 22-24, and the selected VL CDR1, 2,3 amino acid sequences are set forth in SEQ ID NOs: 28-30.

In some embodiments, the VH comprises a VH having the amino acid sequences as set forth in SEQ ID NOs: 22. 23, 24, and the VL comprises CDRs 1, 2,3 having the amino acid sequences set forth in SEQ ID NOs: 28. 29, 30, and CDR1, 2, and 3 of the amino acid sequences shown in SEQ ID NO.

In some embodiments, the antibody or antigen-binding fragment thereof specifically binds human CD 3.

In some embodiments, the antibody or antigen-binding fragment thereof is a bispecific antibody.

In another aspect, the present disclosure also provides an antibody or antigen-binding fragment thereof that binds to PD-L1, comprising: a heavy chain variable region (VH) comprising Complementarity Determining Regions (CDRs) 1, 2 and 3, wherein the VH CDR1 region comprises an amino acid sequence at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence at least 80% identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2 and 3, wherein the VL CDR1 region comprises an amino acid sequence at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence at least 80% identical to a selected VL CDR3 amino acid sequence; wherein the selected VH CDR1, 2, and 3 amino acid sequences and the selected VL CDR1, 2, and 3 amino acid sequences are one of:

(1) the selected VH CDR1, 2 and 3 amino acid sequences are respectively shown in SEQ ID NO: 41-43, and the selected VL CDR1, 2,3 amino acid sequences are set forth in SEQ ID NOs: 53-55;

(2) the selected VH CDR1, 2 and 3 amino acid sequences are respectively shown in SEQ ID NO: 41-43, and the selected VL CDR1, 2,3 amino acid sequences are set forth in SEQ ID NOs: 59-61.

In some embodiments, the VH comprises a VH having the amino acid sequences as set forth in SEQ ID NOs: 41-43, and the VL comprises CDRs 1, 2,3 having amino acid sequences set forth in SEQ ID NOs: 59-61, and CDR1, 2, and 3 of the amino acid sequence shown in the sequence table.

In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to human CD 3.

In some embodiments, the antibody or antigen-binding fragment thereof is a bispecific antibody.

In another aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to CD55, comprising:

a heavy chain variable region (VH) comprising Complementarity Determining Regions (CDRs) 1, 2 and 3, wherein the VH CDR1 region comprises an amino acid sequence at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence at least 80% identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2 and 3, wherein the VL CDR1 region comprises an amino acid sequence at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence at least 80% identical to a selected VL CDR3 amino acid sequence; wherein the selected VH CDR1, 2, and 3 amino acid sequences and the selected VL CDR1, 2, and 3 amino acid sequences are one of:

(1) the selected VH CDR1, 2 and 3 amino acid sequences are respectively shown in SEQ ID NO: 47-49, and the selected VL CDR1, 2,3 amino acid sequences are set forth in SEQ ID NOs: 53-55;

(2) the selected VH CDR1, 2 and 3 amino acid sequences are respectively shown in SEQ ID NO: 47-49, and the selected VL CDR1, 2,3 amino acid sequences are set forth in SEQ ID NOs: 59-61.

In some embodiments, the VH comprises a VH having the amino acid sequences as set forth in SEQ ID NOs: 47-49, and the VL comprises CDRs 1, 2,3 having the amino acid sequences set forth in SEQ ID NOs: 59-61, and CDR1, 2, and 3 of the amino acid sequence shown in the sequence table.

In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to human CD 3.

In some embodiments, the antibody or antigen-binding fragment thereof is a bispecific antibody.

In one aspect, the present disclosure provides a nucleic acid comprising a polynucleotide encoding a polypeptide comprising: an immunoglobulin light chain or fragment thereof comprising a VL comprising CDRs 1, 2, and 3, said CDRs 1, 2, and 3 comprising, respectively, the amino acid sequences set forth in SEQ ID NOs: 53-55. In some embodiments, when the VL is substantially identical to a VL as comprising SEQ ID NO:4, the VL binds PD-L1, and/or when the VL is paired with a VH comprising an amino acid sequence as set forth in SEQ ID NO:5, said VL binds to CD 55.

In one aspect, the present disclosure provides a nucleic acid comprising a polynucleotide encoding a polypeptide comprising: an immunoglobulin light chain or fragment thereof comprising a VL comprising CDRs 1, 2, and 3, said CDRs 1, 2, and 3 comprising, respectively, the amino acid sequences set forth in SEQ ID NOs: 59-61. In some embodiments, when the VL is substantially identical to a VL as comprising SEQ ID NO:4, the VL binds PD-L1, and/or when the VL is paired with a VH comprising an amino acid sequence as set forth in SEQ ID NO:5, said VL binds to CD 55.

In some embodiments, the nucleic acid encodes a bispecific antibody. In some embodiments, the nucleic acid is cDNA.

In one aspect, the disclosure provides a vector comprising one or more of the nucleic acids described herein.

In one aspect, the present disclosure provides a cell comprising a vector described herein. In some embodiments, the cell is a CHO cell.

In one aspect, the present disclosure provides a cell comprising one or more nucleic acids described herein.

In one aspect, the present disclosure provides a bispecific antibody or antigen-binding fragment thereof that binds to CD20 and CD3, comprising: a first polypeptide comprising a first heavy chain variable region (VH) comprising a sequence identical to SEQ ID NO:1 amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical; a second polypeptide comprising a second heavy chain variable region (VH) comprising a second amino acid sequence substantially identical to SEQ ID NO:2 an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical; a third polypeptide comprising a first light chain variable region (VL) comprising a sequence identical to SEQ ID NO:3 amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical; a fourth polypeptide comprising a second light chain variable region (VL) comprising a sequence identical to SEQ ID NO:3 amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical.

In some embodiments, the first heavy chain variable region (VH) comprises SEQ ID NO: 1; the second heavy chain variable region (VH) comprises SEQ ID NO: 2; the first light chain variable region (VL) comprises SEQ ID NO: 3; and the second light chain variable region (VL) comprises SEQ ID NO: 3.

in some embodiments, the first polypeptide comprises a sequence identical to SEQ ID NO: 34. 35 or 36 at least 90%, 95%, 96%, 97%, 98% or 99% identical; the second polypeptide comprises a sequence identical to SEQ ID NO: 37. 38 or 39, at least 90%, 95%, 96%, 97%, 98% or 99% identical; the third polypeptide comprises a sequence identical to SEQ ID NO: 40 amino acid sequences that are at least 90%, 95%, 96%, 97%, 98%, or 99% identical; and the fourth polypeptide comprises a sequence identical to SEQ ID NO: 40 amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical.

In some embodiments, the first polypeptide comprises SEQ ID NO: 35; and the second polypeptide comprises SEQ ID NO: 38, or a pharmaceutically acceptable salt thereof.

In one aspect, the present disclosure provides a bispecific antibody or antigen-binding fragment thereof that binds to PD-L1 and CD55, comprising: a first polypeptide comprising a first heavy chain variable region (VH) comprising a sequence identical to SEQ ID NO:4 an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical; a second polypeptide comprising a second heavy chain variable region (VH) comprising a second amino acid sequence substantially identical to SEQ ID NO:5 amino acid sequences that are at least 90%, 95%, 96%, 97%, 98%, or 99% identical; a third polypeptide comprising a first light chain variable region (VL) comprising a sequence identical to SEQ ID NO:6 or 7, at least 90%, 95%, 96%, 97%, 98% or 99% identical; a fourth polypeptide comprising a second light chain variable region (VL) comprising a sequence identical to SEQ ID NO:6 or 7, at least 90%, 95%, 96%, 97%, 98% or 99% identical.

In some embodiments, the first heavy chain variable region (VH) comprises SEQ ID NO: 4; the second heavy chain variable region (VH) comprises SEQ ID NO: 5; the first light chain variable region (VL) comprises SEQ ID NO: 7; and the second light chain variable region (VL) comprises SEQ ID NO: 7.

in some embodiments, the first polypeptide comprises a sequence identical to SEQ ID NO: 65 an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical; the second polypeptide comprises a sequence identical to SEQ ID NO: 66 an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical; the third polypeptide comprises a sequence identical to SEQ ID NO: 67 or 68, at least 90%, 95%, 96%, 97%, 98% or 99% identical; and the fourth polypeptide comprises a sequence identical to SEQ ID NO: 67 or 68, or at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of said polypeptide.

In some embodiments, the first polypeptide comprises SEQ ID NO: 65; the second polypeptide comprises SEQ ID NO: 66; the third polypeptide comprises SEQ ID NO: 68; and the fourth polypeptide comprises SEQ ID NO: 68.

In one aspect, the present disclosure provides an antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof described herein covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent.

In one aspect, the present disclosure provides a method of treating a subject having cancer. The method comprises administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein or an antibody-drug conjugate described herein. In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is melanoma, pancreatic cancer, or a hematologic malignancy. In some embodiments, the cancer is non-hodgkin's lymphoma, or chronic lymphocytic leukemia.

In one aspect, the present disclosure provides a method of reducing the rate of tumor growth. The method comprises the following steps: for a subject, contacting tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein or an antibody-drug conjugate described herein.

In one aspect, the present disclosure provides a method of killing tumor cells. The method comprises the following steps: for a subject, contacting tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein or an antibody-drug conjugate described herein.

In one aspect, the present disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described herein, and a pharmaceutically acceptable carrier.

In one aspect, the present disclosure provides a pharmaceutical composition comprising an antibody drug conjugate described herein and a pharmaceutically acceptable carrier.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims.

Drawings

Fig. 1A is a graph showing that antibody a redesigned in example 1 binds to Raji cells expressing CD 20.

Fig. 1B is a diagram showing that antibody B redesigned in example 1 binds to Jukart cells expressing CD 3.

Fig. 2A is the results from the CD20+ Raji cell binding assay.

Figure 2B is the result of the CD3+ Jurkat cell binding assay.

FIG. 3 is a T cell activation assay (CD 20/3 in the figure is CD20/CD3 BsMab; A10 and A11 represent different elution fractions; isotype is IgG1 antibody, which is used as a control).

FIG. 4 is a titration curve of T cell activation for different antibodies.

Figure 5 is antibody-mediated killing of CD20+ Raji cells in the presence of Peripheral Blood Mononuclear Cells (PBMCs).

FIG. 6 is a graph of antibody-mediated CD20+ Raji cell killing in the presence of PBMCs in which T cells were activated for 4 days by IL-2 and CD3/CD28 magnetic beads.

FIG. 7 is a graph of antibody-mediated CD20+ Raji cell killing in the presence of PBMCs in which T cells were activated for 7 days by IL-2 and CD3/CD28 magnetic beads.

Figure 8 is the antibody-mediated killing of CD3+ Jurkat cells in the presence of PBMCs.

FIG. 9 is antibody-mediated killing of CD3+ Jurkat cells in the presence of PBMCs in which T cells were activated for 4 days by IL-2 and CD3/CD28 beads.

FIG. 10 is a graph of antibody-mediated killing of CD3+ Jurkat cells in the presence of PBMCs in which T cells were activated for 7 days by IL-2 and CD3/CD28 beads.

Figure 11 is antibody-induced depletion of activated T cells in PBMC (depletion).

Figure 12 is antibody-induced depletion of inactivated T cells in PBMC.

FIG. 13 is Complement Dependent Cytotoxicity (CDC) mediated Raji cell lysis as determined by FACS.

Fig. 14 shows CDC mediated Raji cell lysis as determined by calcein release.

Figure 15 is CDC mediated lysis of Jurkat cells as determined by FACS (a1 and B3 are different elution fractions).

Figure 16 is T cell depletion in PBMCs in human complement-rich serum.

Figures 17-19 are rituximab-resistant cell lysis mediated by T cell activation, based on PBMCs from 3 different donors.

Fig. 20 is a graph used to evaluate T cell activation of purified antibodies.

FIG. 21A is the average body weight of each group of mice injected with phosphate buffered saline PBS (G1), CD20/CD3BsMab (G2; "BIS") or rituximab (G3; "RTX").

FIG. 21B is the mean imaging intensity of luciferase-tagged Raji cells of each group following injection with phosphate buffered saline PBS (G1), CD20/CD3BsMab (G2; "BIS") or rituximab (G3; "RTX").

FIG. 22A is the result of reduced capillary electrophoresis sodium dodecyl sulfate (Re-CE-SDS) of purified CD20/CD3 bispecific antibody samples.

FIG. 22B is the Non-reduced CE (Non-Re-CE-SDS) results for the purified CD20/CD3 bispecific antibody sample.

FIG. 23A is the binding affinity of avizumab (PD-L1 wt) and a designed PD-L1 mono-dimer IgG antibody (PD-L1V 1) with PD-L1V 1 comprising VHa and common VL (SEQ ID NO: 6) to PD-L1(SEQ ID NO: 4).

FIG. 23B is the binding affinity of a parent anti-CD55 antibody (CD55 wt) and a designed CD55 mono-dimer IgG antibody (CD 55V 1), the CD 55V 1 comprising a VHb to CD55(SEQ ID NO: 5) and a common VL (SEQ ID NO: 6).

FIG. 24 is an alignment of the common light chain of BsMab v 1(SEQ IN NO: 67) and BsMab v2(SEQ ID NO: 68).

FIG. 25A is the binding affinity of avilumab (PD-L1 wt) and a redesigned PD-L1 mono-dimer IgG antibody (PD-L1V 2), the PD-L1V 2 comprising VHa to PD-L1(SEQ ID NO: 4) and common VL V2(SEQ ID NO: 7).

FIG. 25B is the binding affinity of a parent anti-CD55 antibody (CD55 wt) and a redesigned CD55 homodimer IgG antibody (CD 55V 2) comprising VHb for CD55(SEQ ID NO: 5) and common VL V2(SEQ ID NO: 7).

FIGS. 26A-26B are antibody-mediated CDC in MDA231 cells.

Fig. 27A is the result of antibody internalization assay using MDA231 cells.

Fig. 27B is the results of an antibody internalization assay for SIHA cells.

Figure 28 is a schematic showing how bispecific antibodies that bind CD3 and a cancer antigen (e.g., a cancer-specific antigen) recognize and kill tumor cells.

Figure 29 is a schematic showing how bispecific antibodies that bind to cancer-specific antigens and cancer-associated antigens recognize and kill tumor cells.

Detailed Description

Bispecific antibodies or antigen-binding fragments thereof are artificial proteins that can bind two different types of antigens simultaneously. In some embodiments, a bispecific antibody or antigen-binding fragment thereof can have two arms (arms a and B). Each arm has one heavy chain variable region and one light chain variable region.

Bispecific antibodies or antigen-binding fragments thereof can be IgG-like and non-IgG-like. An IgG-like bispecific antibody can have two Fab arms and one Fc region, and the two Fab arms bind different antigens. The non-IgG-like bispecific antibody or antigen binding fragment can be, for example, a chemically linked Fab (e.g., two Fab regions chemically linked) and a single chain variable fragment (scFV). For example, a scFV may have two heavy chain variable regions and two light chain variable regions.

In an unbalanced bispecific antibody or antigen-binding fragment thereof, the two arms (arms: A and B) or the two antigen-binding regions (antigen-binding regions: A and B) can bind to the respective target antigens with different affinities. Binding affinity can be expressed as an association constant (Ka):

ka ═ antibody-antigen ]/[ antibody ] [ antigen ]

Antibodies with high affinity generally have a Ka>10⁷M^-1. The Ka of an arm or an antigen binding region may be greater than 10⁵M^-1、10⁶M^-1、10⁷M^-1、10⁸M^-1、10⁹M^-1、10¹⁰M^-1、10¹¹M^-1Or 10¹²M^-1. In some embodiments, Ka may be less than 10⁵M^-1、10⁶M^-1、10⁷M^-1、10⁸M^-1、10⁹M^-1、10¹⁰M^-1、10¹¹M^-1Or 10¹²M^-1。

The binding affinity of the first arm or first antigen-binding region (a) may be greater than the binding affinity of the second arm or second antigen-binding region (B). Bispecific antibodies with unbalanced affinities can have a variety of advantages. For example, bispecific antibodies with unbalanced affinities may be used to target a cancer-specific antigen on cancer cells and CD3 on T cells. In this case, high affinity for cancer specific antigens may lead to better capture of cancer cells by T cells, while low affinity for CD3 may avoid triggering T cell signaling by CD3 (fig. 28). Only when bispecific antibodies are presented to T cells in a multivalent manner by target cancer cells, T cells can be activated and kill the target cancer cells. In addition, bispecific antibodies with unbalanced affinities can also be used to target cancer-specific and cancer-associated antigens (fig. 29). In this case, the bispecific antibody binds only weakly to non-cancer cells expressing low levels of the cancer-associated antigen, and strongly to cancer cells expressing low levels of the cancer-specific antigen and high levels of the cancer-associated antigen.

For bispecific antibodies with unbalanced affinity, the Ka of the first arm or first antigen-binding region (a) may be greater than 0⁷M^-1、10⁸M^-1、10⁹M^-1、10¹⁰M^-1、10¹¹M^-1Or 10¹²M^-1. In some embodiments, the Ka of the first arm or first antigen-binding region (a) may be 10, 100, 1000, 10000, or 100000 times greater than the Ka of the second arm or second antigen-binding region (B). Thus, in some embodiments, the Ka of the second arm or second antigen binding region (B) may be less than 10⁵M^-1、10⁶M^-1、10⁷M^-1、10⁸M^-1Or 10⁹M^-1. In some embodiments, the Ka of the second arm or second antigen-binding region (B) is still with reasonable affinity, e.g., greater than 10⁴M^-1、10⁵M^-1Or 10⁶M^-1Specifically binds to the target antigen.

Binding affinity can also be expressed by the dissociation constant (Kd).

Kd ═ antibody ] [ antigen ]/[ antibody-antigen ]

Kd may be less than 10^-5M、10^-6M、10^-7M、10^-8M、10^-9M、10^-10M、10^-11M, or 10^-12And M. In some embodiments, Kd may be greater than 10^-5M、10^-6M、10^-7M、10^-8M、10^-9M、10^-10M、10^-11M, or 10^-12M。

In some embodiments, the binding affinity of the first arm or first antigen-binding region (a) is greater than the binding affinity of the second arm or second antigen-binding region (B). For example, the Kd of the second arm or second antigen-binding region (B) may be 10, 100, 1000, 10000 or 100000 times greater (and thus less affinity) than the Kd of the first arm or first antigen-binding region (a). Thus, in some embodiments, the Kd of the first arm or first antigen-binding region (a) may be less than 10^-7M、10^-8M、10^-9M、10^-10M、10^- ¹¹M, or 10^-12M; the Kd of the second arm or second antigen-binding region (B) may be greater than 10^-5M、10^-6M、10^-7M、10^-8M, or 10^-9M。

In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises two light chains and two heavy chains. Each of the two light chains has a light chain variable region (VL) and a light chain constant region (CL).

Each of the two heavy chains has one heavy chain variable region (VH) and three heavy chain constant regions (CH1, CH2, and CH 3). In some embodiments, the two light chains of arm a and arm B are identical. Thus, the CDRs in the VL of both light chains may be identical. In some embodiments, the two heavy chains in the bispecific antibody or antigen-binding fragment thereof are different. Thus, the CDRs in the VH of the two heavy chains are different.

When making bispecific antibodies, various methods can be used to ensure that identical heavy chains do not bind to each other. For example, the "knob-hole" (Knobs-into-holes) method introduced mutations of the larger amino acid on the side chain of one heavy chain and the smaller amino acid on the side chain of the other heavy chain. Thus, the same heavy chain is less likely to be associated with each other, whereas two different heavy chains are more likely to be associated with each other. For example, 'Knobs-into-holes' engineering of anti CH3 domains for heavy chain heiderodeionization "in Ridgway, John BB, Leonard G.Presta and Paul Carter"

Protein Engineering, Design and Selection 9.7(1996), which is incorporated by reference herein in its entirety.

Unbalanced bispecific antibodies that bind to T cell-specific and cancer antigens

Bispecific antibodies (BsAb or BsMab) with T cell specific antigen binding arms (e.g., CD3, CD4, or CD8) that recruit and activate T cells have been extensively studied. However, for safety reasons, the effector functions of many of these bispecific antibodies are eliminated. Since effector functions of antibodies (e.g., ADCC and CDC) have been shown to play a critical role in cancer cell killing, maintaining effector functions of antibodies "safely" would expand the mechanism of action of therapeutic antibodies and improve the cancer killing function of antibodies. In order to "safely" maintain effector functions and expand the application of these bispecific antibodies, an unbalanced bispecific antibody technology platform has been developed based on computational antibody design.

In this design, the first antigen binding region targets a cancer specific antigen and the second antigen binding region targets a T cell specific antigen (e.g., CD3, CD4, or CD8) to recruit T cells to attack the cancer with the cancer specific antigen (fig. 28).

As used herein, the term "cancer specific antigen" refers to an antigen that is specifically expressed on the surface of a cancer cell. These antigens can be used to identify tumor cells. Normal cells rarely express cancer-specific antigens. Some exemplary cancer specific antigens include, for example, CD20, PSA, PSCA, PD-L1, Her2, Her3, Her1, β -catenin, CD19, CEACAM3, EGFR, c-Met, EPCAM, PSMA, CD40, MUC1, IGFIR, and the like. PSA is expressed predominantly on prostate cancer cells, whereas Her2 is expressed predominantly on breast cancer cells.

Bispecific antibodies that bind CD20 and CD3 are provided in the present disclosure. The bispecific antibodies can be used to target a variety of CD 20-positive cancers, such as CD 20-positive non-hodgkin's lymphoma (NHL), and thus can be used to treat non-hodgkin's lymphoma in a subject. Because the bispecific antibody has a different mechanism of action to treat cancer than a therapeutic antibody targeting CD20 alone, it can be used as a complementary therapy to CD 20-positive cancers, especially for those CD 20-positive cancers that do not respond well to current CD20 therapy (e.g., NHL with rituximab resistance).

Antibodies with high affinity for CD3 trigger T cell signaling and elicit adverse immune responses. Thus, there is a need to reduce the low affinity for CD3 (e.g., Ka may be less than 10)⁵M^-1、10⁶M^-1Or 10⁷M^-1). CD3 triggers T cell signaling while "safely" maintaining the risk of antibody effector function. As used herein, the term "safely maintaining effector function of an antibody" refers to an antibody that does not induce ADCC or CDC on normal cells (e.g., non-cancer cells). When multiple bispecific antibodies are present on target cancer cells (e.g., in clusters) and bridge the cancerUpon interaction between cells and T cells, these bispecific antibodies can trigger T cell signaling in a multivalent manner through CD3, and activate T cells which then kill the target cancer cells.

Accordingly, the present disclosure provides a bispecific antibody or antigen-binding fragment thereof comprising two heavy chain variable regions and two light chain variable regions, wherein the first heavy chain variable region comprises a heavy chain variable region comprising a heavy chain variable region that differs from the amino acid sequence of SEQ ID NO:1, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and a second heavy chain variable region comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.

In some embodiments, the CDR sequences used to bind CD20 include the CDRs of the variable domain of the heavy chain as defined by Kabat numbering, SEQ ID NO: 16-18, and the CDRs of the light chain variable domain, SEQ ID NO: 28-30. Under Chothia numbering, the CDR sequences of the heavy chain variable domains are set forth in SEQ ID NOs: 19-21, and the CDRs of the light chain variable domain are set forth in SEQ ID NOs: 31-33.

In some embodiments, the CDR sequences used to bind CD3 include the CDRs of the variable domain of the heavy chain as defined by Kabat numbering, SEQ ID NO: 22-24, and the CDRs of the light chain variable domain, SEQ ID NO: 28-30. Under Chothia numbering, the CDR sequences of the heavy chain variable domains are set forth in SEQ ID NOs: 25-27, and the CDRs of the light chain variable domain are set forth in SEQ ID NOs: 31-33.

In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises a first heavy chain amino acid sequence that hybridizes to SEQ ID NO: 34. 35 or 36 are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical; a second heavy chain amino acid sequence that is identical to SEQ ID NO: 37. 38 or 39 are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical; a first light chain amino acid sequence that is identical to SEQ ID NO: 40 are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical; and a second light chain amino acid sequence that is identical to SEQ ID NO: 40 are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical. In some embodiments, the first light chain amino acid sequence and the second light chain amino acid sequence are the same.

Unbalanced bispecific binding to cancer specific and cancer associated antigens

The present disclosure also provides unbalanced bispecific antibodies whose first antigen-binding region targets a cancer-specific antigen and whose second antigen-binding region targets a cancer-associated antigen.

As used herein, the term "cancer-associated antigen" refers to an antigen that is expressed at relatively high levels on cancer cells but at relatively low levels on normal cells. CD55, CD59, CD46, and many adhesion molecules (e.g., N-cadherin, VE-cadherin, NCAM, Mel-CAM, ICAM, NrCAM, VCAM1, ALCAM, MCAM, etc.) are all cancer-associated antigens. Although both cancer-specific and cancer-associated antigens are expressed on the surface of cancer cells, the difference between cancer-specific and cancer-associated antigens is that cancer-associated antigens are also expressed on normal cells, but at relatively low levels compared to cancer cells. In contrast, cancer-specific antigens are rarely expressed on normal cells, and even if they are expressed on normal cells, the amounts thereof are extremely low. Antibodies directed against cancer-specific antigens do not typically induce antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) against normal cells. In contrast, antibodies that target cancer-associated antigens with high affinity may cause cytotoxic effects between normal cells. Therefore, it is important that bispecific antibodies bind to cancer-associated antigens with relatively low affinity (fig. 29).

Bispecific antibodies that bind to PD-L1 and CD55 are provided in the examples. The antibodies are useful for treating a subject with PD-L1 and CD55 positive cancer by ADCC or CDC and blocking the PD-L1/PD1 interaction to activate T-cell dependent immune responses and reduce inhibition of CDC by CD 55. Furthermore, since cancer cells may become resistant to the PD-L1 antibody, binding between the second arm of the cancer cell and CD55 may provide additional therapeutic effects.

Accordingly, the present disclosure provides a bispecific antibody or antigen-binding fragment thereof comprising two heavy chain variable regions and two light chain variable regions, wherein the first heavy chain variable region comprises a heavy chain variable region comprising a heavy chain variable region that differs from the amino acid sequence of SEQ ID NO:4, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and a second heavy chain variable region comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:5, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6 or 7, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.

In some embodiments, the CDR sequences used to bind PD-L1 include the CDRs of the heavy chain variable domain as defined by Kabat numbering, SEQ ID NO: 41-43, and the CDRs of the light chain variable domain, SEQ ID NO: 53-55 or 59-61. Under Chothia numbering, the CDR sequences of the heavy chain variable domains are set forth in SEQ ID NOs: 44-46, and the CDRs of the light chain variable domain are set forth in SEQ ID NO: 56-58 or 62-64.

In some embodiments, the CDR sequences used to bind CD55 include the CDRs of the variable domain of the heavy chain as defined by Kabat numbering, SEQ ID NO: 47-49, and the CDRs of the light chain variable domain, SEQ ID NO: 53-55 or 59-61. Under Chothia numbering, the CDR sequences of the heavy chain variable domains are set forth in SEQ ID NOs: 50-52, while the CDRs of the light chain variable domain are set forth in SEQ ID NO: 56-58 or 62-64.

In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises a first heavy chain amino acid sequence that hybridizes to SEQ ID NO: 65 are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical; a second heavy chain amino acid sequence that is identical to SEQ ID NO: 66 are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical; a first light chain amino acid sequence that is identical to SEQ ID NO: 67 or 68 are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical; and a second light chain amino acid sequence, a first light chain amino acid sequence thereof that is identical to SEQ ID NO: 67 or 68 are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical. In some embodiments, the first light chain amino acid sequence and the second light chain amino acid sequence are the same.

Production of unbalanced bispecific antibodies or antigens or antigen-binding fragments thereof

Bispecific antibodies or antigens or antigen-binding fragments thereof can be prepared by the following method:

1) two antigens of interest were selected and the heavy chain variable region sequence (VHa) and light chain variable region sequence (VLa) of the antibody (antibody a) that bound to the first antigen were determined, followed by the heavy chain variable region sequence (VHb) and light chain variable region sequence (VLb) of the antibody (antibody B) that bound to the second antigen.

2) VLa and VLb are aligned and if the sequence homology exceeds 80%, a generic VL is designed using computer modeling tools (e.g., BioLuminate from Schordingerm, Cambridge, MA). During the design process, attempts are made to maintain the affinity of VLa, but to some extent the affinity of VLb is sacrificed. The common VL may be VLa, VLb themselves, or a novel VLc with high sequence homology to VLa and VLb. The 3D structure of VLa and VLb may be determined, for example, from a structural model or a crystal structure. The process may start with a sequence of vlas. If based on a 3D structure, an amino acid in the light chain is considered important for binding to the second antigen (e.g. when it is paired with VHb) but not involved in binding to the first antigen (e.g. when it is paired with VHa), then the amino acid in VLa may be changed to the corresponding amino acid in VLb. After repeating this procedure several times, a common VLc can be obtained.

3) If VLa and VLb have less than 80% homology, a human ScFV or Fab phage library is prepared by replacing VL of the existing humanized naive ScFV library with VL of antibody A, then less than 20% of nucleic acid mutations are induced to VL using error-prone PCR, and the antigen against antibody B is panned to give a new antibody B' having VLa or its homolog (> 80% homology) as VL. If the VL is not a VLa, but a VLa homolog (e.g., having > 80% homology), then step 2) is repeated to design a common VL.

4) The VHa and VHb sequences were individually redesigned using computer modeling tools to increase the difference between the biochemical and biophysical properties of a and B, such as the 3D isoelectric Point (PI). In this process, the affinity of A cannot be reduced, while the affinity of B can be reduced to some extent.

5) A buffer system was developed to purify unbalanced bispecific antibodies.

The isoelectric Point (PI) of a peptide is the pH at which a particular molecule does not carry a net charge in the statistical average. The amino acids that make up the peptide can be positive, negative, neutral, or polar in nature, and together provide the overall charge to the protein. However, some amino acids in proteins are buried in the protein and do not interact with the surrounding solution. The 3D PI takes into account the 3D structure of the protein and provides a better estimate of the pH at which the protein, when correctly folded, carries no net charge in its statistical average. (We used the gradient pH buffer in the publication, so this buffer is not our invention.

In some embodiments, a bispecific antibody or antigen-binding fragment thereof can also be prepared by:

(a) selecting a first antigen and a second antigen, and identifying a first antibody or antigen-binding fragment thereof that binds to the first antigen and a second antibody or antigen-binding fragment thereof that binds to the second antigen, wherein the first antibody or antigen-binding fragment thereof comprises a first heavy chain variable region (VHa) and a first light chain variable region (VLa) and the second antibody or antigen-binding fragment thereof comprises a second heavy chain variable region (VHb) and a second light chain variable region (VLb);

(b) determining the amino acid sequences of VHa, VLa and VLb;

(c) aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is less than 80%;

(d) replacing all light chain variable regions in the phage display antibody library with VLa and panning against the second antigen to obtain a third heavy chain variable region (VHc);

(e) redesigning the VHa and VHc sequences, thereby obtaining VHa 'and VHc', to increase the difference in biochemical or biophysical characteristics between a first protein and a second protein, wherein the first protein comprises two polypeptides each comprising VHa 'and two polypeptides each comprising VLa, and the second protein comprises two polypeptides each comprising VHc' and two polypeptides each comprising VLa; and is

(f) A bispecific antibody or antigen-binding fragment thereof is produced having two light chain variable regions each comprising VLa and two heavy chain variable regions comprising VHa 'and VHc', respectively.

In some embodiments, a bispecific antibody or antigen-binding fragment thereof can also be prepared by:

(a) selecting a first antigen and a second antigen, and identifying a first antibody or antigen-binding fragment thereof that binds to the first antigen and a second antibody or antigen-binding fragment thereof that binds to the second antigen, wherein the first antibody or antigen-binding fragment thereof comprises a first heavy chain variable region (VHa) and a first light chain variable region (VLa) and the second antibody or antigen-binding fragment thereof comprises a second heavy chain variable region (VHb) and a second antibody light chain variable region (VLb);

(b) determining the amino acid sequences of VHa, VLa, VHb and VLb;

(c) aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is less than 80%;

(d) replacing all light chain variable regions in a phage display antibody library with a plurality of light chain variable regions, wherein the light chain variable regions are at least 80%, 85%, 90%, 95%, or 99% identical to VLa or VLb;

(e) panning against a first and/or second antigen (e.g., a second antigen);

(f) selecting a common light chain variable region (VLc) and third heavy chain variable region (VHc), wherein VHa-VLc binds the first antigen with a desired affinity and VHc-VLc binds the second antigen with a desired affinity;

redesigning the VHa and VHc sequences, thereby obtaining VHa 'and VHc', to increase the difference in biochemical or biophysical characteristics between a first protein and a second protein, wherein the first protein comprises two polypeptides each comprising VHa 'and two polypeptides each comprising VLc, and the second protein comprises two polypeptides each comprising VHc' and two polypeptides each comprising VLc; and is

(h) Producing a bispecific antibody or antigen-binding fragment thereof having two light chain variable regions and two heavy chain variable regions, wherein the two light chain variable regions each comprise a VLc and the two heavy chain variable regions comprise VHa 'and VHc', respectively.

In some embodiments, if VHa-VLc is unable to bind to the first antigen with the desired affinity, additional steps may be performed. For example, if VLc is at least 80% identical to VLa, a new common light chain can be designed. In some embodiments, the process begins with VLa and the amino acids may be mutated to amino acids in VLc based on the methods described herein (e.g., based on the 3D structure of VLa and VLc).

In some embodiments, designing a common light chain variable region involves aligning VLa and VLb and investigating the difference in residues between VLa and VLb at the same kabat position. If different residues on the VLb do not contact the CDR, interface, canonical, or vernier zone residues on the B Fv structure, the residue on the VLb will be mutated to the residue at the same kabat position on VLa. Otherwise, residues on VLb will be retained.

In some embodiments, redesigning the heavy chain variable region involves calculating the 3D PI of Fv a and Fv B using BioLuminate and mutating non-CDR, non-canonical, non-interface and non-vernier region residues, thereby making the Fv with higher 3D PI higher and the Fv with lower 3D PI even lower.

References to how to use BioLuminate may be found, for example, in the BioLuminate user guide, which is incorporated herein by reference in its entirety.

Antibodies and antigen binding fragments

The present disclosure provides antibodies and antigen-binding fragments thereof comprising Complementarity Determining Regions (CDRs), heavy chain variable regions, light chain variable regions, heavy chains, or light chains as described herein. In some embodiments, the antibodies and antigen-binding fragments thereof are unbalanced bispecific antibodies and antigen-binding fragments thereof.

Generally, antibodies (also referred to as immunoglobulins) are composed of two types of polypeptide chains: light and heavy chains. One non-limiting antibody of the present disclosure can be a full four immunoglobulin chain antibody comprising two heavy chains and two light chains. The heavy chain of an antibody may be of any isotype, including IgM, IgG, IgE, IgA or IgD, and may be of subtypes including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, and the like. The light chain may be a kappa light chain or a lambda light chain. The antibody may comprise two identical copies of the light chain and/or two identical copies of the heavy chain. Heavy chains, each comprising one variable domain (or variable region, VH) and multiple constant domains (or constant regions), are bound to each other within their constant domains by disulfide bonds to form the "stem" of the antibody. Light chains, each comprising one variable domain (or variable region, VL) and one constant domain (or constant region), are each bound to one heavy chain by disulfide bonds. The variable region of each light chain is aligned with the variable region of the heavy chain to which it is bound. The variable regions of both the light and heavy chains contain three hypervariable regions, which are sandwiched between more conserved Framework Regions (FR).

These hypervariable regions, called Complementarity Determining Regions (CDRs), form loops that comprise the primary antigen-binding surface of the antibody. The four framework regions adopt predominantly a β -sheet conformation, and the CDRs form loops that connect and in some cases form part of the β -sheet structure. The CDRs in each chain are tightly linked by framework regions and together with the CDRs in the other chain contribute to the formation of the antigen binding region.

Methods for identifying the CDR regions of an antibody by analyzing the amino acid sequence of the antibody are well known, and many definitions of CDRs are commonly used. The Kabat definition is based on sequence variability and Chothia definition is based on the position of the structural loop regions. Such methods and definitions are described, for example, in Martin, "Protein sequence and structure analysis of Antibody variable domains," Antibody engineering, Springer Berlin Heidelberg, 2001.422-439; abhinandan et al, "analyses and improvements to Kabat and structural core number of antibody variable domains," Molecular immunology 45.14(2008): 3832-; wu, T.T. and Kabat, E.A. (1970) J.exp.Med.132: 211-250; martin et al, Methods enzymol.203:121-53 (1991); morea et al, Biophys chem.68(1-3):9-16 (10 months 1997); morea et al, J Mol biol.275(2):269-94(1 month 1998); chothia et al, Nature 342(6252) 877-83 (12 months 1989); ponomarenko and Bourne, BMC Structural Biology 7:64 (2007); each of these documents is incorporated by reference herein in its entirety. Kabat numbering is used by default in this disclosure unless specifically noted otherwise.

The CDRs are important for recognizing epitopes. As used herein, an "epitope" is the smallest portion of a target molecule that is capable of being specifically bound by an antigen binding domain of an antibody. The minimum size of an epitope may be about 3, 4,5, 6, or 7 amino acids, but these amino acids need not be in a continuous linear sequence of the primary structure of the antigen, as an epitope may depend on the three-dimensional configuration of the antigen based on its secondary and tertiary structure.

In some embodiments, the antibody is an intact immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, differing in their constant regions, particularly their hinge and upper CH2 domains. The sequences and differences of the IgG subclasses are known in the art and are described, for example, in Vidarsson et al, "IgG subclasses and allotypes: from structures to effects functions." Frontiers in immunology 5 (2014); irani et al, "Molecular properties of human IgG subclasses and the same injections for identifying thermal monoclonal antibodies induced diseases". Molecular immunology 67.2(2015): 171-; shakib, Farouk eds, The human IgG subclasses, molecular analysis of structure, function and regulation. Elsevier, 2016; each of these documents is incorporated by reference herein in its entirety.

The antibody may also be an immunoglobulin molecule derived from any species (e.g., human, rodent, mouse, rat, camelid).Antibodies disclosed herein also include, but are not limited to, polyclonal, monoclonal, monospecific, multispecific antibodies and chimeric antibodies comprising an immunoglobulin binding domain fused to another polypeptide. The term "antigen binding domain" or "antigen binding fragment" is a portion of an antibody that retains the specific binding activity of an intact antibody, i.e., any portion of an antibody that is capable of specifically binding to an epitope of the target molecule of an intact antibody. It includes, for example, Fab ', F (ab')₂And variants of these fragments. Thus, in some embodiments, an antibody or antigen-binding fragment thereof can be, for example, an scFv, Fv, Fd, dAb, bispecific antibody, bispecific scFv, diabody, linear antibody, single chain antibody molecule, multispecific antibody formed from antibody fragments, and any polypeptide comprising a binding domain that is the same as or homologous to an antibody binding domain. Non-limiting examples of antigen binding domains include, for example, a heavy and/or light chain CDR of a complete antibody, a heavy and/or light chain variable region of a complete antibody, a full length heavy or light chain of a complete antibody, or a single CDR from a heavy or light chain of a complete antibody.

In some embodiments, the scFV has two heavy chain variable domains and two light chain variable domains. In some embodiments, the scFV has two antigen binding regions (antigen binding regions: a and B), and the two antigen binding regions can bind to respective target antigens with different affinities.

In some embodiments, the antigen-binding fragment may form part of a Chimeric Antigen Receptor (CAR). In some embodiments, the chimeric antigen receptor is a fusion of a single chain variable fragment (scFv) as described herein, fused to the CD 3-zeta transmembrane-and endodomain. In some embodiments, the chimeric antigen receptor further comprises an intracellular signaling domain from multiple co-stimulatory protein receptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimeric antigen receptor comprises multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to increase potency. Accordingly, in one aspect, the present disclosure further provides a cell (e.g., a T cell) expressing a chimeric antigen receptor as described herein.

In some embodiments, an antibody or antigen-binding fragment thereof can bind to two different antigens or two different epitopes.

In some embodiments, the antibody or antigen-binding fragment thereof may comprise one, two, or three heavy chain variable region CDRs selected from table 1, table 2, table 11, and table 12. In some embodiments, the antibody or antigen-binding fragment thereof may comprise one, two, or three light chain variable region CDRs selected from table 3, table 13, and table 14.

In some embodiments, an antibody may have a heavy chain variable region (VH) comprising Complementarity Determining Regions (CDRs) 1, 2,3, wherein the CDR1 region comprises or consists of an amino acid sequence at least 80%, 85%, 90%, or 95% identical to a selected VH CDR1 amino acid sequence; the CDR2 region comprises or consists of an amino acid sequence at least 80%, 85%, 90% or 95% identical to a selected VH CDR2 amino acid sequence; the CDR3 region comprises or consists of an amino acid sequence at least 80%, 85%, 90% or 95% identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising Complementarity Determining Regions (CDRs) 1, 2,3, wherein the CDR1 region comprises or consists of an amino acid sequence at least 80%, 85%, 90%, or 95% identical to a selected VL CDR1 amino acid sequence; the CDR2 region comprises or consists of an amino acid sequence at least 80%, 85%, 90%, or 95% identical to a selected VL CDR2 amino acid sequence; the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR3 amino acid sequence. The selected VH CDR1, 2,3 amino acid sequences are shown in table 1, table 2, table 11 and table 12, and the selected VL CDR1, 2,3 amino acid sequences are shown in table 3, table 13, table 14.

In some embodiments, an antibody or antigen-binding fragment described herein may comprise a heavy chain variable domain comprising one, two, or three CDRs selected from table 1, table 2, table 11, and table 12 with zero, one, or two amino acid insertions, deletions, or substitutions.

In some embodiments, an antibody or antigen-binding fragment described herein can comprise a light chain variable domain comprising one, two, or three CDRs selected from table 3, table 13, table 14 with zero, one, or two amino acid insertions, deletions, or substitutions.

The insertions, deletions and substitutions may be within the CDR sequences, or at one or both ends of the CDR sequences.

Antibodies and antibody fragments of the present disclosure may be modified in the Fc region to provide a desired effector function or serum half-life.

Multimerization of antibodies can be achieved by natural aggregation of antibodies or by chemical or recombinant linking techniques known in the art. For example, a certain percentage of purified antibody preparations (e.g., purified IgG1 molecules) spontaneously form protein aggregates comprising antibody homodimers and other higher order antibody multimers.

Any of the antibodies or antigen-binding fragments described herein can be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or solution). Non-limiting examples of stabilizing molecules include: a polymer (e.g., polyethylene glycol) or a protein (e.g., serum albumin, such as human serum albumin). Conjugation of the stabilizing molecule can extend the half-life of the antibody or antigen-binding fragment or extend the biological activity of the antibody or antigen-binding fragment thereof in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in a human).

In some embodiments, an antibody or antigen-binding fragment (e.g., a bispecific antibody) described herein can be conjugated to a therapeutic agent. An antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof can be covalently or non-covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., cytochalasin B, gramicidin D, ethidium bromide, emidine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, maytansinoids (e.g., DM-1 and DM-4), diketones, serine, mitomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide, and analogs thereof).

Antibody characterization

The antibodies or antigen-binding fragments thereof (e.g., bispecific antibodies) described herein can increase the immune response. In some embodiments, an antibody or antigen-binding fragment thereof described herein can increase an immune response, activity, or number of T cells (e.g., CD3+ cells, CD8+ and/or CD4+ cells) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold.

In some embodiments, an antibody or antigen-binding fragment thereof described herein can reduce the activity or number of T cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold.

In some embodiments, an antibody or antigen-binding fragment thereof described herein does not induce an immune response in normal cells (e.g., non-tumor cells) or in the absence of tumor cells.

In some embodiments, the antibody or antigen-binding fragment thereof (e.g., a bispecific antibody) can bind PD-L1 or PD-L2. Thus, the antibodies or antigen-binding fragments thereof described herein can block binding between PD-1 and PD-L1 and/or between PD-1 and PD-L2. In some embodiments, the antibody may inhibit the PD-1 signaling pathway and up-regulate the immune response by binding to PD-L1 or PD-L2. Thus, in some embodiments, an antibody or antigen-binding fragment thereof described herein is a PD-1 antagonist. In some embodiments, the antibody or antigen-binding fragment thereof is a PD-1 agonist.

In some embodiments, the antibody or antigen-binding fragment thereof (e.g., a bispecific antibody) can bind CD 3. Thus, an antibody or antigen-binding fragment thereof described herein can recruit T cells to a target cell.

In some embodiments, the antibody (or antigen-binding fragment thereof) is administered in less than 0.1s^-1Less than 0.01s^-1Less than 0.001s^-1Less than 0.0001s^-1Or less than 0.00001s^-1Specific binding of an antigen (e.g., human protein, monkey protein, and/orMouse protein). In some embodiments, the off-rate (koff) is greater than 0.01s^-1Greater than 0.001s^-1Greater than 0.0001s^-1Greater than 0.00001s^-1Or greater than 0.000001s^-1. In some embodiments, the kinetic association rate (kon) is greater than 1x10²/Ms, greater than 1x10³/Ms, greater than 1x10⁴/Ms, greater than 1x10⁵/Ms, or greater than 1x10⁶and/Ms. In some embodiments, the kinetic association rate (kon) is less than 1x10⁵/Ms, less than 1x10⁶/Ms, or less than 1x10⁷/Ms。

Affinity can be deduced from the quotient of kinetic rate constants (Kd ═ koff/kon). In some embodiments, Kd is less than 1x10^-4M, less than 1x10^-5M, less than 1x10^-6M, less than 1x10^-7M, less than 1x10^-8M, less than 1x10^-9M, or less than 1x10^-10And M. In some embodiments, the Kd is less than 50nM, 30nM, 20nM, 15nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, or 1 nM. In some embodiments, Kd is greater than 1x10^-4M, greater than 1x10^-5M, greater than 1x10^-6M, greater than 1x10^-7M, greater than 1x10^-8M, greater than 1x10^-9M, greater than 1x10^-10M, greater than 1x10^-11M, or greater than 1x10^-12And M. Further, Ka can be derived from Kd by the formula Ka ═ 1/Kd.

Common techniques for measuring the affinity of an antibody for an antigen include, for example, ELISA, RIA, and Surface Plasmon Resonance (SPR).

In some embodiments, thermal stability is determined. The Tm of an antibody or antigen-binding fragment described herein can be greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 ℃.

Since IgG can be described as a multidomain protein, the melting curve sometimes shows two transitions or three transitions, with a first denaturation temperature Tm D1 and a second denaturation temperature Tm D2, and optionally a third denaturation temperature Tm D3.

In some embodiments, the antibody or antigen-binding fragment described herein has a Tm D1 greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 ℃. In some embodiments, the antibody or antigen-binding fragment described herein has a Tm D2 greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 ℃. In some embodiments, the antibody or antigen-binding fragment described herein has a Tm D3 greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 ℃.

In some embodiments, Tm D1, Tm D2, Tm D3 is less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 ℃.

In some embodiments, an antibody or antigen-binding fragment described herein does not begin to form aggregates when the temperature is less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 ℃. In some embodiments, Tagg266 or Tagg473 are less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 ℃ c

In some embodiments, the antibody or antigen-binding fragment described herein has a pI greater than 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, or 9.9. In some embodiments, the antibody or antigen-binding fragment described herein has a pI of less than 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, or 9.9.

In some embodiments, the antibody has a percent tumor growth inhibition (TGI%) greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the antibody has a percent tumor growth inhibition of less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. The TGI% can be determined, for example, at 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after initiation of treatment or at 1, 2,3, 4,5, 6, 7, 8, 9, 10, 11, or 12 months after initiation of treatment. As used herein, percent tumor growth inhibition (TGI%) was calculated using the following formula:

TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100

ti is the mean tumor volume of the day i treated group. T0 is the mean tumor volume at day 0 in the treatment group. Vi is the average tumor volume of the day i control group. V0 is the mean tumor volume at day 0 in the control group.

In some embodiments, the antibody or antigen-binding fragment may increase Complement Dependent Cytotoxicity (CDC) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold.

In some embodiments, the antibody or antigen-binding fragment can increase antibody-dependent cell-mediated cytotoxicity (ADCC) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold.

In some embodiments, the antibody or antigen binding fragment may increase the internalization rate by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold.

In some embodiments, the antibody or antigen-binding fragment can increase phagocytosis rate by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold.

In some embodiments, the antibody or antigen-binding fragment may enhance T cell function (e.g., as compared to proliferation and/or cytokine production of a previous treatment with the antibody or antigen-binding fragment), for example, by increasing effector T cell proliferation and/or increasing interferon-gamma production by effector T cells.

In some embodiments, the antibody or antigen binding fragment enhances CD4+ effector T cell function (e.g., as compared to proliferation and/or cytokine production prior to treatment with the antibody or antigen binding fragment), for example, by increasing CD4+ effector T cell proliferation and/or increasing gamma interferon production by CD4+ effector T cells. In some embodiments, the cytokine is gamma interferon. In some embodiments, for example, the antibody or antigen binding fragment increases the number of (infiltrating) CD4+ effector T cells within the tumor (e.g., the total number of CD4+ effector T cells, or the percentage of CD4+ cells, e.g., CD45+ cells). Intratumoral (infiltrating) CD4+ T cells were detected prior to treatment with the antibody or antigen binding fragment. In some embodiments, the antibody or antigen-binding fragment increases the number of (infiltrating) CD4+ effector T cells within the interferon-gamma expressing tumor (e.g., total interferon-gamma expressing CD4+ cells, or, e.g., the percentage of interferon-gamma expressing CD4+ cells in total CD4+ cells), e.g., as compared to the number of (infiltrating) CD4+ T cells within the interferon-gamma expressing tumor prior to treatment.

In some embodiments, the antibody or antigen binding fragment increases the number of (infiltrating) CD8+ effector T cells within the tumor (e.g., the total number of CD8+ effector T cells, or, e.g., the percentage of CD8+ in CD45+ cells), e.g., as compared to (infiltrating) CD8+ T effector cells within the tumor prior to treatment. In some embodiments, the antibody or antigen-binding fragment increases the number of γ -interferon expressing intratumoral (infiltrating) CD8+ effector T cells (e.g., the percentage of γ -interferon expressing CD8+ cells out of total CD8+ cells), e.g., as compared to γ -interferon expressing intratumoral (infiltrating) CD8+ T effector cells prior to treatment with the antibody.

In some embodiments, the antibody or antigen binding fragment enhances memory T cell function, e.g., by increasing memory T cell proliferation and/or increasing cytokine (e.g., gamma interferon) production by memory cells.

In some embodiments, the antibody or antigen binding fragment has a functional Fc region. In some embodiments, the effector function of the functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector functions of the functional Fc region are ADCC and phagocytosis. In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG 4.

In some embodiments, the antibody or antigen binding fragment may induce apoptosis. In some embodiments, the antibody or antigen binding fragment does not have a functional Fc region. For example, antibodies or antigen binding fragments are Fab, Fab ', F (ab') 2 and Fv fragments.

In some embodiments, the antibody or antigen-binding fragment is a humanized antibody. Percent humanization refers to the percent identity of the heavy or light chain variable region sequence compared to the human antibody sequences in the international immunogenetic information system (IMGT) database. In some embodiments, the percent humanization is greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%. Detailed descriptions of how to determine The percent humanization are known in The art and are described, for example, in Jones, Tim d. et al, "The ins and amounts of anti nonproprietary names," mabs. vol.8, No. 1, Taylor & Francis,2016, The entire contents of which are incorporated herein by reference. A high humanization percentage generally has various advantages, for example, being safer in humans, more effective, more likely to be tolerated by human subjects, and/or having fewer side effects. In some embodiments, the antibody or antigen-binding fragment is a human antibody.

Recombinant vector

The present disclosure also provides recombinant vectors (e.g., expression vectors) comprising an isolated polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein), introducing the recombinant vector into a host cell (i.e., such that the host cell comprises the polynucleotide and/or comprises the polynucleotide), and producing the recombinant antibody polypeptide or fragment thereof by recombinant techniques.

As used herein, a "vector" is any construct capable of delivering one or more. When the vector is introduced into a host cell, the host cell is interested in one or more polynucleotides. An "expression vector" is capable of delivering and expressing one or more polynucleotides of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, in an expression vector, a polynucleotide of interest is expressed in the vector by being operably linked to regulatory elements within the vector or within the genome, such as a promoter, enhancer, and/or poly-a tail, to position the polynucleotide of interest for expression in the vector, whether within the vector or within the genome of the host cell at or near or flanking the insertion site of the polynucleotide of interest, such that the polynucleotide of interest will be translated in the host cell into which the expression vector is introduced.

The vector may be introduced into the host cell by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and/or transduction (e.g., recombinant viruses). Thus, non-limiting examples of vectors include viral vectors (useful for the production of recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

In some embodiments, a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus) is used to introduce a polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein), which may involve the use of a non-pathogenic (defective) replication competent virus, or the use of a replication defective virus. In the latter case, viral propagation will generally only occur in compatible viral packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al, 1989, Proc. Natl. Acad. Sci. USA86: 317-; flexner et al, 1989, Ann.N.Y.Acad Sci.569: 86-103; flexner et al, 1990, Vaccine,8: 17-21; U.S. patent nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. patent No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner-Biotechniques,6:616-627, 1988; rosenfeld et al, 1991, Science,252: 431-; kolls et al, 1994, Proc. Natl. Acad. Sci. USA,91: 215-219; Kass-Eisler et al, 1993, Proc.Natl.Acad.Sci.USA,90: 11498-; guzman et al, 1993, Circulation,88: 2838-; and Guzman et al, 1993, cir.Res.,73: 1202-1207. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be "naked", as described, for example, in Ulmer et al, 1993, Science,259: 1745-. Uptake of naked DNA can be increased by coating the DNA on biodegradable beads, which can be efficiently transported into the cell.

For expression, a DNA insert comprising an antibody-encoding or polypeptide-encoding polynucleotide disclosed herein can be operably linked to a suitable promoter (e.g., a heterologous promoter), such as the phage lambda PL promoter, the e.coli lac, trp, and tac promoters, the SV40 early and late promoters, and the promoters of retroviral LTRs, to name a few. Other suitable promoters are known to the skilled person. The expression construct may further comprise sites for transcription initiation, termination, and a ribosome binding site for translation in the transcribed region. The coding portion of the mature transcript expressed by the construct may include translation initiated at the beginning and a stop codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated.

As indicated, the expression vector may include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, as well as tetracycline or ampicillin resistance genes for culture in E.coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells, such as E.coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells, if fly S2 and spodoptera Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells; and plant cells. Suitable media and conditions for the host cells described herein are known in the art.

Non-limiting vectors for bacteria include pQE70, pQE60 and pQE-9, available from Qiagen. pBS vector, Phagescript vector, Bluescript vector, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5, available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG, available from Stratagene. And pSVK3, pBPV, pMSG, and pSVL, available from Pharmacia. Other suitable vectors will be apparent to the skilled person.

Non-limiting bacterial promoters suitable for use include the E.coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters, and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the SV40 early and late promoters, the promoter of the retroviral LTR, such as the Rous Sarcoma Virus (RSV), and the promoter of metallothionein, such as the mouse metallothionein-I promoter.

In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters may be used, such as alpha factor, alcohol oxidase and PGH. For review see Ausubel et al (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., and Grant et al, Methods enzymol, 153:516-544 (1997).

The construct can be introduced into the host cell by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, or other methods. Such Methods are described In a number of standard laboratory manuals, such as Davis et al, Basic Methods In Molecular Biology (1986), which is incorporated herein by reference In its entirety.

Transcription of DNA encoding the antibodies of the disclosure by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300bp, that act to increase the transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer, which is located at base pairs 100 to 270 posterior to the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer posterior to the origin of replication, and adenovirus enhancers.

For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, an appropriate secretion signal may be incorporated into the expressed polypeptide. The signal may be endogenous to the polypeptide or may be heterologous.

The polypeptide (e.g., antibody) may be expressed in modified form, such as a fusion protein (e.g., a GST-fusion) or with a histidine tag, and may include not only secretion signals, but also other heterologous functional regions. For example, additional regions of amino acids, particularly charged amino acids, can be added at the N-terminus of the polypeptide to improve stability and persistence in the host cell during purification or during subsequent handling and storage. Likewise, peptide moieties may be added to the polypeptide to facilitate purification. These regions may be removed prior to final preparation of the polypeptide. The addition of a peptide moiety to a polypeptide to cause secretion or excretion to improve stability and facilitate purification is well known and routine in the art.

The present disclosure also provides a nucleic acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of the nucleotide sequences described herein, and provides an amino acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of the amino acid sequences described herein.

The present disclosure also provides nucleic acid sequences having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology to any of the nucleotide sequences described herein, and provides nucleic acid sequences having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, homology to any of the amino acid sequences described herein, and provides nucleic acid sequences having at least 1%, 2%, 3%, 4%, 5%, 93%, 94%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% and 99% homology.

In some embodiments, the present disclosure relates to a nucleotide sequence encoding any of the peptides described herein, or any amino acid sequence encoded by any of the nucleotide sequences described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, or 400 amino acid residues.

In some embodiments, the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any sequence described herein.

In some embodiments, nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any sequence described herein.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for comparison purposes, and optimal alignment and non-homologous sequences can be ignored). The length of a reference sequence aligned for comparison purposes is at least 80%, and in some embodiments at least 90%, 95%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein, "identity" of an amino acid or nucleic acid is equivalent to "homology" of an amino acid or nucleic acid). The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which needs to be introduced to achieve optimal alignment of the two sequences. For the purposes of the present invention, comparison of two sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

The percentage of sequence homology (e.g., amino acid sequence homology or nucleic acid homology) can also be determined. How to determine the percentage of sequence homology is known in the art. In some embodiments, conserved amino acid residues (percent homology) with similar physicochemical properties, such as leucine and isoleucine, may be used to measure sequence similarity. Families of amino acid residues with similar physicochemical properties have been defined in the art. These families include, for example, amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). In many cases, the percentage homology is higher than the percentage identity.

Method for producing antibody

Isolated fragments of human proteins (e.g., CD55, CD3, cancer-specific antigen, or cancer-associated antigen) can be used as immunogens to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be produced in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of an antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected with at least one adjuvant. In some embodiments, the antigenic peptide or protein may be conjugated to an agent that is immunogenic in the species to be immunized. The antigenic peptide or protein can be injected into the animal more than once (e.g., two, three, or four times).

Full-length polypeptides or proteins or antigenic peptide fragments thereof may be used as immunogens. An antigenic peptide of a protein comprises at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence of the protein, and comprises an epitope of the protein such that antibodies directed against the peptide form specific immune complexes with the protein.

Immunogens are typically used to produce antibodies by immunizing a suitable subject (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). Suitable immunogenic preparations may comprise, for example, recombinantly expressed or chemically synthesized polypeptides. The formulation may further comprise an adjuvant, such as freund's complete or incomplete adjuvant, or a similar immunostimulant.

As described above, polyclonal antibodies can be prepared by immunizing a suitable subject with a polypeptide or an antigenic peptide thereof (e.g., a portion of a protein) as an immunogen. Antibody titers in immunized subjects can be monitored over time by standard techniques, such as enzyme-linked immunosorbent assays (ELISAs) using immobilized polypeptides or peptides. If desired, the antibody molecules can be isolated from the mammal (e.g., from blood) and further purified by well-known techniques such as protein A chromatography on protein G to obtain the IgG fraction. At an appropriate time after immunization, for example, when the specific antibody titer is highest, antibody-producing cells can be obtained from the subject and Monoclonal Antibodies can be prepared by standard techniques, such as the hybridoma technique first described by Kohler et al (Nature 256: 495-. Techniques for generating hybridomas are well known (see generally, Current Protocols in Immunology,1994, Coligan et al (eds.), John Wiley & Sons, Inc., New York, NY). Monoclonal antibody-producing hybridoma cells are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide or epitope of interest, e.g., using a standard ELISA assay.

Variants of the antibodies or antigen-binding fragments thereof described herein can be prepared by introducing appropriate nucleotide changes into DNA encoding the human, humanized or chimeric antibodies or antigen-binding fragments thereof described herein, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acid sequence of the antigen binding site that makes up the antibody or antigen binding domain. In a population of such variants, some antibodies or antigen-binding fragments will have increased affinity for the target protein. Any combination of deletions, insertions, and/or combinations can be made to obtain an antibody or antigen-binding fragment thereof with increased binding affinity for the target. Amino acid changes introduced into an antibody or antigen-binding fragment can also alter or introduce new post-translational modifications into the antibody or antigen-binding fragment, such as altering (e.g., increasing or decreasing) the number, type, or glycosylation sites (e.g., altering the amino acid sequence to attach a different sugar to an enzyme present in the cell), or introducing new glycosylation sites.

The antibodies disclosed herein can be derived from any animal species, including mammals. Non-limiting examples of natural antibodies include antibodies derived from humans, primates (e.g., monkeys and apes), cows, pigs, horses, sheep, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits), including transgenic rodents that have been genetically engineered to produce human antibodies.

Phage display (panning) can be used to optimize antibody sequences with a desired binding affinity. In this technique, a gene encoding a single chain Fv (comprising VH or VL) can be inserted into the phage coat protein gene, allowing the phage to "display" the scFv on its outside while containing the gene for that protein on its inside, thereby linking between genotype and phenotype. These displayed phage can then be screened against the target antigen in order to detect the interaction between the displayed antigen binding site and the target antigen. Thus, large protein libraries can be screened and amplified in a process called in vitro selection, and antibody sequences with the desired binding affinity can be obtained.

Human and humanized antibodies include antibodies having variable and constant regions derived from human germline immunoglobulin sequences (or having amino acid sequences identical to those derived from human germline immunoglobulin sequences). Human antibodies can include, for example, amino acid residues in the CDRs that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

Humanized antibodies typically have a human Framework (FR) into which non-human CDRs are grafted. Thus, a humanized antibody has one or more amino acid sequences introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed by, for example, replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence. Such methods are described, for example, in Jones et al, Nature,321:522-525 (1986); riechmann et al, Nature,332: 323-; verhoeyen et al, Science,239:1534-1536(1988), each of which is incorporated herein by reference in its entirety. Thus, a "humanized" antibody is a chimeric antibody in which substantially less than the entire human V domain has been substituted with the corresponding sequence from a non-human species. In practice, humanized antibodies are typically mouse antibodies in which some CDR residues and some FR residues are substituted by residues from analogous sites in human antibodies.

It is further important to humanize antibodies while retaining high specificity and affinity for antigens and other favorable biological properties. To achieve this goal, humanized antibodies can be made by a process of analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and familiar to those skilled in the art. Computer programs can be used to illustrate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these displays allows analysis of the likely role of the residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this manner, FR residues can be selected from the acceptor and donor sequences and combined to achieve a desired antibody characteristic, such as increased affinity for the target antigen.

Identity or homology to the original sequence is typically the percentage of amino acid residues in the candidate sequence that are identical to the sequence present in the human, humanized or chimeric antibody or fragment that are identical after alignment and the introduction of gaps, if necessary, to achieve the maximum percent sequence identity and without regard to any conservative substitutions as part of the sequence identity.

In some embodiments, the antibody or antigen-binding fragment thereof may be covalently modified. These covalent modifications can be made by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of antibodies or antibody fragments are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with organic derivatizing agents capable of reacting with selected side chains or N-or C-terminal residues.

In some embodiments, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The content of fucose was determined by calculating the average content of fucose in the Asn297 sugar chain relative to the sum of all sugar structures (e.g., complex, hybrid and high mannose structures) attached to Asn297 as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to an asparagine residue located at about position 297 in the Fc region (Eu numbering of residues in the Fc region; or position 314 in Kabat numbering). However, due to minor sequence variations in the antibody, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e. between positions 294 and 300. Such fucosylated variants may have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody may be further engineered to replace the asparagine at position 297 with alanine (N297A).

In some embodiments, to facilitate production efficiency by avoiding Fab arm exchange, the Fc region of the antibody is further engineered to replace serine at position 228 (EU numbering) of IgG4 with proline (S228P). A detailed description of The S228 mutation is described, for example, in Silva et al, "The S228P mutation predictions in vivo and in vitro IgG4 Fab-arm ex change as purified using a combination of novel quantitative immunological assays and physical matrix prediction," Journal of Biological Chemistry 290.9(2015): 5462-.

In some embodiments, the methods described herein are designed to make bispecific antibodies. Bispecific antibodies can be prepared by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers recovered from recombinant cell culture. For example, the interface may comprise at least a portion of the CH3 domain of the antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing larger amino acid side chains with smaller ones (e.g., alanine or threonine), compensatory "cavities" of the same or similar size to the larger side chains are created at the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers relative to other undesired end products (like dimers). This process is described, for example, in WO 96/27011, which is incorporated by reference in its entirety.

In some embodiments, one or more amino acid residues in the CH3 portion of the IgG are substituted. In some embodiments, one heavy chain has one or more of substitutions Y349C and T366W. The other heavy chain may have one or more of the following substitutions: E356C, T366S, L368A and Y407V. Furthermore, substitutions (-ppcpSCp- - > -ppcpPcp-) may also be introduced at the hinge region of two substituted IgGs. In some embodiments, one heavy chain has a T366Y (knob) substitution, while the other heavy chain has a Y407T (hole) substitution.

In addition, anion exchange chromatography can be used to purify bispecific antibodies. Anion exchange chromatography is a process of separating substances according to their charges using an ion exchange resin containing positively charged groups such as Diethylaminoethyl (DEAE). In solution, the resin is coated with positively charged counter ions (cations). The anion exchange resin will bind to the negatively charged molecule, replacing the counter ion. Anion exchange chromatography can be used to purify proteins based on their isoelectric point (pi). The isoelectric point is defined as the pH at which the protein has no net charge. When pH > pi, the protein carries a net negative charge; when pH < pi, the protein carries a net positive charge. Thus, in some embodiments, different amino acid substitutions may be introduced into the two heavy chains, such that pi for a homodimer comprising two arms a and pi for a homodimer comprising two arms B are different. The pi of a bispecific antibody with arm a and arm B will be between two pi of a homodimer.

Thus, the two homodimers and bispecific antibodies can be released under different pH conditions. The present disclosure shows that some amino acid residue substitutions can be introduced into the heavy chain to modulate the pI.

Thus, in some embodiments, the amino acid residue at Kabat numbering position 83 is lysine, arginine, or histidine. In some embodiments, the amino acid residue at one or more of positions 1, 6, 43, 81 and 105(Kabat numbering) is aspartic acid or glutamic acid.

In some embodiments, the amino acid residue at one or more of positions 13 and 105(Kabat numbering) is aspartic acid or glutamic acid. In some embodiments, the amino acid residue at one or more of positions 13 and 42 (Kabat numbering) is lysine, arginine, histidine, or glycine.

Bispecific antibodies can also include, for example, cross-linked or "heteroconjugated" antibodies. For example, one antibody in the heterologous conjugate can be coupled to avidin and the other to biotin. Heteroconjugate antibodies can also be prepared using any convenient cross-linking method. Suitable crosslinking agents and crosslinking techniques are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, which is incorporated herein by reference in its entirety.

Methods for producing bispecific antibodies from antibody fragments are also known in the art. For example, bispecific antibodies can be prepared using chemical ligation. Brennan et al (Science 229:81,1985) describe methods for proteolytic cleavage of intact antibodies to generate F (ab') 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize the vicinal dithiols and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted to Thionitrobenzoate (TNB) derivatives. One of the Fab ' TNB derivatives is then converted to the Fab ' thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab ' TNB derivative to form the bispecific antibody.

Method of treatment

The methods described herein include methods for treating diseases associated with cancer. Typically, the method comprises administering a therapeutically effective amount of an engineered bispecific antibody (e.g., an unbalanced bispecific antibody) or antigen-binding fragment thereof as described herein to a subject in need or determined to be in need of such treatment.

As used herein, "treating" refers to ameliorating at least one symptom of a condition associated with cancer. Often, cancer leads to death. Thus, treatment may result in an increase in life expectancy (e.g., at least 1, 2,3, 4,5, 6, 7, 8, 9, 10, 11, 12 months, or at least 1, 2,3, 4,5, 6, 7, 8, 9, 10 years). Administration of a therapeutically effective amount of an agent described herein (e.g., an unbalanced bispecific antibody) to treat a disorder associated with cancer will result in a reduction in the number of cancer cells and/or a reduction in symptoms.

As used herein, the term "cancer" refers to a cell that has the ability to grow autonomously, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. The term is intended to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, regardless of histopathological type or invasive stage. The term "tumor" as used herein refers to a cancerous cell, e.g., a mass of cancerous cells. Cancers that may be treated or diagnosed using the methods described herein include malignancies of various organ systems, such as adenocarcinoma cancers affecting the lung, breast, thyroid, lymphoid, gastrointestinal and genitourinary tracts, as well as including malignancies (e.g., most colon), renal cell carcinoma, prostate cancer and/or testicular tumors, lung non-small cell carcinoma, small bowel cancer, and esophageal cancer. In some embodiments, the agents described herein are designed to treat or diagnose cancer in a subject. The term "cancer" is art recognized and refers to malignancies of epithelial or endocrine tissue, including cancers of the respiratory system, gastrointestinal system, genitourinary system, testicular, breast, prostate, endocrine system, and melanoma. In some embodiments, the cancer is renal cancer or melanoma. Exemplary cancers include those formed from cervical, lung, prostate, breast, head and neck, colon, and ovarian cancer tissues. The term also includes carcinosarcomas, for example, which include malignant tumors composed of cancerous and sarcoma tissues. "adenocarcinoma" refers to a cancer derived from glandular tissue or in which tumor cells form recognizable glandular structures. The term "sarcoma" is art-recognized and refers to mesenchymal-derived malignancies.

In some embodiments, the cancer is rituximabResistant cancer.

In one aspect, the disclosure also provides methods for treating cancer in a subject, methods of reducing the rate of increase in tumor volume over time in a subject, methods of reducing the risk of developing metastasis, or methods of reducing the risk of developing additional metastasis in a subject. In some embodiments, the treatment can stop, slow, delay, or inhibit the progression of the cancer. In some embodiments, the treatment may result in a reduction in the number, severity, and/or duration of one or more cancers in the subject.

In one aspect, the disclosure features methods that include administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof or antibody drug conjugate disclosed herein to a subject in need thereof, e.g., having, identified, or diagnosed with: such as breast cancer (e.g., triple negative breast cancer), carcinoids, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urinary tract cancer, or hematological malignancies.

As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification and describe an animal, human or non-human, that provides treatment according to the methods of the invention. The present invention contemplates veterinary and non-veterinary applications. The human patient may be an adult human or a minor human (e.g., a human under 18 years of age). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates (including, e.g., non-human primates such as monkeys, chimpanzees, gorillas, and the like), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorpha, swine (e.g., pigs, mini-pigs) horses, dogs, cats, cows, and other domestic, farm, and zoo animals.

In some embodiments, the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung cancer (NSCLC), Small Cell Lung Cancer (SCLC), bladder cancer, or metastatic hormone refractory prostate cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is squamous cell carcinoma of the head and neck (SCCHN), Renal Cell Carcinoma (RCC), Triple Negative Breast Cancer (TNBC), or colorectal cancer. In some embodiments, the subject has hodgkin's lymphoma. In some embodiments, the subject has Triple Negative Breast Cancer (TNBC), gastric cancer, urothelial cancer, merkel cell cancer, or head and neck cancer. In some embodiments, the cancer is melanoma, pancreatic cancer, mesothelioma, hematologic malignancies, in particular non-hodgkin's lymphoma, chronic lymphocytic leukemia, or advanced solid tumors.

In some embodiments, the compositions and methods disclosed herein can be used to treat a patient at risk for developing cancer. Cancer patients can be identified by various methods known in the art.

As used herein, "effective amount" refers to an amount or dose sufficient to produce a beneficial or desired result, including halting, slowing, delaying or inhibiting the progression of a disease, such as cancer. The effective amount will vary depending on the age and weight of the subject to which the antibody, antigen-binding fragment, antibody-drug conjugate, antibody-encoding polynucleotide, polynucleotide-containing vector, and/or composition thereof is administered, the severity of the symptoms, and the route of administration, and thus the mode of administration can be determined on an individual basis.

An effective amount may be administered in one or more administrations. For example, an effective amount of an antibody, antigen-binding fragment, or antibody-drug conjugate is an amount sufficient to ameliorate, halt, stabilize, reverse, inhibit, slow, and/or delay the progression of an autoimmune disease or cancer in a subject, or an amount sufficient to ameliorate, halt, stabilize, reverse, slow, and/or delay the proliferation of a cell (e.g., a biopsy cell, any cancer cell or cell line described herein (e.g., a cancer cell line)) in vitro. As understood in the art, the effective amount of antibody, antigen-binding fragment, or antibody-drug conjugate can vary, depending on, inter alia, the patient's medical history and other factors, such as the type (and/or dosage) of antibody used.

Effective amounts and dosing regimens for administering the antibodies, antibody-encoding polynucleotides, antibody-drug conjugates, and/or compositions disclosed herein can be determined empirically, and making such determinations is within the skill of the art. One skilled in the art will appreciate that the dosage that must be administered will depend on, for example, the mammal that will receive the antibody, antibody-encoding polynucleotide, antibody-drug conjugate and/or composition disclosed herein, the route of administration, the particular type of antibody, the polynucleotide encoding the antibody, the antigen-binding fragment, the antibody-drug conjugate and/or the composition disclosed herein, as well as other drugs that will be administered to the mammal. Guidance for selecting appropriate dosages of Antibodies or antigen-binding fragments can be found in the literature for therapeutic uses of Antibodies and antigen-binding fragments, e.g., Handbook of Monoclonal Antibodies, Ferrone et al, Nos. Publications, Park Ridge, N.J.,1985, Chapter 22, p.303-357; smith et al, Antibodies in Human diagnostics and Therapy, Haber et al, Raven Press, New York,1977, pp 365-.

A typical daily dose of an effective amount of antibody is 0.01mg/kg to 100 mg/kg. In some embodiments, the dose may be less than 100mg/kg, 10mg/kg, 9mg/kg, 8mg/kg, 7mg/kg, 6mg/kg, 5mg/kg, 4mg/kg, 3mg/kg, 2mg/kg, 1mg/kg, 0.5mg/kg, or 0.1 mg/kg. In some embodiments, the dose may be greater than 10mg/kg, 9mg/kg, 8mg/kg, 7mg/kg, 6mg/kg, 5mg/kg, 4mg/kg, 3mg/kg, 2mg/kg, 1mg/kg, 0.5mg/kg, 0.1mg/kg, 0.05mg/kg, or 0.01 mg/kg. In some embodiments, the dose is about 10mg/kg, 9mg/kg, 8mg/kg, 7mg/kg, 6mg/kg, 5mg/kg, 4mg/kg, 3mg/kg, 2mg/kg, 1mg/kg, 0.9mg/kg, 0.8mg/kg, 0.7mg/kg, 0.6mg/kg, 0.5mg/kg, 0.4mg/kg, 0.3mg/kg, 0.2mg/kg, or 0.1 mg/kg.

In any of the methods described herein, the at least one antibody, antigen-binding fragment thereof, antibody-drug conjugate, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding fragments, antibody-drug conjugates, or pharmaceutical compositions described herein), and optionally the at least one other therapeutic agent, can be administered to the subject at least once per week (e.g., once per week, twice per week, three times per week, four times per week, once per day, twice per day, or three times per day). In some embodiments, at least two different antibodies and/or antigen-binding fragments are administered in the same composition (e.g., a liquid composition). In some embodiments, the at least one antibody, antigen-binding fragment, antibody-drug conjugate, and at least one other therapeutic agent are administered in the same composition (e.g., a liquid composition). In some embodiments, the at least two antibodies or antigen-binding fragments and the at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition comprising the at least one antibody or antigen-binding fragment and a solid oral composition comprising the at least one additional therapeutic agent). In some embodiments, the at least one additional therapeutic agent is administered in the form of a pill, tablet, or capsule. In some embodiments, the at least one additional therapeutic agent is administered in a sustained release oral formulation.

In some embodiments, one or more additional therapeutic agents may be administered to the subject before or after administration of at least one antibody, antigen-binding antibody fragment, antibody-drug conjugate, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding fragments, antibody-drug conjugates, or pharmaceutical compositions described herein). In some embodiments, one or more additional therapeutic agents and at least one antibody, antigen-binding antibody fragment, antibody-drug conjugate, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding fragments, antibody-drug conjugates, or pharmaceutical compositions described herein) are administered to a subject such that the biological activity phases of the one or more additional therapeutic agents and the at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) overlap.

In some embodiments, at least one antibody, antigen-binding antibody fragment, antibody-drug conjugate, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) can be administered to a subject over an extended period of time (e.g., a period of time that exceeds at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years). The length of the treatment period can be determined by a skilled medical professional using any of the methods described herein to diagnose or track the effectiveness of the treatment (e.g., observing at least one symptom of cancer). As described herein, the skilled medical professional can also alter the confirmation and quantity (e.g., increase or decrease) of the antibody or antigen-binding antibody fragment, antibody-drug conjugate (and/or one or more other therapeutic agents) administered to the subject. And the dose or frequency of administration of the at least one antibody or antigen-binding antibody fragment (and/or one or more other therapeutic agents) to the subject can also be adjusted (e.g., increased or decreased) based on the assessment of the therapeutic effect (e.g., using any of the methods described herein and known in the art).

In some embodiments, one or more additional therapeutic agents may be administered to the subject. The additional therapeutic agent may comprise one or more inhibitors selected from the group consisting of: B-Raf inhibitors, EGFR inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, Anaplastic Lymphoma Kinase (ALK) inhibitors, phosphatidylinositol 3 kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, PI3K/mTOR dual inhibitors, Bruton's Tyrosine Kinase (BTK) inhibitors, and isocitrate dehydrogenase 1(IDH1) and/or isocitrate dehydrogenase 2(IDH2) inhibitors. In some embodiments, the additional therapeutic agent is an indoleamine 2, 3-dioxygenase-1 (IDO1) inhibitor (e.g., epacadostat).

In some embodiments, the additional therapeutic agent may comprise one or more inhibitors selected from the group consisting of: HER3 inhibitors, LSD 1 inhibitors, MDM2 inhibitors, BCL2 inhibitors, CHK1 inhibitors, inhibitors of the activation hedgehog signaling pathway, and drugs that selectively degrade estrogen receptors.

In some embodiments, the additional therapeutic agent may comprise one or more therapeutic agents selected from the group consisting of: trabectedin, nabumeclidin, Trebananib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, petasitin, ceritinib, sotriptan, temsirolimus, acitinib, everolimus, sorafenib, voltrient, pazopanib, IMA-901, AGS-003, cabozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temozolomide, IL-2, IFNa, vinblastine, thalidomide, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomib, amrubicin, carfilzomib, pralatrexate, and enzastaurin.

In some embodiments, the additional therapeutic agent may comprise one or more therapeutic agents selected from the group consisting of: adjuvants, TLR agonists, Tumor Necrosis Factor (TNF) alpha, IL-1, HMGB1, IL-10 antagonists, IL-4 antagonists, IL-13 antagonists, IL-17 antagonists, HVEM antagonists, ICOS agonists, CX3CL1 targeted therapeutics, CXCL9 targeted therapeutics, CXCL10 targeted therapeutics, CCL5 targeted therapeutics, LFA-1 agonists, ICAM1 agonists, and selectin agonists.

In some embodiments, carboplatin, nabumetone, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI is administered to the subject.

In some embodiments, the additional therapeutic agent is an anti-OX 40 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, or an anti-GITR antibody.

Pharmaceutical compositions and routes of administration

Also provided herein are pharmaceutical compositions comprising at least one (e.g., one, two, three, or four) of the antibodies, antigen-binding fragments, or antibody-drug conjugates described herein. Two or more (e.g., two, three, or four) of any of the antibodies, antigen-binding fragments, or antibody-drug conjugates described herein can be present in the pharmaceutical composition in any combination. The pharmaceutical compositions may be formulated in any manner known in the art.

The pharmaceutical composition is formulated to be compatible with its intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The composition may include a sterile diluent (e.g., sterile water or saline), a fixed oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvent, an antibacterial or antifungal agent, such as benzyl alcohol or methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, an antioxidant, such as ascorbic acid or sodium bisulfite, a chelating agent, such as ethylenediaminetetraacetic acid, a buffering agent, such as an acetate, citrate, or phosphate, an isotonic agent, such as a sugar (e.g., glucose), a polyol (e.g., mannitol or sorbitol), or a salt (e.g., sodium chloride), or any combination thereof. Liposomal suspensions can also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Pat. No. 4,522,811). The compositions may be formulated and enclosed in ampoules, disposable syringes or multiple dose vials. Proper fluidity can be maintained, when required (e.g., in injectable formulations), by the use of a coating, for example, lecithin or a surfactant. Absorption of the antibody, or antigen-binding fragment thereof, can be prolonged by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin. Alternatively, controlled release may be achieved by implants and microencapsulated delivery systems, which may include biodegradable biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.).

Compositions comprising any one or more of the antibodies, antigen-binding fragments, antibody-drug conjugates described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (e.g., physically dispersed units containing a predetermined amount of the active compound to facilitate administration and uniformity of dosage).

Toxicity and therapeutic efficacy of the compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). LD50 (the dose lethal to 50% of the population) and ED50 (the therapeutic dose effective in 50% of the population) can be determined: the therapeutic index is the ratio of LD50 to ED 50. Agents that exhibit high therapeutic indices are preferred. If the drug exhibits adverse side effects, care should be taken to minimize potential damage (i.e., reduce adverse side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

The data obtained from cell culture assays and animal studies can be used to formulate an appropriate dose of any given agent for use in a subject (e.g., a human). A therapeutically effective amount of one or more (e.g., one, two, three, or four) antibodies or antigen-binding fragments thereof (e.g., any of the antibodies or antibody fragments described herein) will be an amount that treats a disease in a subject (e.g., a human subject identified as having cancer) (e.g., kills cancer cells) or in a subject identified as at risk for the disease (e.g., a subject that has previously had cancer but is now cured), reduces the severity, frequency, and/or duration of one or more disease symptoms in the subject (e.g., a human). The effectiveness and dosage of any antibody or antigen-binding fragment described herein can be determined by a healthcare professional or veterinary professional using methods known in the art, and by observing one or more symptoms of a disease in a subject (e.g., a human). Certain factors may affect the dosage and time required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the overall health and/or age of the subject, and the presence of other diseases).

Exemplary doses include milligrams or microgram amounts of any of the antibodies or antigen-binding fragments or antibody-drug conjugates described herein per kilogram body weight of the subject (e.g., about 1 μ g/kg to about 500 mg/kg; about 100 μ g/kg to about 50 mg/kg; about 10 μ g/kg to about 5 mg/kg; about 10 μ g/kg to about 0.5 mg/kg; or about 1 μ g/kg to about 50 μ g/kg). Although these dosages cover a wide range, one of ordinary skill in the art will appreciate that the efficacy of the therapeutic agents, including antibodies and antigen-binding fragments thereof, varies and that an effective amount can be determined by methods known in the art. Typically, a relatively low dose is administered first, and then the healthcare or veterinary professional (in a therapeutic application) or researcher at visit (while still in development) can gradually increase the dose until an appropriate response is obtained. In addition, it will be understood that the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, and diet of the subject, time, mode of administration, route of administration, rate of excretion, and half-life of the antibody or antibody fragment in vivo.

The pharmaceutical composition may be contained in a container, package or dispenser together with instructions for administration. The present disclosure also provides methods of making antibodies or antigen-binding fragments thereof or antibody-drug conjugates for the various uses described herein.

Examples

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1: method and material

The following assays were used in the examples below.

Binding assays

a) 50 μ l of 5X 10 in medium⁵Cells were dispensed into each well of a 96-well plate.

b) Different dilutions of 100 μ L antibody were added to the wells.

c) Incubate the plate at Room Temperature (RT) for 60 min.

d) Cells were centrifuged quickly and cell x3 was washed with Phosphate Buffered Saline (PBS) containing 0.1 Bovine Serum Albumin (BSA).

e) The cell pellet was resuspended in 100. mu.L PBS containing 0.1% BSA in 1:500 Cy 3-conjugated goat anti-human IgG.

f) Incubate for 30 minutes in the dark at room temperature.

g) Cells were washed 3 times and resuspended in Fluorescence Activated Cell Sorting (FACS) buffer.

h) Cells were analyzed on a flow cytometer.

Antibody Dependent Cellular Cytotoxicity (ADCC) assay

a) Target cells were washed once with PBS before calcein AM labeling.

b) Target cells were labeled with 2.5mM 1:333 calcein AM stock.

c) Cells were incubated at 37 ℃ for 30 minutes in the absence of light.

d) Cells were washed 3 times with PBS.

e) 50 μ L of 1X10⁴Calcein AM-labeled target cells were dispensed into each well.

f) Mu.l of diluted antibody was added to the wells.

g) The plates were incubated at room temperature for 60 minutes.

h) Adding into 50 μ L of culture medium5X 10 of⁴PBMS (Effector cells) (E/T ratio 5).

i) The plates were incubated at 37 ℃ for 4 hours.

j) Cells were centrifuged quickly and 180. mu.l of supernatant was transferred to another 96-well plate with a translucent bottom and black walls.

k) The plate was read at a 485 excitation wavelength and a 520 emission wavelength.

Complement Dependent Cytotoxicity (CDC) assay

a) Target cells were collected and stained with calcein AM as ADCC (calcein release assay only).

b) 50. mu.L of target cells (1X 10)⁵Individual cells) were seeded in wells of a 96-well plate.

c) 100 μ L of antibody was added to the wells at different concentrations.

d) The plates were incubated at room temperature for 15 minutes.

e) To each well 50 μ L of 10% complement-enriched human serum was added (final 5%).

f) The plates were incubated at room temperature for 45 minutes.

g) Transfer 180 μ L of supernatant to another 96-well plate with translucent bottom and black walls. (for calcein release assay only).

h) Cells were washed 3 times with PBS containing 0.1% BSA.

i) Cells were stained with 2. mu.L of 7-amino-actinomycin D (7AAD) per well in the dark at room temperature for 15 minutes.

j) Cells were washed three times and analyzed on a flow cytometer.

T cell activation

Pre-activated Peripheral Blood Mononuclear Cells (PBMCs) were used in some ADCC experiments.

a) PBMCs were activated using Dynabeads (human T activator CD3/CD 28).

b) After washing with buffer, Dynabeads were added to PBMC at a ratio of 1:1 with 30U/mL of interleukin-2 (IL 2).

c) The cell mixture is incubated for a sufficient time.

d) At the end of incubation, the magnetic beads were removed by magnet and the activated PBMCs were used for ADCC analysis.

Cell binding assays involving MDA231 cells

a) 1X10 in the culture medium⁶Concentration of cells/mL MDA231 cells were prepared.

b) The antibody sample was diluted to the appropriate concentration.

c) 50 μ L of cells were transferred to each well of a 96-well V-shaped plate.

d) 50 μ L of antibody was transferred to wells of a 96-well V-shaped bottom plate.

e) The cell mixture was incubated at room temperature for 60 minutes.

f) Cells were centrifuged quickly and washed twice with FACS buffer.

g) Cells were resuspended in 100. mu.L of FACS buffer containing Cy 3-conjugated goat anti-human (GAH) IgG (1:500) per well.

h) Incubate for 30 min at room temperature and wash x 2 with FACS buffer.

i) FACS analysis.

Internalization assays involving MDA231 and SIHA cells

a) Add 50. mu.L to each well of 96-well plate at 1X10⁶Suspension cells (MDA231 or SIHA cells) in an amount of/mL.

b) Add 50. mu.L Ab to the corresponding wells.

c) Incubate at 37 ℃ for 30 minutes.

d) mu.L of pHrodo Red-labeled GAH IgG was added to each well and incubated at 37 ℃ for 24 hours.

e) Cells were trypsinized and harvested, washed twice, and then FACS was run.

Complement Dependent Cytotoxicity (CDC) assays involving MDA231 cells

a) Target cell: MDA231 cells were washed twice with PBS and then adjusted to 0.5X 10 in PBS⁶Concentration in/mL.

b) Seed cells were plated in flat bottom 24-well plates at 300. mu.L per well.

c) To the corresponding wells, 300. mu.L of 20. mu.g/mL antibody was added to give a final concentration of 10. mu.g/mL.

d) The plates were incubated at 37 ℃ for 48 hours.

e) After the incubation was completed, the cells were digested with trypsin and washed twice with a common medium.

f) The cell pellet was resuspended in 100. mu.L of normal medium, and the cells were transferred to a 96-well plate.

g) 100 μ L of 10% complement-enriched serum was added to each well.

h) Cells were incubated at 37 ℃ for 4 hours.

i) Cells were washed twice with FACS buffer.

j) Cells were detached by adding 100 μ L trypsin for 3 minutes.

k) Resuspending the cell pellet in FACS buffer containing 7AAD (1:50 dilution)

1) After incubation for 15 min at room temperature, the cells were washed twice.

m) running FACS analysis.

Example 2: bispecific antibodies that bind to CD20 and CD3

Bispecific antibodies were designed to bind CD20 and CD 3. The bispecific antibody has two common light chains (identical in sequence) and two different heavy chains. The sequences of the two heavy chain and common light chain variable regions are shown below.

VHa against CD20 (designed from rituximab VH):

VHb against CD3 (designed from MAb 12F6 VH):

common VL (VL of rituximab)

12F6 antibodies are described, for example, in the Construction and characterization of a humanized anti-human CD3monoclonal antibodies 12F6 with effective immunization functions, Immunology,116(4),487-498(2005), which is incorporated herein by reference in its entirety. For comparison purposes, the sequences of the parent antibodies are also shown below:

parent CD20VH (VH of rituximab):

parent CD20VL (rituximab):

parent CD3VH (MAb 12F6 VH):

parent CD3VL (MAb 12F6 VL):

the following table also summarizes the CDR sequences of the redesigned VH and VL:

TABLE 1 VHa for the heavy chain of CD20

TABLE 2 VHb for the heavy chain of CD3

TABLE 3VL of common light chains

Rituximab Fv (VH + VL) has a 3D (three-dimensional) isoelectric Point (PI) of 9.9, whereas 12F6 Fv has a 3D PI of 9.8. After redesign of the sequence, the 3D PI for VHa + common VL was 10.0 and the 3D PI for VHb + common VL was 9.1. PI changes do not affect binding affinity to CD20, and the second antigen-binding region still retains reasonable binding affinity to CD 3. Mutations in both VH chains are shown in the table below.

TABLE 4 modified amino acids in VH (CD20)

Kabat numbering	Amino acids in the parent	Modified amino acids
			83	T	R

TABLE 5 modified amino acids in VH (CD3)

Kabat numbering	Amino acids in the parent	Modified amino acids
			1	Q	E
6	Q	E
			43	Q	E
81	Q	E
			105	Q	E

Referring to fig. 1A and 1B, the antigen binding ability of redesigned rituximab (antibody a) and redesigned 12F6 (antibody B), respectively, was tested. Figure 1A shows binding of redesigned rituximab (antibody a) to CD20 positive Raji cells. Antibody A is a homodimer with two VHas (SEQ ID NO: 1) and two common VLs (SEQ IN NO: 3). FIG. 1B shows binding of redesigned 12F6 (antibody B) to CD3 positive Jurkart cells. Antibody B is also a homodimer with two VHbs (SEQ ID NO: 2) and two common VLs (SEQ ID NO: 3). These data indicate that the redesigned rituximab heavy chain, the redesigned 12F6 heavy chain, and the common light chain can be combined into a functional bispecific antibody, for example, by a "knob and hole structure.

Thus, a "bispecific antibody unbalanced" of CD20/CD3 was designed. Mutations in the knob structure were also introduced into the constant region of the heavy chain to facilitate bispecific antibody formation.

The full length sequences of the heavy and light chains are shown below:

full length of heavy chain variant 1 against CD20 (wild-type IgG1 Fc):

full length of heavy chain variant 2 against CD20 (IgG1 Fc with Y407T (hole) mutation):

full length of heavy chain variant 3 against CD20 (IgG1 Fc with T366Y (knob) mutation):

full length of heavy chain variant 1 against CD3 (wild-type IgG1 Fc):

full length of heavy chain variant 2 against CD3 (IgG1 Fc with T366Y (knob) mutation):

full length of heavy chain variant 3 against CD3 (IgG1 Fc with Y407T (hole) mutation):

full length for common light chain:

the IgG1 heavy chain against CD20 with Y407T (EU numbering) (variant 2; SEQ ID NO: 35) and the IgG1 heavy chain against CD3 with the T366Y (EU numbering) mutation (variant 2; SEQ ID NO: 38) were selected to make bispecific antibodies for further experiments. The bispecific antibody also has two common light chains (SEQ ID NO: 40).

This unbalanced bispecific antibody also has the following characteristics: (1) CD3 binding affinity was significantly reduced to improve safety; (2) maintaining ADCC/CDC effector function to expand clinical applications; (3) biochemical and biophysical characteristics of the CD20 binding arm and the CD3 binding arm were distinguished in order to better isolate the bispecific antibody during downstream purification.

As shown in the examples below, the antibody had better CD20+ Raji cell killing efficacy compared to CD20 homodimer and rituximab in the presence of human PBMC. At the same time, the antibody does not kill CD3+ Jurkat cells or deplete normal T cells under the same conditions. Therefore, the antibody has a wide clinical application prospect compared with the current anti-CD 20 cancer therapy: 1) the antibody has a T cell recruitment function compared to rituximab; 2) the antibody retains functional effector function compared to CAR-T/other T cell recruitment therapy; 3) the antibody has no any potential safety hazard in vitro. In view of the above, the CD20/CD3 bispecific antibodies and platforms described in the present disclosure can address an unmet need in the field of targeted cancer therapy.

The bispecific antibodies disclosed herein were purified by two steps: affinity purification using protein a (round 1) and anion exchange purification using monoQ5/50 (round 2). In the second round, the antibody is eluted using a gradient pH buffer (e.g. PBS). T cell activation assays were performed to evaluate different fractions after elution. In fig. 20, the numbers indicate different fractions. Only the CD20/CD3 bispecific antibody can activate T cells, so the T cell activation assay can assess the purity and content of the CD20/CD3 bispecific antibody in each fraction. The results indicate that fractions 4-7 have relatively pure CD20/CD3 bispecific antibody and demonstrate that the CD20/CD3 bispecific antibody can be purified by the methods described herein.

In addition, the pI of the antibodies described herein has also been determined. This information is useful for selecting a suitable pH elution.

TABLE 6

	PI
		CD20/CD3 BsAb variant 1	8.48
CD20 homodimer variant 1	8.72
		CD3 homodimer variant 1	8.09
CD20/CD3 BsAb variant 2	8.48
		CD20 homodimer variant 2	8.73
CD3 homodimer variant 2	8.09
		CD20/CD3 BsAb variant 3	8.48
CD20 homodimer variant 3	8.72
		CD3 homodimer variant 3	8.09
CD20 parent Ab	8.66
		CD3 parent Ab	8.59

Example 3: binding affinity of bispecific antibodies

The CD20 homodimer IgG comprising the designed VH sequence (SEQ ID NO: 1) and the common VL sequence (SEQ ID NO: 3) had similar binding ability to CD20 as compared to the parent anti-CD 20 IgG (parent CD20) as calculated and designed. The cell binding affinity assay was performed using Raji cells (expressing CD 20). The binding results are shown in FIG. 2A.

The binding capacity of CD3 homodimer IgG to CD3 comprising the designed VH sequence (SEQ ID NO: 2) and the common VL sequence (SEQ ID NO: 3) was reduced compared to the parental anti-CD 3 IgG (parental CD 3). The cell binding affinity assay was performed with Jurkat cells (expressing CD 3). The binding results are shown in FIG. 2B.

Example 4: unbalanced CD20/CD3 bispecific antibodies activate T cells

The unbalanced CD20/CD3 bispecific monoclonal antibody (BsMab) activated T cells only in the presence of target tumor cells. The following experiment was performed by using Raji cells as CD20+ target tumor cells, 293 cells as CD 20-control cells and Jurkat cells as T cell models to test whether CD20/CD3BsMab can activate T cells in the presence of target tumor cells because of the cluster formed by multiple bsmabs that simultaneously bind to T cells and target tumor cells. In contrast, CD20/CD3BsMab failed to activate T cells in the presence of CD 20-control cells due to weak binding of one arm to CD3 on T cells.

The following experimental procedure was used in this example:

1) raji and Jurkat, Jurkat and 2931X 10 were inoculated in U-shaped bottom 96-well plates, respectively⁵。

2) Test antibody was added and incubated overnight (19 hours).

3) Cells were washed once with PBS + 0.1% BSA.

4) Anti-human CD69 antibody (labeled with PE) (1.5 ul/well) was added and incubated at room temperature for 30 minutes.

5) The cells were washed once.

6) And (6) reading.

The results are shown in FIG. 3. The T cell activation capacity in the presence of Raji cells with different concentrations of test antibody is shown in fig. 4. Isotype antibodies (non-specific IgG1 antibodies) were used as controls. T cell activation was measured by expressing CD69 on the surface of Jurkat cells.

Example 5: unbalanced CD20/CD3BsMab induces PBMC-mediated cell killing

Unbalanced CD20/CD3BsMab induced better PBMC-mediated cell killing compared to rituximab and CD20 homodimer antibodies before and after T cell activation.

T-precursor cell activation: fresh Peripheral Blood Mononuclear Cells (PBMCs) from healthy donors were incubated overnight at 37 ℃ for 4 hours with calcein-labeled CD20+ Raji cells in the presence of the different antibodies shown in the figure. Cell mortality was measured by calcein release. The results are shown in figure 5 of the drawings,

after T cell activation (4 days): fresh PBMCs from healthy donors were incubated with recombinant IL-2 and CD3/CD28 magnetic beads for 4 days to activate T cells, and then with calcein labeled CD20+ Raji cells and the different antibodies shown in the figure for 2 hours. Cell mortality was measured by calcein release. The results are shown in FIG. 6.

After T cell activation (7 days): fresh PBMCs from healthy donors were incubated with recombinant IL-2 and CD3/CD28 magnetic beads for 7 days to activate T cells, and then with calcein labeled CD20+ Raji cells and the different antibodies shown in the figure for 2 hours. Cell mortality was measured by calcein release. The results are shown in FIG. 7.

To address the safety issue of whether unbalanced CD20/CD3 bsmabs also killed CD3+ T cells under the same conditions as those shown in the PBMC killing assay, CD3+ Jurkat cells were used as controls for each experiment. Only the highest antibody concentration (10ug/ml) was tested. The results of T-cell activation are shown in FIG. 8. The results after T cell activation (4 days) are shown in figure 9. The results after T cell activation (7 days) are shown in figure 10. The number of Jurkat cells in the group treated with PBS was set as baseline. Thus, if the number of Jurkat cells is equal to the number of groups treated with PBS, the percent killing will be zero. If the number of cells is greater than the group treated with PBS, the percent killing is negative.

The results show that no killing of Jurkat cells was observed before and 4 days after T cell activation. However, Jurkat killing was observed at day 7 after T cell activation, in which case rituximab and CD20 homodimer IgG did not induce Raji cell killing. This suggests that 7-day Jurkat cell killing may be caused by T cell hyperactivation. To further test whether 7-day activated native T cells themselves were also killed in the presence of unbalanced CD20/CD3BsMab, the same 7-day activated PBMC were incubated overnight with different antibodies shown in the figure, and the exhaustion status of T cells was examined. The results are shown in FIG. 11. LALA in the figure is CD20/CD3BsMab with mutations L234A and L235A (EU numbering). Antibodies with the L234A and L235A mutations had no Fc effector function and were used as negative controls.

Example 6: unbalanced bispecific CD20/CD3 antibody induced T cell failure

Experiments were also performed to test whether unbalanced CD20/CD3BsMab would deplete pre-activated T cells.

FIG. 12 shows that unbalanced CD20/CD3BsMab did not deplete unactivated T cells in PBMCs after overnight incubation.

Example 7: induction of complement dependent cytotoxicity

Since CD20/CD3BsMab has an arm that binds CD20 with high affinity, experiments were performed to test whether CD20 arm binding was sufficient to induce complement dependent cytotoxicity. Antibodies were incubated with human complement-rich serum and CD20+ Raji cells. Unbalanced CD20/CD3BsMab reduced CDC efficacy compared to rituximab and CD20 homodimer antibodies. The results of detection by FACS (7AAD) are shown in FIG. 13. The results of the assay for calcein release are shown in fig. 14.

Example 8: security assessment

Unbalanced CD20/CD3BsMab was also tested for the ability to kill CD3+ Jurkat cells and normal T cells. FIG. 15 shows that high doses of unbalanced CD20/CD3BsMab do not induce CDC on Jurkat cells.

FIG. 16 shows that unbalanced CD20/CD3BsMab does not induce T cell death after co-incubation of PBMC with human serum enriched with human complement serum.

Example 9: unbalanced CD20/CD3BsMab can kill Raji cells resistant to rituximab

To test whether unbalanced CD20/CD3BsMab can kill rituximab-Resistant Raji Cells (RRCL), RRCL with 7-day activated PBMCs from three different donors were incubated in the presence of the antibodies shown in the figure. Significant RRCL killing was observed in the presence of unbalanced CD20/CD3BsMab (FIGS. 17-19).

Example 10: animal study of unbalanced CD20/CD3BsMab

Experiments were performed to evaluate the effect of CD20/CD3BsMab in animals.

Raji cells, human PBMC and unbalanced CD20/CD3BsMab were mixed and then injected into mice by intravenous injection. These Raji cells are luciferase-labeled. Each mouse in the treatment group (B-NDG, Biocytogen, Beijing, Cat. No. 201811808) received 5X 10⁵Raji cell, 2.5X 10⁶Human PBMC cells and 60 μ g antibody. Mice were imaged every three days after day 0, day 2, day 3 and day 3 to follow the depletion of Raji cells.

On day 0, luciferase-labeled Raji cells and human PBMC cells were incubated with phosphate buffered saline PBS (G1 group; control group; n ═ 4), CD20/CD3BsMab (G2 group; n ═ 4) or rituximab (anti-CD 20 antibody; G3 group; n ═ 4). Mice were imaged first 15 minutes after intravenous (i.v.) injection and then every 3 days after day 2, day 3 and day 3.

FIG. 21A shows that CD20/CD3BsMab and rituximab have no significant toxic effects. FIG. 21B shows that both CD20/CD3BsMab and rituximab have tumor suppressive effects, and that rituximab is less effective than CD20/CD3 BsMab. Differences in tumor suppression were observed starting on day 16 post-injection.

Example 11: characterization of unbalanced bispecific antibodies

Experiments were performed to characterize purified CD20/CD3 bispecific antibody samples.

First, purified CD20/CD3 bispecific antibody samples were subjected to reduced capillary electrophoresis with sodium dodecyl sulfate (Re-CE-SDS). The results show the presence of three major peaks. Based on molecular size, peak #1 was a common Light Chain (LC), and peaks #2 and #3 were two different Heavy Chains (HC) (fig. 22A).

Non-reducing CE (Non-Re-CE-SDS) was also performed. The results indicated that there was one major peak for the CD20/CD3 bispecific IgG (FIG. 22B). The results in FIGS. 22A and 22B show that the CD20/CD3 bispecific antibody sample has good purity.

Next, Differential Scanning Fluorescence (DSF) was performed to measure the melting temperature (Tm) of the protein, and Static Light Scattering (SLS) was performed to measure the aggregation temperature at 266nm (Tagg 266) and 473nm (Tagg 473). The samples were submitted to the Uncle System for analysis. The monitored temperature of DSF and SLS was raised from 20 ℃ to 95 ℃ at 1 ℃/min. Uncle measures SLS at 266nm and 473 nm. Tm and Tagg were calculated and analyzed using Uncle analysis software.

Some of the test antibodies had two Tm's, some had three Tm's. This is because IgG is a multidomain structure, the Tm of the CH2 domain in PBS is typically about 70 ℃, while CH3 is more stable, with a Tm of about 80 ℃. The Tm of Fab is in a wide range (about 50-85 ℃ C.) due to its large sequence difference. Thus, the Tm value measured by various analytical techniques is typically the "apparent" transition temperature rather than the formal melting temperature. For an antibody intact IgG, there are typically 2-3 Tm values in DSF measurements. It is not easy to determine which Tm represents which domain.

For such bispecific antibodies, the 86.7 ℃ Tm may represent only the CH3 domain. The other lower 1 or 2 Tm's indicate Fab, CH2, or Fab + CH 2.

For Tagg, this is the temperature at which SLS begins to detect aggregation. Tagg266 measures SLS at 266nm, which is more sensitive and suitable for detecting smaller particles. The measurement wavelength of Tagg473 is 473nm, which allows better detection of larger particles.

Both DSF and SLS data indicate that the CD20/CD3 bispecific antibody has good thermostability.

TABLE 7

Third, Dynamic Light Scattering (DLS) detects only molecular particles of one size (10.15 nm). The results showed no aggregation in the sample.

TABLE 8

These characterization data indicate that the CD20/CD3 bispecific antibody has good developability (developability) as a therapeutic antibody.

Example 12: bispecific antibodies that bind to PD-L1 and CD55

Two forms of bispecific antibodies were designed to bind to PD-L1 and CD55(PD-L1/CD55 BsMab v1 and PD-L1/CD55BsMab v 2). These bispecific antibodies have two common light chains and two different heavy chains.

The variable region sequences of the two heavy chains and the common light chain of the first variant of the bispecific antibody (PD-L1/CD55 BsMab v1) are shown below.

VHa for PD-L1 (designed from Avermemab)

VHb for CD55 (designed from CD55 ScFV)

Common VL (designed from CD55 ScFV)

CD55 ScFV is described in, for example, Identification of a human anti-CD55 single-chain Fv by negative working of a phase library and non-master cell lines, Cancer Res.59(11),2718-2723(1999), which is incorporated herein by reference in its entirety. For comparison purposes, the sequences of the parent antibodies are also shown below:

parent PD-L1 VH:

parent PD-L1 VL:

parent CD55 VH:

parent CD55 VL:

the 3D PI of the Ablumumab Fv (VH + VL) is 9.4, and the 3D PI of the anti-CD55 Fv is 9.8. After redesign of the sequence, the 3D PI for VHa + common VL was 9.9 and the 3D PI for VHb + common VL was 9.3. Mutations of both VH chains are shown in the table below.

Modified amino acids in VH (PD-L1)

Kabat numbering	Amino acids in the parent	Modified amino acids
			13	Q	E
105	Q	E

TABLE 10 modified amino acids in VH (CD55)

Kabat numbering	Amino acids in the parent	Modified amino acids
			13	Q	K
42	D	G

Example 13: binding affinity for newly designed PD-L1 and CD55 antibodies

Experiments were performed to determine the binding affinity of the newly designed PD-L1 and CD55 antibodies.

The binding affinity of the anti-PD-L1 homodimer IgG (PD-L1 VL) comprising the designed VH sequence (SEQ ID NO: 4) and the common VL sequence (SEQ ID NO: 6) was weaker than the parent anti-PD-L1 antibody (PD-L1 wt) (FIG. 23A). anti-CD55 homodimer IgG (CD55 v1) comprising the designed VH sequence (SEQ ID NO: 5) and the common VL sequence (SEQ ID NO: 6) had similar binding affinity compared to the parental anti-CD55 antibody (CD55 wt) (FIG. 23B).

Antibodies (CD55 vl and PD-L1 vl) do not meet this requirement, since bispecific antibodies should bind with high affinity to the cancer-specific antigen (PD-L1) and the other arm of bispecific antibodies should bind with low affinity to the cancer-associated antigen (CD 55).

Thus, the second variant of the bispecific antibody was designed to bind to PD-L1 and CD55(PD-L1/CD55 BsMab v 2). The VHa and VHb of the second bispecific antibody are identical to the VHa and VHb of the first bispecific antibody. However, the common light chain was redesigned based on the methods described herein. The sequence of the redesigned common light chain is shown below:

common VL2 (redesigned from SEQ ID NO: 6):

an alignment of common VL (SEQ IN NO: 6) and common VL2(SEQ ID NO: 7) is shown IN FIG. 24. The underlined sequences are those of the light chain constant region.

The CDR sequences of these redesigned VH and VL are shown below:

TABLE 11 VHa for the heavy chain of PD-L1

TABLE 12 VHb for the heavy chain of CD55

TABLE 13 VL variant 1 against common VL

TABLE 14 VL variant 2 against common VL

Furthermore, since lambda light chains are less common than kappa light chains in human serum, the constant region of the lambda light chains is replaced by the constant region of the kappa light chains in the examples.

Experiments were performed to determine the binding affinity of the second antibody. anti-PD-L1 homodimer IgG (PD-L1 v2) containing the designed VH sequence (SEQ ID NO: 4) and the common VL2 sequence (SEQ ID NO: 7) had similar binding affinity compared to the parental anti-PD-L1 antibody (PD-L1 wt) (FIG. 25A). anti-CD55 homodimer IgG (CD55 v2) comprising the designed VH sequence (SEQ ID NO: 5) and the common VL2 sequence (SEQ ID NO: 7) had weaker binding affinity compared to the parental anti-CD55 antibody (CD55 wt) (FIG. 25B). Thus, the binding affinity of the antibody with the redesigned sequence was satisfactory and PD-L1/CD55BsMab v2 was selected for further experiments. PD-L1/CD55BsMab v2 has two amino acid sequences comprising SEQ ID NO: 7 (kappa chain), and one IgG1 heavy chain comprising SEQ ID NO:4, one IgG1 heavy chain comprises SEQ ID NO: 5. furthermore, the heavy chain directed to PD-L1 had the Y407T mutation (EU numbering), while the IgG1 heavy chain directed to CD55 had the T366Y (EU numbering) mutation.

The full length sequences of the heavy and light chains are shown below:

full length for the heavy chain of PD-L1

Full length for the heavy chain of CD55

Variant 1 against the common light chain of CD55

Variant 2 of the common light chain to CD55

In addition, the pI of the antibodies is also determined herein. This information is useful for selecting a suitable pH elution.

Watch 15

	PI
		PDL1/CD55BsAb variant 1	8.64
PDL1 homodimer variant 1	8.36
		CD55 homodimer variant 1	8.83
PDL1/CD55BsAb variant 2	8.57
		PDL1 homodimer variant 2	8.24
CD55 homodimer variant 2	8.78
		PDL1 parent Ab	8.36
CD55 parent Ab	8.6

Example 14: complement Dependent Cytotoxicity (CDC) of PD-L1/CD55 bispecific antibody

Experiments were performed to test the complement dependent cytotoxicity of the PD-L1/CD55 bispecific antibody. The parent antibody was included for comparison. The concentration of each antibody was 10ug/ml, as determined on MDA231 cells based on the protocol described herein. The results are shown in FIGS. 26A-26B.

As shown, CDC may be induced by both anti-PD-L1 (PD-L1 wt) and anti-CD55 (CD55 wt) parent antibodies. The PD-L1/CD55 bispecific antibody v1 has a much lower CDC compared to the parent anti-PD-L1 antibody (PD-L1 wt) and the parent anti-CD55 antibody (CD55 wt). In contrast, the PD-L1/CD55 bispecific antibody v2 has a much higher CDC compared to the first variant, the parent anti-PD-L1 antibody (PD-L1 wt) and the parent anti-CD55 antibody (CD55 wt). The CDC effect of the PD-L1/CD55 bispecific antibody v2 was about 4.5 times higher than that of the bispecific antibody of the first variant.

Example 15: internalization induced by PD-L1/CD55 bispecific antibody

Experiments were performed to assess the internalization induced by the PD-L1/CD55 bispecific antibody.

Internalization assays were performed for both variants of the PD-L1/CD55 bispecific antibody and its parent antibody. MDA231 cells were used in the first internalization experiment (fig. 27A) and SIHA cells were used in the second internalization experiment (fig. 27B). Cells were mixed with 20ug/ml antibody and incubated at 37 ℃ for 30 min. The pHrodo-labeled secondary antibody was then added and incubated with the cells for 24 hours at 37 ℃. Cells were then collected and analyzed on FACS.

CD55 is a receptor for infection by echoviruses and coxsackieviruses, and is known to have internalizing ability. Thus, the parent anti-CD55 monoclonal antibody (CD55 wt) can trigger rapid internalization of CD 55. As shown in fig. 27A, internalization triggered by anti-PD-L1 antibodies was much slower than CD55 in MDA231 cells with similar expression levels of PD-L1 and CD 55. However, both PD-L1/CD55 bispecific antibodies v1 and v2 induced internalization at a rate comparable to the internalization rate of the parent anti-CD55 monoclonal antibody.

In FIG. 27B, CD55 expression was higher in SIHA cells than in PD-L1. Both PD-L1/CD55 bispecific antibodies v1 and v2 induced better internalization compared to the parent anti-PD-L1 antibody and the parent anti-CD55 antibody. Nevertheless, in view of the reduced binding of PD-L1/CD55BsMab v2 to CD55 compared to v1, PD-L1/CD55BsMab v2 should have a better efficacy/safety balance in vivo and should be safer than PD-L1/CD55BsMab v 1. Thus, PD-L1/CD55BsMab is expected to induce target cancer cell death at three different levels: (1) block the PD1/PD-L1 interaction; (2) (ii) induces internalization of PD-L1; 3) when conjugated with a drug, the antibody drug conjugate can kill cancer cells.

Other embodiments

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the appended claims.

Sequence listing

<110> AB STUDIO, Inc. (AB STUDIOs INC.)

<120> bispecific antibody and use thereof

<130> 44836-0002WO1

<140> PCT/US2018/044778

<141> 2018-08-01

<150> 62/654,112

<151> 2018-04-06

<150> 62/539,970

<151> 2017-08-01

<160> 68

<170> PatentIn version 3.5

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Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

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Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

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Met Gln Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys

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Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

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Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

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Gln Val Lys Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

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Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

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Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ala

85 90 95

Ser Thr Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 16

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 16

Ser Tyr Asn Met His

1 5

<210> 17

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 17

Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 18

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 18

Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val

1 5 10

<210> 19

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 19

Gly Tyr Thr Phe Thr Ser Tyr

1 5

<210> 20

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 20

Tyr Pro Gly Asn Gly Asp

1 5

<210> 21

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 21

Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val

1 5 10

<210> 22

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 22

Ser Tyr Thr Met His

1 5

<210> 23

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 23

Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Lys Tyr Asn Gln Lys Phe Lys

1 5 10 15

Asp

<210> 24

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 24

Trp Gln Asp Tyr Asp Val Tyr Phe Asp Tyr

1 5 10

<210> 25

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 25

Gly Tyr Thr Phe Thr Ser Tyr

1 5

<210> 26

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 26

Asn Pro Ser Ser Gly Tyr

1 5

<210> 27

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 27

Trp Gln Asp Tyr Asp Val Tyr Phe Asp Tyr

1 5 10

<210> 28

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 28

Arg Ala Ser Ser Ser Val Ser Tyr Ile His

1 5 10

<210> 29

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 29

Ala Thr Ser Asn Leu Ala Ser

1 5

<210> 30

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 30

Gln Gln Trp Thr Ser Asn Pro Pro Thr

1 5

<210> 31

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 31

Arg Ala Ser Ser Ser Val Ser Tyr Ile His

1 5 10

<210> 32

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 32

Ala Thr Ser Asn Leu Ala Ser

1 5

<210> 33

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 33

Gln Gln Trp Thr Ser Asn Pro Pro Thr

1 5

<210> 34

<211> 451

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 34

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

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

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

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

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

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

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

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

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

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

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 35

<211> 451

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 35

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

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

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

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

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

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

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

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

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

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

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 36

<211> 451

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 36

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

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

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

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

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

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

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

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

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

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

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 37

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 37

Glu Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

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

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Glu Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Lys Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Trp Gln Asp Tyr Asp Val Tyr Phe Asp Tyr Trp Gly Glu Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

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

130 135 140

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

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

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

195 200 205

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

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

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

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

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

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

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

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

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

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

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

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 38

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 38

Glu Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

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

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Glu Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Lys Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Trp Gln Asp Tyr Asp Val Tyr Phe Asp Tyr Trp Gly Glu Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

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

130 135 140

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

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

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

195 200 205

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

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

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

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

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

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

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

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Tyr

355 360 365

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

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

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

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 39

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 39

Glu Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

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

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Glu Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Lys Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Trp Gln Asp Tyr Asp Val Tyr Phe Asp Tyr Trp Gly Glu Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

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

130 135 140

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

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

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

195 200 205

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

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

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

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

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

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

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

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

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

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

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

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 40

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 40

Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

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

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 41

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 41

Ser Tyr Ile Met Met

1 5

<210> 42

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 42

Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val Lys

1 5 10 15

Gly

<210> 43

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 43

Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr

1 5 10

<210> 44

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 44

Gly Phe Thr Phe Ser Ser Tyr

1 5

<210> 45

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 45

Tyr Pro Ser Gly Gly Ile

1 5

<210> 46

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 46

Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr

1 5 10

<210> 47

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 47

Gly Tyr Gly Met Ser

1 5

<210> 48

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 48

Thr Ile Asn Ser Gly Gly Ser Tyr Thr Tyr Tyr Ser Asp Ser Val Lys

1 5 10 15

Gly

<210> 49

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 49

Arg Asn Gly Thr Leu Tyr Tyr Tyr Leu Met Asp Tyr

1 5 10

<210> 50

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 50

Gly Phe Thr Phe Ser Gly Tyr

1 5

<210> 51

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 51

Asn Ser Gly Gly Ser Tyr

1 5

<210> 52

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 52

Arg Asn Gly Thr Leu Tyr Tyr Tyr Leu Met Asp Tyr

1 5 10

<210> 53

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 53

Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser

1 5 10

<210> 54

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 54

Asp Val Ser Lys Arg Pro Ser

1 5

<210> 55

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 55

Ser Ser Tyr Thr Ser Ala Ser Thr Arg Ile

1 5 10

<210> 56

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 56

Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser

1 5 10

<210> 57

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 57

Asp Val Ser Lys Arg Pro Ser

1 5

<210> 58

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 58

Ser Ser Tyr Thr Ser Ala Ser Thr Arg Ile

1 5 10

<210> 59

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 59

Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser

1 5 10

<210> 60

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 60

Asp Val Ser Asn Arg Pro Ser

1 5

<210> 61

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 61

Ser Ser Tyr Thr Ser Ser Ser Thr Arg Ile

1 5 10

<210> 62

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 62

Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser

1 5 10

<210> 63

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 63

Asp Val Ser Asn Arg Pro Ser

1 5

<210> 64

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 64

Ser Ser Tyr Thr Ser Ser Ser Thr Arg Ile

1 5 10

<210> 65

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 65

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Glu Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val

50 55 60

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

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Glu

100 105 110

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

115 120 125

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

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

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

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

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

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

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

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

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

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

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

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

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

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 66

<211> 451

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 66

Gln Val Lys Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

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

20 25 30

Gly Met Ser Trp Ile Arg Gln Thr Pro Gly Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Asn Ser Gly Gly Ser Tyr Thr Tyr Tyr Ser Asp Ser Val

50 55 60

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

65 70 75 80

Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Arg Asn Gly Thr Leu Tyr Tyr Tyr Leu Met Asp Tyr Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

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

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

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

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

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

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

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

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 67

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 67

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Asp Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ala

85 90 95

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

100 105 110

Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu

115 120 125

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

130 135 140

Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

145 150 155 160

Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr

165 170 175

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

180 185 190

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

195 200 205

Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 68

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 68

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

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

100 105 110

Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu

115 120 125

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

130 135 140

Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

145 150 155 160

Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr

165 170 175

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

180 185 190

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

195 200 205

Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

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