WO2021098851A1

WO2021098851A1 - Anti-ctla4/ox40 bispecific antibodies and uses thereof

Info

Publication number: WO2021098851A1
Application number: PCT/CN2020/130615
Authority: WO
Inventors: Baihong Liu; Yi Yang; Fang Yang; Yuelei SHEN; Yanan GUO; Yunyun CHEN; Jingshu XIE
Original assignee: Eucure (Beijing) Biopharma Co., Ltd
Priority date: 2019-11-20
Filing date: 2020-11-20
Publication date: 2021-05-27

Abstract

Provided are antigen-binding protein constructs (e.g., bispecific antibodies or antigen-binding fragments thereof), wherein the antigen-binding protein constructs specifically bind to two different antigens (e.g., CTLA4 and OX40).

Description

ANTI-CTLA4/OX40 BISPECIFIC ANTIBODIES AND USES THEREOF

CLAIM OF PRIORITY

This application claims priority to international Application No. PCT/CN2019119682, filed on November 20, 2019. The entire contents of the foregoing application are incorporated herein by reference.

TECHNICAL FIELD

This disclosure relates to antigen-binding protein constructs (e.g., bispecific antibodies or antigen-binding fragments thereof) .

BACKGROUND

A bispecific antibody is an artificial protein that can simultaneously bind to two different types of antigens or two different epitopes. This dual specificity opens up a wide range of applications, including redirecting T cells to tumor cells, dual targeting of different disease mediators, and delivering payloads to targeted sites. The approval of catumaxomab (anti-EpCAM and anti-CD3) and blinatumomab (anti-CD19 and anti-CD3) has become a major milestone in the development of bispecific antibodies.

As bispecific antibodies have various applications, there is a need to continue todevelop various therapeutics based on bispecific antibodies.

SUMMARY

This disclosure relates toantigen-binding protein constructs, wherein the antigen-binding protein construct specifically bind to two different antigens (e.g., CTLA4 and OX40) .

In one aspect, the disclosure relates to an antigen-binding protein construct, including a first heavy chain variable region and a first light chain variable region. In some embodiments, the first heavy chain variable region and the first light chain variable region associate with each other, forming a first antigen binding region that specifically binds to CTLA4; and a second heavy chain variable region and a second light chain variable region. In some embodiments, 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 to OX40.

In some embodiments, the antigen-binding protein construct includes a first polypeptide that includes the first heavy chain variable region, a first heavy chain constant region 2 (CH2) , and a first heavy chain constant region 3 (CH3) ; and a second polypeptide that includes the second heavy chain variable region, a second heavy chain constant region 2 (CH2) , and a second heavy chain constant region 3 (CH3) .

In some embodiments, either one or both of the first polypeptide and the second polypeptide includes one or both of the following:

(i) Asp at position 239 (EU numbering) ; and

(ii) Glu at position 332 (EU numbering) .

In some embodiments, the antigen-binding protein construct includes a third polypeptide comprising the first light chain variable region; and a fourth polypeptide comprising the second light chain variable region.

In some embodiments, the amino acid sequence of the first light chain variable region and the amino acid sequence of the second light chain variable region are at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical.

In some embodiments, the second polypeptide further includes the second light chain variable region. In some embodiments, the first polypeptide further includes the first light chain variable region.

In some embodiments, the first polypeptide and the second polypeptide associate with each other by knobs into holes.

In some embodiments, the first polypeptide has one or more of the following: (i) Cys at position 354 (EU numbering) ; and (ii) Trp at position 366 (EU numbering) .

In some embodiments, the second polypeptide has one or more of the following: (i) Cys at position 349 (EU numbering) ; (ii) Ser at position 366 (EU numbering) ; (iii) Ala at position 368 (EU numbering) ; and (iv) Val at position 407 (EU numbering) .

In some embodiments, the second polypeptide has one or more of the following: (i) Cys at position 354 (EU numbering) ; and (ii) Trp at position 366 (EU numbering) . In some embodiments, the first polypeptide has one or more of the following: (i) Cys at position 349 (EU numbering) ; (ii) Ser at position 366 (EU numbering) ; (iii) Ala at position 368 (EU numbering) ; and (iv) Val at position 407 (EU numbering) .

In some embodiments, the first heavy chain variable region (VH1) includes complementarity determining regions (CDRs) 1, 2, and 3. In some embodiments, the VH1 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR3 amino acid sequence.

In some embodiments, the first light chain variable region (VL1) includes

CDRs

1, 2, and 3. In some embodiments, the VL1 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL1 CDR3 amino acid sequence.

In some embodiments, the selected VH1

CDRs

1, 2, and 3 amino acid sequences, the selected

VL1 CDRs

1, 2, and 3 amino acid sequences are one of the following:

(1) the selected VH1

CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 85, 86, 87, respectively, and the selected

VL1 CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 88, 89, 90, respectively, according to the Kabat numbering scheme;

(2) the selected VH1

CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 109, 110, 111, respectively, and the selected

VL1 CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 112, 113, 114, respectively, according to the Chothia numbering scheme;

(3) the selected VH1

CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 91, 92, 93, respectively, and the selected

VL1 CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 94, 95, 96, respectively, according to the Kabat numbering scheme;

(4) the selected VH1

CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 115, 116, 117, respectively, and the selected

VL1 CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 118, 119, 120, respectively, according to the Chothia numbering scheme;

(5) the selected VH1

CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 97, 98, 99, respectively, and the selected

VL1 CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 100, 101, 102, respectively, according to the Kabat numbering scheme;

(6) the selected VH1

CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 121, 122, 123, respectively, and the selected

VL1 CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 124, 125, 126, respectively, according to the Chothia numbering scheme;

(7) the selected VH1

CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 103, 104, 105, respectively, and the selected

VL1 CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 106, 107, 108, respectively, according to the Kabat numbering scheme; and

(8) the selected VH1

CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 127, 128, 129, respectively, and the selected

VL1 CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 130, 131, 132, respectively, according to the Chothia numbering scheme.

In some embodiments, the second heavy chain variable region (VH2) includes

CDRs

1, 2, and 3. In some embodiments, the VH2 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH2 CDR3 amino acid sequence.

In some embodiments, the second light chain variable region (VL2) includes

CDRs

1, 2, and 3. In some embodiments, the VL2 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL2 CDR3 amino acid sequence.

In some embodiments, the selected

VH2 CDRs

1, 2, and 3 amino acid sequences, and the selected

VL2 CDRs

1, 2, and 3 amino acid sequences are one of the following:

(1) the selected

VH2 CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 2, 3, respectively, and the selected

VL2 CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, 6, respectively, according to the Kabat numbering scheme;

(2) the selected

VH2 CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 26, 27, 28, respectively, and the selected

VL2 CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 29, 30, 31, respectively, according to the Chothia numbering scheme;

(3) the selected

VH2 CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 7, 8, 9, respectively, and the selected

VL2 CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10, 11, 12, respectively, according to the Kabat numbering scheme;

(4) the selected

VH2 CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 32, 33, 34, respectively, and the selected

VL2 CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 35, 36, 37, respectively, according to the Chothia numbering scheme;

(5) the selected

VH2 CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 13, 14, 15, respectively, and the selected

VL2 CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, 18, respectively, according to the Kabat numbering scheme;

(6) the selected

VH2 CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 38, 39, 40, respectively, and the selected

VL2 CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 41, 42, 43, respectively, according to the Chothia numbering scheme;

(7) the selected

VH2 CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 19, 20, 21, respectively, and the selected

VL2 CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22, 23, 24, respectively, according to the Kabat numbering scheme;

(8) the selected

VH2 CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 44, 45, 46, respectively, and the selected

VL2 CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 47, 48, 49, respectively, according to the Chothia numbering scheme;

(9) the selected

VH2 CDRs

VL2 CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 25, 6, respectively, according to the Kabat numbering scheme; and

(10) the selected

VH2 CDRs

VL2 CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 29, 50, 31, respectively, according to the Chothia numbering scheme.

In some embodiments, provided herein is the antigen-binding protein construct, wherein

(1) the selected VH1

CDRs

VL1 CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 94, 95, 96, respectively, the selected

VH2 CDRs

VL2 CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 25, 6, respectively, according to the Kabat numbering scheme; or

(2) the selected VH1

CDRs

VL1 CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 118, 119, 120, respectively, the selected

VH2 CDRs

VL2 CDRs

In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 133, 134, 135, 136, 137, or 149, and the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 138, 139, 140, or 150.

In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 141, 142, 143, 144, or 151, and the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 145, 146, 147, 148, or 152.

In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 153, and the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 154.

In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 155, and the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 156.

In some embodiments, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 51, 52, 53, 77, or 170, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 54, 55, 56, 78, or 171.

In some embodiments, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 57, 58, 59, or 79, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 60, 61, 62, 63, or 80.

In some embodiments, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 64, 65, 66, or 81, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 67, 68, 69, 70, or 82.

In some embodiments, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 71, 72, 73, or 83, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 74, 75, 76, or 84.

In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 141, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 145, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 53, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 54.

In some embodiments, the first polypeptide comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 158, and the third polypeptide comprise a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 157.

In some embodiments, the second polypeptide comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 160, and the fourth polypeptide comprise a sequence that is at least80%, 85%, 90%, or 95%identical to SEQ ID NO: 159.

In some embodiments, the first polypeptide comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 162, and the third polypeptide comprise a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 161.

In some embodiments, the second polypeptide comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 164, and the fourth polypeptide comprise a sequence that is at least80%, 85%, 90%, or 95%identical to SEQ ID NO: 163.

In some embodiments, the second polypeptide comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 169.

In some embodiments, the disclosure relates to a bispecific antibody or antigen-binding fragment thereof. In some embodiments, the bispecific antibody has a common light chain. In some embodiments, the VL for the two or more antigen-binding sites are identical.

In some embodiments, the antigen-binding protein construct has two, three, four, five, six, or more light chain variable regions. In some embodiments, the light chain variable regions are at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical. In some embodiments, the light chain variable regions are at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 138, 139, 140, or 150. In some embodiments, the light chain variable regions are at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 145, 146, 147, 148, or 152. In some embodiments, the light chain variable regions are at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 154. In some embodiments, the light chain variable regions are at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 156. In some embodiments, the light chain variable regions are at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 54, 55, 56, 78, or 171. In some embodiments, the light chain variable regions are at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 60, 61, 62, 63, or 80. In some embodiments, the light chain variable regions are at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 67, 68, 69, 70, or 82. In some embodiments, the light chain variable regions are at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 74, 75, 76, or 84.

In one aspect, the disclosure relates to a bispecificantibody or antigen-binding fragment thereof that includes: a first heavy chain polypeptide comprising a first heavy chain variable region; a first light chain polypeptide comprising a first light chain variable region; a second heavy chain polypeptide comprising a second heavy chain variable region; and a second light chain polypeptide, comprising a second light chain variable region.

In some embodiments, the first heavy chain variable region and the first light chain variable region associate with each other, forming a first antigen binding region that specifically binds to CTLA4, 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 to OX40.

In some embodiments, the first heavy chain variable region (VH1) includes

CDRs

1, 2, and 3. In some embodiments, the VH1 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR3 amino acid sequence.

In some embodiments, the first light chain variable region (VL1) includes

CDRs

In some embodiments, the second heavy chain variable region (VH2) includes

CDRs

In some embodiments, the second light chain variable region (VL2) includes

CDRs

In some embodiments, the selected

VH1 CDRs

1, 2, and 3 amino acid sequences, the selected

VL1 CDRs

1, 2, and 3 amino acid sequences, the selected

VH2 CDRs

1, 2, and 3 amino acid sequences, and the selected

VL2 CDRs

1, 2, and 3 amino acid sequences are one of the following:

(1) the selected

VH1 CDRs

VL1 CDRs

VH2 CDRs

VL2 CDRs

(2) the selected

VH1 CDRs

VL1 CDRs

VH2 CDRs

VL2 CDRs

In some embodiments, provided herein is the antibody or antigen-binding fragment that:

(1) the first heavy chain polypeptide comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 158, and the first light chain polypeptide comprise a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 157; and

(2) the second heavy chain polypeptide comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 160, and the second light chain polypeptide comprise a sequence that is at least80%, 85%, 90%, or 95%identical to SEQ ID NO: 159.

(1) the first heavy chain polypeptide comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 162, and the first light chain polypeptide comprise a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 161; and

(2) the second heavy chain polypeptide comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 164, and the second light chain polypeptide comprise a sequence that is at least80%, 85%, 90%, or 95%identical to SEQ ID NO: 163.

In one aspect, the disclosure relates to a bispecificantibody or antigen-binding fragment thereof, including a heavy chain polypeptide comprising the first heavy chain variable region; a light chain polypeptide comprising the first light chain variable region; a single-chain variable fragment polypeptide comprising a second heavy chain variable region, and a second light chain variable region.

In some embodiments, either one or both of the heavy chain polypeptide and the single-chain variable fragment polypeptide comprises one or both of the following:

(iii) Asp at position 239 (EU numbering) ; and

(iv) Glu at position 332 (EU numbering) .

In some embodiments, the first heavy chain variable region (VH1) includes

CDRs

In some embodiments, the first light chain variable region (VL1) includes

CDRs

In some embodiments, the second heavy chain variable region (VH2) includes

CDRs

In some embodiments, the second light chain variable region (VL2) includes

CDRs

1, 2, and 3. In some embodiments, the VL2 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL2 CDR3 amino acid sequence;

In some embodiments, the selected

VH1 CDRs

1, 2, and 3 amino acid sequences, the selected

VL1 CDRs

1, 2, and 3 amino acid sequences, the selected

VH2 CDRs

1, 2, and 3 amino acid sequences, and the selected

VL2 CDRs

1, 2, and 3 amino acid sequences are one of the following:

(1) the selected

VH1 CDRs

VL1 CDRs

VH2 CDRs

VL2 CDRs

(2) the selected

VH1 CDRs

VL1 CDRs

VH2 CDRs

VL2 CDRs

In some embodiments, the first heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 141, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 145, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 53 or 170, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 54 or 171.

(1) the heavy chain polypeptide comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 168, and the light chain polypeptide comprise a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 167; and

(2) the single-chain variable fragment polypeptide comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 169.

In one aspect, the disclosure relates to an antibody or antigen-binding fragment thereof that binds to OX40 that includes: a heavy chain variable region (VH) that includes

CDRs

1, 2, and 3; and a light chain variable region (VL) that includes

CDRs

1, 2, and 3.

In some embodiments, the VH CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR3 amino acid sequence.

In some embodiments, the VL CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR3 amino acid sequence.

(1) the selected

VH CDRs

1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1, 2, 3, respectively, and the selected VL CDRs, 1, 2, and 3 amino acid sequences are set forth in SEQ IN NOs: 4, 25, 6, respectively, according to the Kabat numbering scheme; and

(2) the selected

VH CDRs

1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 26, 27, 28, respectively, and the selected VL CDRs, 1, 2, and 3 amino acid sequences are set forth in SEQ IN NOs: 29, 50, 31, respectively, according to the Chothia numbering scheme.

In some embodiments, the antibody or antigen-binding fragment specifically binds to human OX40.

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

In some embodiments, the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) .

In some embodiments, the heavy chain variable region (VH) includes an amino acid sequence that is at least 90%identical to a selected VH sequence, and the light chain variable region (VL) includes an amino acid sequence that is at least 90%identical to a selected VL sequence,

In some embodiments, the selected VH sequence and the selected VL sequence are one of the following: the selected VH sequence is SEQ ID NOs: 51, 52, 53, 77, or 170, and the selected VL sequence is SEQ ID NOs: 54 or 171.

In one aspect, the disclosure provides an antigen-binding protein construct, comprising a first antigen binding region that specifically binds to CTLA4; and a second antigen binding region that specifically binds to OX40. In some embodiments, the first antigen binding region is a VHH. In some embodiments, the second antigen binding region is a VHH.

In one aspect, the disclosure relates to an antibody-drug conjugate that includes the antibody or antigen-binding fragment thereof as described herein, the bispecific antibody or antigen-binding fragment thereof as described herein, or the antibody or antigen-binding fragment thereof as described herein, that covalently bound to a therapeutic agent.

In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent.

In one aspect, the disclosure relates to a method of treating a subject having cancer, the method includes administering a therapeutically effective amount of a composition comprising the antigen-binding protein constructs (e.g., bispecific antibodies or antigen-binding fragments thereof) as described herein, the bispecific antibody or antigen-binding fragment thereof as described herein, the antibody or antigen-binding fragment thereof as described herein, or the antibody-drug conjugate as described herein, to the subject.

In some embodiments, the subject has a solid tumor.

In some embodiments, the cancer is breast cancer, oropharyngeal cancer, ovarian cancer, bladder cancer, colon cancer, pancreas cancer, B cell lymphoma, or Non-Hodgkin's lymphoma.

In some embodiments, the method further includes administering an anti-PD1 antibody to the subject.

In some embodiments, the method further includes administering a chemotherapy to the subject.

In one aspect, the disclosure relates to a method of decreasing the rate of tumor growth, the method includes administering to a subject in need thereof an effective amount of a composition comprising theantigen-binding protein constructs (e.g., bispecific antibodies or antigen-binding fragments thereof) as described herein, the bispecific antibody or antigen-binding fragment thereof as described herein, the antibody or antigen-binding fragment thereof as described herein, or the antibody-drug conjugate as described herein, to the subject.

In one aspect, the disclosure relates to a method of killing a tumor cell, the method includes administering to a subject in need thereof an effective amount of a composition comprising the antigen-binding protein constructs (e.g., bispecific antibodies or antigen-binding fragments thereof) as described herein, the bispecific antibody or antigen-binding fragment thereof as described herein, the antibody or antigen-binding fragment thereof as described herein, or the antibody-drug conjugate as described herein, to the subject.

In one aspect, the disclosure relates to a pharmaceutical composition that includes the antigen-binding protein constructs (e.g., bispecific antibodies or antigen-binding fragments thereof) as described herein, the bispecific antibody or antigen-binding fragment thereof as described herein, or the antibody or antigen-binding fragment thereof as described herein, and a pharmaceutically acceptable carrier.

In one aspect, the disclosure relates to a pharmaceutical composition that includes the antibody drug conjugate as described herein, and a pharmaceutically acceptable carrier.

In one aspect, the disclosure provides a nucleic acid comprising a polynucleotide encoding any polypeptide as described herein. 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 disclosure provides a cell comprising the vector described herein. In some embodiments, the cell is a CHO cell. In one aspect, the disclosure provides a cell comprising one or more of the nucleic acids described herein.

As used herein, the term “antigen-binding protein construct” is (i) a single polypeptide that includes at least two different antigen-binding domains or (ii) a complex of two or more polypeptides (e.g., the same or different polypeptides) that together form at least two different antigen-binding domains. Non-limiting examples and aspects of antigen-binding protein constructs are described herein. Additional examples and aspects of antigen-binding protein constructs are known in the art.

As used herein, the term “antigen-binding domain” refers to one or more protein domain (s) (e.g., formed from amino acids from a single polypeptide or formed from amino acids from two or more polypeptides (e.g., the same or different polypeptides) that is capable of specifically binding to one or more different antigen (s) (e.g., an effector antigen or control antigen) . In some examples, an antigen-binding domain can bind to an antigen or epitope with specificity and affinity similar to that of naturally-occurring antibodies. In some embodiments, the antigen-binding domain can be an antibody or a fragment thereof. One example of an antigen-binding domain is an antigen-binding domain formed by a VH -VL dimer. In some embodiments, an antigen-binding domain can include an alternative scaffold. In some embodiments, the antigen-binding domain is a VHH. Non-limiting examples of antigen-binding domains are described herein. Additional examples of antigen-binding domains are known in the art. In some examples, an antigen-binding domain can bind to a single antigen (e.g., one of an effector antigen and a control antigen) . In other examples, an antigen-binding domain can bind to two different antigens (e.g., an effector antigen and a control antigen) .

The term “antibody” is used herein in its broadest sense and includes certain types of immunoglobulin molecules that include one or more antigen-binding domains that specifically bind to an antigen or epitope. An antibody specifically includes, e.g., intact antibodies (e.g., intact immunoglobulins) , antibody fragments, bispecific antibodies, and multi-specific antibodies. One example of an antibody is a protein complex that includes two heavy chains and two light chains. Additional examples of an antibody are described herein.

As used herein, the term “multispecific antigen-binding protein construct” is an antigen-binding protein construct that includes two or more different antigen-binding domains that collectively specifically bind two or more different epitopes. The two or more different epitopes may be epitopes on the same antigen (e.g., a single polypeptide present on the surface of a cell) or on different antigens (e.g., different proteins present on the surface of the same cell or present on the surface of different cells) . In some aspects, a multi-specific antigen-binding protein construct binds two different epitopes (i.e., a “bispecific antigen-binding protein construct” ) . In some aspects, a multi-specific antigen-binding protein construct binds three different epitopes (i.e., a “trispecific antigen-binding protein construct” ) . In some aspects, a multi-specific antigen-binding protein construct binds four different epitopes (i.e., a “quadspecific antigen-binding protein construct” ) . In some aspects, a multi-specific antigen-binding protein construct binds five different epitopes (i.e., a “quintspecific antigen-binding protein construct” ) . Each binding specificity may be present in any suitable valency. Non-limiting examples of multispecific antigen-binding protein constructs are described herein.

As used herein, the term “bispecific antibody” refers to an antibody that binds to two different epitopes. The epitopes can be on the same antigen or on different antigens.

As used herein, a “VHH” refers to the variable domain of a heavy chain antibody. In some embodiments, the VHH is a humanized VHH.

As used herein, the term “common light chain” refers to a light chain that can interact with two or more different heavy chains, forming different antigen-binding sites, wherein these different antigen-binding sites can specifically bind to different antigens or epitopes. Similarly, the term “common light chain variable region” refers to a light chain variable region that can interact with two or more different heavy chain variable regions, forming different antigen-binding sites, wherein these different antigen-binding sites can specifically bind to different antigens or epitopes. In some embodiments, the antigen-binding construct can have a common light chain. In some embodiments, the antigen-binding construct can have a common light chain variable region.

Unless otherwise defined, 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 are described herein for use in the present invention; other, suitable methods and materials known in the art can 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 figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1Ais a schematic diagram showing components of an exemplary vector (Vector 1) that can be used in makingbispecific antibodies.

FIG. 1Bis a schematic diagram showing components of another exemplary vector (Vector 2) that can be used in makingbispecific antibodies.

FIG. 2 is a schematic diagram showing the structure of bispecific antibody BCG-93412-1 and BCG-93412-2 (BCG-93412-1 with additional mutations) .

FIG. 3 is a graph showing the sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) results for purified bispecific antibody BCG-93412-2 in a 6%separation gel. Numbers 1-5 are lane numbers. M is the Marker.

FIG. 4 is a graph showing the blocking effects of three tested antibodieson CTLA4/B7-1 binding.

FIG. 5A is a graph showing the activity of 9H3-H3K1-IgG1 antibody against OX40 reporter cells.

FIG. 5B is a graph showing the activity of BCG-93412-1 antibody against OX40 reporter cells.

FIG. 6 is a graph showing binding avidities against CHO-K1-hOX40-hCTLA4 cells for five tested antibodies.

FIG. 7 is a graph showing antibody-dependent cellular cytotoxicity (ADCC) activities of five tested antibodies.

FIG. 8A is a graph showing body weight over time of mice that were injected with mouse colon cancer cells MC38, and were treated with monoclonal or bispecific antibodies.

FIG. 8Bis a graph showing percentage change of body weight over time of mice that were injected with mouse colon cancer cells MC38, and were treated with monoclonal or bispecific antibodies.

FIG. 8C is a graph showing average tumor volume in different groups of mice that were injected with mouse colon cancer cells MC38, and were treated with monoclonal or bispecific antibodies. Saline solution was injected as a control.

FIG. 9A is a graph showing body weight over time of mice that were injected with mouse colon cancer cells MC38, and were treated with monoclonal or bispecific antibodies.

FIG. 9B is a graph showing percentage change of body weight over time of mice that were injected with mouse colon cancer cells MC38, and were treated with monoclonal or bispecific antibodies.

FIG. 9C is a graph showing average tumor volume in different groups of mice that were injected with mouse colon cancer cells MC38, and were treated with monoclonal or bispecific antibodies. Saline solution was injected as a control.

FIG. 10 is a schematic diagram showing the structure of bispecific antibody BCG-93412-3.

FIG. 11 is a graph showing the SDS results for the purified bispecific antibody BCG-93412-3 in a 6%separation gel. Numbers 1-2 are lane numbers. M is the Marker.

FIG. 12 is a graph showing the blocking effect of tested antibodies on CTLA4/B7-1 binding.

FIG. 13 is a graph showing binding avidities against CHO-K1-hOX40-hCTLA4 cells.

FIG. 14 lists CDR sequences of anti-OX40 antibodies 07-9H3, 07-9A4, 11-5C1, 17-5D10 and modified 07-9H3 as defined by Kabat numbering.

FIG. 15 lists CDR sequences of anti-OX40 antibodies 07-9H3, 07-9A4, 11-5C1, 17-5D10 and modified 07-9H3 as defined by Chothia numbering.

FIG. 16 lists amino acid sequences of heavy chain variable regions and light chain variable regions of humanized and mouse anti-OX40 antibodies.

FIG. 17 lists CDR sequences of anti-CTLA4 antibodies 04-13A4, 03-4G12, 20-6D2, and 20-7E12 antibodies as defined by Kabat numbering.

FIG. 18 lists CDR sequences of anti-CTLA4 antibodies 04-13A4, 03-4G12, 20-6D2, and 20-7E12 antibodies as defined by Chothia numbering.

FIG. 19 lists amino acid sequences of heavy chain variable regions and light chain variable regions of humanized and mouse anti-CTLA4 antibodies.

DETAILED DESCRIPTION

A bispecificantibody or antigen-binding fragment thereofis an artificial protein that can simultaneously bind to two different epitopes (e.g., on two different antigens) . In some embodiments, abispecificantibody or antigen-binding fragment thereofcan have two arms (Arms A and B) . Each arm has one heavy chain variable region and one light chain variable region, forming an antigen-binding domain (or an antigen-binding region) .

The bispecificantibody or antigen-binding fragment thereofcan be IgG-like and non-IgG-like. The IgG-like bispecific antibodycan have two Fab arms and one Fc region, and the two Fab arms bind to different antigens. The non-IgG-like bispecific antibody or antigen-binding fragmentcan be e.g., chemically linked Fabs (e.g., two Fab regions are chemically linked) , and single-chain variable fragments (scFVs) . For example, a scFV can have two heavy chain variable regions and two light chain variable regions. In some embodiments, one arm is a scFV polypeptide. In some embodiments, both arms are scFV polypeptides.

Anti-CTLA4/OX40 Antigen-Binding Protein Construct

The immune system can differentiate between normal cells in the body and those it sees as “foreign, ” which allows the immune system to attack the foreign cells while leaving the normal cells alone. This mechanism sometimes involves proteins called immune checkpoints. Immune checkpoints are molecules in the immune system that either turn up a signal (co-stimulatory molecules) or turn down a signal.

The immune checkpoint pathway involves a series of cellular interactions that prevents excessive effector activity by T cells under normal conditions. Cytotoxic T-lymphocyte antigen-4 (CTLA4 or CD152) is involved in this pathway. CTLA4 is a member of the immunoglobulin superfamily that is expressed exclusively on T-cells. CTLA4 acts to inhibit T-cell activation and is reported to inhibit helper T-cell activity and enhance regulatory T-cell immunosuppressive activity. Thus, CTLA-4 acts as an “off” switch, and turns down the immune response. Once a cytotoxic T cell becomes active, it expresses CTLA-4 on its cell surface, which then competes with the costimulatory molecule CD28 for their mutually shared ligands, B7-1 (CD80) or B7-2 (CD86) on the APC. This “yin-yang” balance holds cytotoxic activity in check, while allowing T-cell function to proceed in a self-limited manner (Creelan, Benjamin C. "Update on immune checkpoint inhibitors in lung cancer. " Cancer Control 21.1 (2014) : 80-89) .

In 2011, the first anti-CTLA4 antibody inhibitor Ipilimumab (trade name

) was approved for treatment of melanoma. However,

presented strong immunotherapy-related side effects (Larkin, James, et al. "Combined nivolumab and ipilimumab or monotherapy in untreated melanoma. " New England journal of medicine 373.1 (2015) : 23-34) , such as anemia, myocarditis, pneumonia, hepatitis, kidney and other multi-organ inflammations, which limited the clinical use of anti-CTLA4 antibodies.

The anti-tumor effects and the side effects of anti-CTLA4 antibody are not necessarily concomitant. The side effects may arise from the expansion of autoreactive T cells and the activation of systemic T cells (or blocking effect) . In recent years, some studies have found that anti-CTLA4 antibody Ipilimumab may not exert anti-tumor effect by blocking CTLA4/B7 interaction, but by antibody Fc domain-mediated effector function to eliminate Regulatory T cells (Tregs) (Selby, Mark J., et al. "Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells. " Cancer immunology research 1.1 (2013) : 32-42) .

Thus, it is hypothesized that the therapeutic effects can be increased if the blocking effects of the anti-CTLA4 antibody is attenuated and the targeting and killing activity of Treg in the tumor microenvironment is enhanced.

Anantigen-binding protein construct that target OX40 and CTLA4 can be developed for this purpose. Tumor necrosis factor receptor superfamily, member 4 (TNFRSF4) , also known as CD134 and OX40, is a member of the TNFR-superfamily of receptors which is not constitutively expressed on resting

T cells. OX40 is a secondary co-stimulatory immune checkpoint molecule, expressed after 24 to 72 hours following activation; its ligand, OX40L, is also not expressed on resting antigen presenting cells, but is following their activation. The expression of OX40 on the surface of mouse T-cells typically occurs between 24 h and 96 h after cognate antigen recognition. Engagement of the OX40 receptor on T-cells (in vitro) , using anti-OX40 agonistic antibodies, directly promotes an increase in survival of different effector T-cell subsets. Moreover, the immunosuppressive subset of CD4+ T-cells called regulatory T-cells (Tregs) also express high levels of OX40. Tregs can inhibit effector T-cells through the secretion of immunosuppressive cytokines such as transforming growth factor-beta (TGFb) and interleukin-10 (IL-10) . These negative regulators can be counter balanced by the stimulation of OX40 on effector T-cells and other TNFRSF co-stimulatory receptors such as 41BB (CD137) and glucocorticoid-induced tumor necrosis factor receptor (GITR) (CD357) .

Both OX40 and CTLA4 are highly expressed specifically in Tregs from tumor infiltrating lymphocytes (TIL) (Vargas, Frederick Arce, et al. "Fc effector function contributes to the activity of human anti-CTLA-4 antibodies. " Cancer Cell 33.4 (2018) : 649-663) . The elimination effect of Tregs is also regarded as a mechanism for anti-OX40 antibody in cancer immunotherapy. Accordingly, an antigen-binding protein construct (e.g., bispecific antibody) that target OX40 and CTLA4 can effectively eliminate Tregs, increase the anti-tumor effects, and reduce potential side effects.

Onebispecific antibody in present disclosure is designed to be 1+1 (monovalent for each target) and has an IgG1 subtype structure. This can reduce the avidityto cells with low expression levels of CTLA4 and OX40, and increase the avidity to Treg cells in TIL that co-express CTLA4 and OX40 with high expression levels, to achieve enhanced targeting function. Mutations S239D and/or I332E (SI mutations) can also be introduced in antibody heavy chains to enhance the antibody affinity to FcγRIIIA. Meanwhile, monovalent antibodies has a weaker blocking effect than bivalent anti-CTLA4 antibodies, thereby further reducing the side effects of anti-CTLA4 antibodies.

In some embodiments, the bispecific antibody or antigen-binding fragment thereof described herein has a common light chain.

Anti-OX40 Antibodies and Antigen-Binding Fragments

The disclosure provides several antibodies and antigen-binding fragments thereof that specifically bind to OX40. The anti-CTLA4/OX40antigen-binding protein constructs (e.g., bispecific antibodies) can include an antigen binding region that is derived from these antibodies.

The antibodies and antigen-binding fragments described herein are capable of binding to OX40. The disclosure provides e.g., mouse anti-OX40 antibodies 07-9H3 ( “9H3” ) , 07-9A4 ( “9A4” ) , 11-5C1 ( “5C1” ) , and 17-5D10 ( “5D10” ) , and chimeric antibodies, the humanized antibodies thereof.

The CDR sequences for 9H3, and 9H3 derived antibodies (e.g., humanized antibodies) include CDRs of the heavy chain variable domain, SEQ ID NOs: 1-3, and CDRs of the light chain variable domain, SEQ ID NOs: 4-6 as defined by Kabat numbering. The CDRs can also be defined by Chothia system. Under the Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 26-28, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 29-31.

CDR2 of VL can be further modified. Thus, the antibody can have CDRs of the heavy chain variable domain, SEQ ID NOs: 1-3, and CDRs of the light chain variable domain, SEQ ID NOs: 4, 25, 6 as defined by Kabat numbering. Under the Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 26-28, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 29, 50, 31.

Similarly, the CDR sequences for 9A4, and 9A4 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 7-9, and CDRs of the light chain variable domain, SEQ ID NOs: 10-12, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 32-34, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 35-37.

The CDR sequences for 5C1, and 5C1 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 13-15, and CDRs of the light chain variable domain, SEQ ID NOs: 16-18, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 38-40, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 41-43.

The CDR sequences for 5D10, and 5D10 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 19-21, and CDRs of the light chain variable domain, SEQ ID NOs: 22-24, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 44-46, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 47-49.

The amino acid sequence for heavy chain variable region and light variable region of humanized antibodies are also provided. As there are different ways to humanize the mouse antibody (e.g., sequence can be substituted by different amino acids) , the heavy chain and the light chain of an antibody can have more than one versions of humanized sequences. The amino acid sequences for the heavy chain variable region of humanized 9H3 antibody are set forth in SEQ ID NOs: 51-53. The amino acid sequences for the light chain variable region of humanized 9H3 antibody are set forth in SEQ ID NOs: 54-56. The heavy chain variable region and the light chain variable region can also be modified to increase the stability or interaction. 9H3-H3 can be further modified to obtain SEQ ID NO: 170.9H3-K1 can be further modified to obtain SEQ ID NO: 171. Any of these heavy chain variable region sequences (SEQ ID NOs: 51-53, 170) can be paired with any of these light chain variable region sequences (SEQ ID NOs: 54-56, 171) .

Similarly, the amino acid sequences for the heavy chain variable region of humanized 9A4 antibody are set forth in SEQ ID NOs: 57-59. The amino acid sequences for the light chain variable region of humanized 9A4 antibody are set forth in SEQ ID NOs: 60-63. Any of these heavy chain variable region sequences (SEQ ID NOs: 57-59) can be paired with any of these light chain variable region sequences (SEQ ID NOs: 60-63) .

The amino acid sequences for the heavy chain variable region of humanized 5C1 antibody are set forth in SEQ ID NOs: 64-66. The amino acid sequences for the light chain variable region of humanized 5C1 antibody are set forth in SEQ ID NOs: 67-70. Any of these heavy chain variable region sequences (SEQ ID NOs: 64-66) can be paired with any of these light chain variable region sequences (SEQ ID NOs: 67-70) .

The amino acid sequences for the heavy chain variable region of humanized 5D10 antibody are set forth in SEQ ID NOs: 71-73. The amino acid sequences for the light chain variable region of humanized 5D10 antibody are set forth in SEQ ID NOs: 74-76. Any of these heavy chain variable region sequences (SEQ ID NOs: 71-73) can be paired with any of these light chain variable region sequences (SEQ ID NOs: 74-76) .

As shown in FIG. 16, humanization percentage means the percentage identity of the heavy chain or light chain variable region sequence as compared to human antibody sequences in International Immunogenetics Information System (IMGT) database. The top hit means that the heavy chain or light chain variable region sequence is closer to a particular species than to other species. For example, top hit to human means that the sequence is closer to human than to other species. Top hit to human and Macacafascicularis means that the sequence has the same percentage identity to the human sequence and the Macacafascicularis sequence, and these percentages identities are highest as compared to the sequences of other species. In some embodiments, humanization percentage is greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%. A detailed description regarding how to determine humanization percentage and how to determine top hits is known in the art, and is described, e.g., in Jones, Tim D., et al. "The INNs and outs of antibody nonproprietary names. " MAbs. Vol. 8. No. 1. Taylor &Francis, 2016, which is incorporated herein by reference in its entirety. A high humanization percentage often has various advantages, e.g., more safe and more effective in humans, more likely to be tolerated by a human subject, and/or less likely to have side effects.

Furthermore, in some embodiments, the antibodies or antigen-binding fragments thereof described herein can also contain one, two, or three heavy chain variable region CDRs selected from the group of SEQ ID NOs: 1-3, SEQ ID NOs: 7-9, SEQ ID NOs: 13-15, SEQ ID NOs: 19-21, SEQ ID NOs: 26-28, SEQ ID NOs: 32-34, SEQ ID NOs: 38-40, and SEQ ID NOs: 44-46; and/or one, two, or three light chain variable region CDRs selected from the group of SEQ ID NOs: 4-6, SEQ ID NOs: 4, 25, 6, SEQ ID NOs: 10-12, SEQ ID NOs: 16-18, SEQ ID NOs: 22-24, SEQ ID NOs: 29-31, SEQ ID NO: 29, 50, 31, SEQ ID NOs: 35-37, SEQ ID NOs: 41-43, and SEQ ID NOs: 47-49.

In some embodiments, the antibodies can 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 that is 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 that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR3 amino acid sequence, and a light chain variable region (VL) comprising CDRs 1, 2, 3, wherein the CDR1 region comprises or consists ofan amino acid sequence that is 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 that is at least 80%, 85%, 90%, or 95%identical to a selected VL CDR2 amino acid sequence, and 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 CDRs

1, 2, 3 amino acid sequences and the selected VL CDRs, 1, 2, 3 amino acid sequences are shown in FIG. 14 (Kabat CDR) and FIG. 15 (Chothia CDR) .

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 1 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 2 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 3 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 7 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 8 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 9 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 13 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 14 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 15 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 19 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 20 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 21 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 26 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 27 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 28 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 32 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 33 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 34 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 38 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 39 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 40 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 44 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 45 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 46 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 4 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 5 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 6 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 10 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 11 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 12 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 16 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 17 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 18 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 22 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 23 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 24 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 4 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 25 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 6 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 29 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 30 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 31 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 35 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 36 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 37 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 41 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 42 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 43 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 47 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 48 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 49 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 29 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 50 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 31 with zero, one or two amino acid insertions, deletions, or substitutions.

The insertions, deletions, and substitutions can be within the CDR sequence, or at one or both terminal ends of the CDR sequence.

The disclosure also provides antibodies or antigen-binding fragments thereof that bind to OX40. The antibodies or antigen-binding fragments thereof contain a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH sequence, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NOs: 51, 52, 53, 77, 170, and the selected VL sequence is SEQ ID NOs: 54, 55, 56, 78, or 171. In some embodiments, the selected VH sequence is SEQ ID NOs: 57, 58, 59, or 79, and the selected VL sequence is SEQ ID NOs: 60, 61, 62, 63, or 80. In some embodiments, the selected VH sequence is SEQ ID NOs: 64, 65, 66, or 81, and the selected VL sequence is SEQ ID NOs: 67, 68, 69, 70, or 82. In some embodiments, the selected VH sequence is SEQ ID NOs: 71, 72, 73, or 83, and the selected VL sequence is SEQ ID NOs: 74, 75, 76, or 84.

In some embodiments, the antibody or antigen binding fragment thereof can have 3 VH CDRs that are identical to the CDRs of any VH sequences as described herein. In some embodiments, the antibody or antigen binding fragment thereof can have 3 VL CDRs that are identical to the CDRs of any VL sequences as described herein.

The disclosure also provides nucleic acid comprising a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin heavy chain. The immunoglobulin heavy chain or immunoglobulin light chain comprises CDRs as shown in FIG. 14 or FIG. 15, or have sequences as shown in FIG. 16. When the polypeptides are paired with corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region) , the paired polypeptides bind to OX40 (e.g., human OX40) .

The anti-OX40 antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments and multi-specific (e.g., bi-specific) antibodies or antibody fragments. Additional antibodies provided herein are polyclonal, monoclonal, multi-specific (multimeric, e.g., bi-specific) , human antibodies, chimeric antibodies (e.g., human-mouse chimera) , single-chain antibodies, intracellularly-made antibodies (i.e., intrabodies) , and antigen-binding fragments thereof. The antibodies or antigen-binding fragments thereof can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) , class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) , or subclass. In some embodiments, the antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof.

Fragments of antibodies are suitable for use in the methods provided so long as they retain the desired affinity and specificity of the full-length antibody. Thus, a fragment of an antibody that binds to OX40 will retain an ability to bind to OX40. An Fv fragment is an antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight association, which can be covalent in nature, for example in scFv. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs or a subset thereof confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) can have the ability to recognize and bind antigen, although usually at a lower affinity than the entire binding site.

Anti-CTLA4 Antibodies and Antigen-Binding Fragments

The disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to CTLA4. The anti-CTLA4/OX40antigen-binding protein construct (e.g., bispecific antibodies) can include an antigen binding region that is derived from these antibodies.

The antibodies and antigen-binding fragments described herein are capable of binding to CTLA4. The disclosure provides mouse anti-CTLA4 antibodies 04-13A4 ( “13A4” ) , 03-4G12 ( “4G12” ) , 20-6D2 ( “6D2” ) , and 20-7E12 ( “7E12” ) , and the humanized antibodies thereof.

The CDR sequences for 13A4, and 13A4 derived antibodies (e.g., humanized antibodies) include CDRs of the heavy chain variable domain, SEQ ID NOs: 85-87, and CDRs of the light chain variable domain, SEQ ID NOs: 88-90 as defined by Kabat numbering. The CDRs can also be defined by Chothia system. Under the Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 109-111, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 112-114.

Similarly, the CDR sequences for 4G12, and 4G12 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 91-93, and CDRs of the light chain variable domain, SEQ ID NOs: 94-96, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 115-117, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 118-120.

The CDR sequences for 6D2, and 6D2 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 97-99, and CDRs of the light chain variable domain, SEQ ID NOs: 100-102, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 121-123, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 124-126.

The CDR sequences for 7E12, and 7E12 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 103-105, and CDRs of the light chain variable domain, SEQ ID NOs: 106-108, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 127-129, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 130-132.

The amino acid sequence for heavy chain variable region and light variable region of humanized antibodies are also provided. As there are different ways to humanize the mouse antibody (e.g., sequence can be substituted by different amino acids) , the heavy chain and the light chain of an antibody can have more than one versions of humanized sequences. FIG. 19 provides humanization percentages for these humanized sequences.

The amino acid sequences for the heavy chain variable region of humanized 13A4 antibody are set forth in SEQ ID NOs: 133-137. The amino acid sequences for the light chain variable region of humanized 13A4 antibody are set forth in SEQ ID NOs: 138-140. Any of these heavy chain variable region sequences (SEQ ID NOs: 133-137) can be paired with any of these light chain variable region sequences (SEQ ID NOs: 138-140) .

Similarly, the amino acid sequences for the heavy chain variable region of humanized 4G12 antibody are set forth in SEQ ID NOs: 141-144. The amino acid sequences for the light chain variable region of humanized 4G12 antibody are set forth in SEQ ID NOs: 145-148. Any of these heavy chain variable region sequences (SEQ ID NOs: 141-144) can be paired with any of these light chain variable region sequences (SEQ ID NOs: 145-148) .

Furthermore, in some embodiments, the antibodies or antigen-binding fragments thereof described herein can also contain one, two, or three heavy chain variable region CDRs selected from the group of SEQ ID NOs: 85-87, SEQ ID NOs: 91-93, SEQ ID NOs: 97-99, SEQ ID NOs: 103-105, SEQ ID NOs: 109-111, SEQ ID NOs: 115-117, SEQ ID NOs: 121-123, and SEQ ID NOs: 127-129; and/or one, two, or three light chain variable region CDRs selected from the group of SEQ ID NOs: 88-90, SEQ ID NOs: 94-96, SEQ ID NOs: 100-102, SEQ ID NOs: 106-108, SEQ ID NOs: 112-114, SEQ ID NOs: 118-120, SEQ ID NOs: 124-126, and SEQ ID NOs: 130-132.

VH CDRs

1, 2, 3 amino acid sequences and the selected VL CDRs, 1, 2, 3 amino acid sequences are shown in FIG. 17 (Kabat CDR) and FIG. 18 (Chothia CDR) .

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 85 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 86 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 87 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 91 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 92 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 93 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 97 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 98 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 99 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 103 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 104 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 105 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 109 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 110 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 111 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 115 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 116 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 117 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 121 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 122 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 123 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 127 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 128 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 129 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 88 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 89 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 90 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 94 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 95 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 96 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 100 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 101 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 102 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 106 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 107 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 108 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 112 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 113 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 114 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 118 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 119 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 120 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 124 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 125 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 126 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 130 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 131 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 132 with zero, one or two amino acid insertions, deletions, or substitutions.

The disclosure also provides antibodies or antigen-binding fragments thereof that binds to CTLA4. The antibodies or antigen-binding fragments thereof contain a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH sequence, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 133, 134, 135, 136, 137, or 149, and the selected VL sequence is SEQ ID NO: 138, 139, 140, or 150. In some embodiments, the selected VH sequence is SEQ ID NO: 141, 142, 143, 144, or 151, and the selected VL sequence is SEQ ID NO: 145, 146, 147, 148, or 152. In some embodiments, the selected VH sequence is SEQ ID NO: 153, and the selected VL sequence is SEQ ID NO: 154. In some embodiments, the selected VH sequence is SEQ ID NO: 155, and the selected VL sequence is SEQ ID NO: 156.

In some embodiments, the antibody or antigen binding fragments thereof can have 3 VH CDRs that are identical to the CDRs of any VH sequences as described herein. In some embodiments, the antibody or antigen binding fragments thereof can have 3 VL CDRs that are identical to the CDRs of any VL sequences as described herein.

The disclosure also provides nucleic acid comprising a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin heavy chain. The immunoglobulin heavy chain or immunoglobulin light chain comprises CDRs as shown in FIG. 17 or FIG. 18, or have sequences as shown in FIG. 19. When the polypeptides are paired with corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region) , the paired polypeptides bind to CTLA4.

The anti-CTLA4 antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments and multi-specific (e.g., bi-specific) antibodies or antibody fragments. Additional antibodies provided herein are polyclonal, monoclonal, multi-specific (multimeric, e.g., bi-specific) , human antibodies, chimeric antibodies (e.g., human-mouse chimera) , single-chain antibodies, intracellularly-made antibodies (i.e., intrabodies) , and antigen-binding fragments thereof. The antibodies or antigen-binding fragments thereof can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) , class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) , or subclass. In some embodiments, the antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof.

Fragments of antibodies are suitable for use in the methods provided so long as they retain the desired affinity and specificity of the full-length antibody. Thus, a fragment of an antibody that binds to CTLA-4 will retain an ability to bind to CTLA-4. An Fv fragment is an antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight association, which can be covalent in nature, for example in scFv. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs or a subset thereof confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) can have the ability to recognize and bind antigen, although usually at a lower affinity than the entire binding site.

Antibody Characteristics

The anti-CTLA4/OX40 antigen-binding protein construct (e.g., antibodies, bispecific antibodies, or antibody fragments thereof) can include an antigen-binding region that is derived from any anti-CTLA4 antibody or anyantigen-binding fragment thereof as described herein.

Theanti-CTLA4 antibodies, or antigen-binding fragments thereof described herein can bind to CTLA4, and block the binding between CTLA4 and CD80, and/or the binding between CTLA4 and CD86. By blocking the binding between CTLA4 and CD80, and/or the binding between CTLA4 and CD86, the anti-CTLA4 antibodies disrupts the CTLA4 inhibitory pathway and upregulates the immune response. In some embodiments, the anti-CTLA4 antibodies, or antigen-binding fragments thereof can bind to Treg that express CTLA4, and induce ADCC.

General techniques can be used to measure the affinity of an antibody for an antigen include, e.g., ELISA, RIA, and surface plasmon resonance (SPR) . Affinities can be deduced from the quotient of the kinetic rate constants (KD=koff/ka) . In some implementations, the antibodies, the antigen-binding fragments thereof, or the antigen-binding protein construct (e.g., bispecific antibody) , canbind to CTLA4 (e.g., human CTLA4, monkey CTLA4, mouse CTLA4, and/or chimeric CTLA4) with a dissociation rate (koff) of less than 0.1 s ^-1, less than 0.01 s ^-1, less than 0.001 s ^-1, less than 0.0001 s ^-1, or less than 0.0001 s ^-1. In some embodiments, the dissociation rate (koff) is greater than 0.01 s ^-1, greater than 0.001 s ^-1, greater than 0.0001 s ^-1, greater than 0.0001 s ^-1, or greater than 0.00001 s ^-1. In some embodiments, the dissociation rate (koff) is less than 7 x 10- ⁴ s ^-1.

In some embodiments, kinetic association rates (ka) is greater than 1 x 10 ²/Ms, greater than 1 x 10 ³/Ms, greater than 1 x 10 ⁴/Ms, greater than 1 x 10 ⁵/Ms, or greater than 1 x 10 ⁶/Ms. In some embodiments, kinetic association rates (ka) is less than 1 x 10 ⁵/Ms, less than 1 x 10 ⁶/Ms, or less than 1 x 10 ⁷/Ms. In some embodiments, kinetic association rates (ka) is greater than 1.3 x 10 ⁵/Ms.

In some embodiments, the antibodies, the antigen-binding fragments thereof, or the antigen-binding protein construct (e.g., bispecific antibody) can bind to CTLA4 (e.g., human CTLA4, monkey CLTA4, mouse CTLA4, and/or chimeric CTLA4) with a KD of less than 1 x 10 ^-6 M, less than 1 x 10 ^-7 M, less than 1 x 10 ^-8 M, less than 1 x 10 ^-9 M, or less than 1 x 10 ^-10 M. In some embodiments, the KD is less than 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, KD is greater than 1 x 10 ^-7 M, greater than 1 x 10 ^-8 M, greater than 1 x 10 ^-9 M, or greater than 1 x 10 ^-10 M. In some embodiments, the antibody binds to human CTLA4 with KD less than or equal to about 5 nM, 4.5 nM, 4 nM, 3 nM or 0.27 nM.

The binding affinity with CTLA4 can also be measured by half maximal inhibitory concentration (IC50) . IC50 is a quantitative measure that indicates how much of a particular inhibitory agent is needed to block the binding between CTLA4 with its ligand by 50%. In some embodiments, the IC50 is less than or about 0.3, 0.25, 0.2, or 0.1 ug/ml.

The anti-CTLA4/OX40 antigen-binding protein construct (e.g., bispecific antibodies) can also include an antigen-binding region that is derived from any anti-OX40 antibody or antigen-binding fragment thereof as described herein. The anti-OX40 antibodies or antigen-binding fragments thereof described herein can block the binding between OX40 and OX40L. In some embodiments, by binding to OX40, the antibody can also promote OX40 signaling pathway and upregulates the immune response. Thus, in some embodiments, the antibodies or antigen-binding fragments thereof as described herein are OX40 agonist. In some embodiments, the antibodies or antigen-binding fragments thereof are OX40 antagonist.

In some implementations, the antibodies, the antigen-binding fragments thereof, or the antigen-binding protein constructs (e.g., bispecific antibody) can bind to OX40 (e.g., human OX40, monkey OX40, mouse OX40, and/or chimeric OX40) with a dissociation rate (koff) of less than 0.1 s ^-1, less than 0.01 s ^-1, less than 0.001 s ^-1, less than 0.0001 s ^-1, or less than 0.0001 s ^-1. In some embodiments, the dissociation rate (koff) is greater than 0.01 s ^-1, greater than 0.001 s ^-1, greater than 0.0001 s ^-1, greater than 0.0001 s ^-1, or greater than 0.00001 s ^-1.

In some embodiments, kinetic association rates (ka) is greater than 1 x 10 ²/Ms, greater than 1 x 10 ³/Ms, greater than 1 x 10 ⁴/Ms, greater than 1 x 10 ⁵/Ms, or greater than 1 x 10 ⁶/Ms. In some embodiments, kinetic association rates (ka) is less than 1 x 10 ⁵/Ms, less than 1 x 10 ⁶/Ms, or less than 1 x 10 ⁷/Ms.

Affinities can be deduced from the quotient of the kinetic rate constants (KD=koff/ka) . In some embodiments, KD is less than 1 x 10 ^-6 M, less than 1 x 10 ^-7 M, less than 1 x 10 ^-8 M, less than 1 x 10 ^-9 M, or less than 1 x 10 ^-10 M. In some embodiments, the KD is less than 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, KD is greater than 1 x 10 ^-7 M, greater than 1 x 10 ^-8 M, greater than 1 x 10 ^-9 M, greater than 1 x 10 ^-10 M, greater than 1 x 10 ^-11 M, or greater than 1 x 10 ^-12 M. In some embodiments, the antibody binds to human OX40 with KD less than or equal to about 15 nM or 10 nM.

The binding affinity with OX40 can also be measured by half maximal inhibitory concentration (IC50) . In some embodiments, the IC50 is less than or about 0.3, 0.2, 0.1, or 0.05 ug/ml.

In some embodiments, the antibodies, the antigen-binding fragments thereof, or the antigen-binding protein constructs (e.g., bispecific antibody) as described herein can increase immune response, activity of OX40, activity or number of T cells (e.g., CD8+ and/or CD4+ cells) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 folds, 3 folds, 5 folds, 10 folds, or 20 folds. In some embodiments, the antibodies, the antigen-binding fragments thereof, or the antigen-binding protein constructs (e.g., bispecific antibody) as described herein can decrease the activity or number of Treg by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 folds, 3 folds, 5 folds, 10 folds, or 20 folds.

In some embodiments, the antibodies, the antigen-binding fragments thereof, or the antigen-binding protein constructs (e.g., bispecific antibody) as described herein are OX40 agonist. In some embodiments, the antibodies, the antigen-binding fragments thereof, or the antigen-binding protein constructs (e.g., bispecific antibody) can increase OX40 signal transduction in a target cell that expresses OX40. In some embodiments, OX40 signal transduction is detected by monitoring NFkB downstream signaling.

The agnostic effects of the antibodies, the antigen-binding fragments thereof, or the antigen-binding protein constructs (e.g., bispecific antibody) can be measured by EC50. Half-maximal effective concentration (EC50) refers to the concentration of the agent which induces a response halfway between the baseline and maximum. In some embodiments, the EC50 is less than or about 0.15, 0.1, 0.01, 0.001, or 0.0006 ug/ml.

Because the antigen-binding protein construct (e.g., bispecific antibody) binds to both OX40 and CTLA4, for cells that express both OX40 and CTLA4, the antigen-binding protein construct has a higher binding affinity to these cells. Avidity can be used to measure the binding affinity of an antigen-binding protein construct to these cells. Avidity is the accumulated strength of multiple affinities of individual non-covalent binding interactions.

Thermal stabilities can also be determined. The antibodies, the antigen-binding fragments thereof, or the antigen-binding protein constructs (e.g., bispecific antibody) as described hereincan have a Tm 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 ℃. As IgG can be described as a multi-domain protein, the melting curve sometimes shows two transitions, with a first denaturation temperature, Tm D1, and a second denaturation temperature Tm D2. The presence of these two peaks often indicate the denaturation of the Fc domains (Tm D1) and Fab domains (Tm D2) , respectively. When there are two peaks, Tm usually refers to Tm D2. Thus, in some embodiments, the antibodies or antigen binding fragments as 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 antibodies or antigen binding fragments as 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, Tm, Tm D1, Tm D2 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 ℃.

In some embodiments, the antibodies, the antigen-binding fragments thereof, or the antigen-binding protein constructs (e.g., the anti-OX40 antibody, the anti-CTLA4 antibody, or the bispecific antibody) has a tumor growth inhibition percentage (TGI%) that is 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 tumor growth inhibition percentage that is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. The TGI%can be determined, e.g., 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 the treatment starts, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the treatment starts. As used herein, the tumor growth inhibition percentage (TGI%) is calculated using the following formula:

TGI (%) = [1- (Ti-T0) / (Vi-V0) ] ×100

Ti is the average tumor volume in the treatment group on day i. T0 is the average tumor volume in the treatment group on day zero. Vi is the average tumor volume in the control group on day i. V0 is the average tumor volume in the control group on day zero.

In some embodiments, the antibody, the antigen-binding fragment thereof, or the antigen-binding protein construct (e.g., bispecific antibody) has a functional Fc region. In some embodiments, effector function of a functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC) . In some embodiments, effector function of a functional Fc region is phagocytosis. In some embodiments, effector function of a functional Fc region is ADCC and phagocytosis. In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, one or both mutations S239D and/or I332E (SI mutations) are introduced in antibody Fc region to enhance the antibody affinity to FcγRIIIA, thereby increasing ADCC effects. A detailed description of SI mutations can be found in US7662925, which is incorporated by reference in their entirety.

In some embodiments, the antibody, the antigen-binding fragment thereof, or the antigen-binding protein construct (e.g., bispecific antibody) does not have a functional Fc region. For example, the antibodies or antigen binding fragments are Fab, Fab’, F (ab’) 2, and Fv fragments.

Antibodies and Antigen Binding Fragments

The present disclosure provides antibodies, the antigen-binding fragments thereof, or the antigen-binding protein constructs (e.g., bispecific antibodies) . The antigen-binding protein construct (e.g., bispecific antibody) can comprise an anti-OX40antibody or antigen-binding fragment thereof, and anti-OX40 antibody or antigen-binding fragment thereof. These antigen-binding protein constructs (e.g., bispecific antibody) , anti-OX40 antibodies, anti-OX40 antibodies, andantigen-binding fragments thereof can have various forms.

In general, antibodies (also called immunoglobulins) are made up of two classes of polypeptide chains, light chains and heavy chains. A non-limiting antibody of the present disclosure can be an intact, four immunoglobulin chain antibody comprising two heavy chains and two light chains. The heavy chain of the antibody can be of any isotype including IgM, IgG, IgE, IgA, or IgD or sub-isotype including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a kappa light chain or a lambda light chain. An antibody can comprise two identical copies of a light chain and/or two identical copies of a heavy chain. The heavy chains, which each contain one variable domain (or variable region, VH) and multiple constant domains (or constant regions) , bind to one another via disulfide bonding within their constant domains to form the “stem” of the antibody. The light chains, which each contain one variable domain (or variable region, VL) and one constant domain (or constant region) , each bind to one heavy chain via disulfide binding. 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 chains and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FR) .

These hypervariable regions, known as the complementary determining regions (CDRs) , form loops that comprise the principle antigen binding surface of the antibody. The four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held in close proximity by the framework regions and, with the CDRs from 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 a number of definitions of the CDRs are commonly used. The Kabat definition is based on sequence variability, and the Chothia definition is based on the location of the structural loop regions. These methods and definitions are described in, e.g., Martin, "Protein sequence and structure analysis of antibody variable domains, " Antibody engineering, Springer Berlin Heidelberg, 2001.422-439; Abhinandan, et al. "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains, " Molecular immunology 45.14 (2008) : 3832-3839; 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 (Oct. 1997) ; Morea et al., J Mol Biol. 275 (2) : 269-94 (Jan . 1998) ; Chothia et al., Nature 342 (6252) : 877-83 (Dec. 1989) ; Ponomarenko and Bourne, BMC Structural Biology 7: 64 (2007) ; each of which is incorporated herein by reference in its entirety. Unless specifically indicated in the present disclosure, Kabat numbering is used in the present disclosure as a default.

The CDRs are important for recognizing an epitope of an antigen. As used herein, an “epitope” is the smallest portion of a target molecule capable of being specifically bound by the antigen binding domain of an antibody. The minimal size of an epitope may be about three, four, five, six, or seven amino acids, but these amino acids need not be in a consecutive linear sequence of the antigen’s primary structure, as the epitope may depend on an antigen’s three-dimensional configuration based on the antigen’s 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, differ in their constant region, particularly in their hinges and upper CH2 domains. The sequences and differences of the IgG subclasses are known in the art, and are described, e.g., in Vidarsson, et al, "IgG subclasses and allotypes: from structure to effector functions. " Frontiers in immunology 5 (2014) ; Irani, et al. "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases. " Molecular immunology 67.2 (2015) : 171-182; Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016; each of which is incorporated herein by reference in its entirety.

The antibody can also be an immunoglobulin molecule that is derived from any species (e.g., human, rodent, mouse, rat, camelid) . Antibodies disclosed herein also include, but are not limited to, polyclonal, monoclonal, monospecific, polyspecific antibodies, and chimeric antibodies that include 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 specific binding activity of the intact antibody, i.e., any portion of an antibody that is capable of specific binding to an epitope on the intact antibody’s target molecule. It includes, e.g., Fab, Fab', F (ab') 2, and variants of these fragments. Thus, in some embodiments, an antibody or an antigen binding fragment thereof can be, e.g., a scFv, a Fv, a Fd, a dAb, a bispecific antibody, a bispecificscFv, a diabody, a linear antibody, a single-chain antibody molecule, a multi-specific antibody formed from antibody fragments, and any polypeptide that includes a binding domain which is, or is homologous to, an antibody binding domain. Non-limiting examples of antigen binding domains include, e.g., the heavy chain and/or light chain CDRs of an intact antibody, the heavy and/or light chain variable regions of an intact antibody, full length heavy or light chains of an intact antibody, or an individual CDR from either the heavy chain or the light chain of an intact 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 the respective target antigens with different affinities.

In some embodiments, the antigen binding fragment can form a part of a chimeric antigen receptor (CAR) . In some embodiments, the chimeric antigen receptor are fusions of single-chain variable fragments (scFv) as described herein, fused to CD3-zeta transmembrane-and endodomain. In some embodiments, the chimeric antigen receptor also comprises intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS) . In some embodiments, the chimeric antigen receptor comprises multiple signaling domains, e.g., CD3z-CD28-41BB or CD3z-CD28-OX40, to increase potency. Thus, in one aspect, the disclosure further provides cells (e.g., T cells) that express the chimeric antigen receptors as described herein.

In some embodiments, the antibodies, the antigen-binding fragments thereof, or the antigen-binding protein constructs (e.g., bispecific antibodies) can bind to two different antigens or two different epitopes.

In some embodiments, the antibodies, the antigen-binding fragments thereof, or the antigen-binding protein constructs (e.g., bispecific antibodies) can comprises one, two, or three heavy chain variable region CDRs selected from FIGS. 14, 15, 17, and 18. In some embodiments, the antibodies, the antigen-binding fragments thereof, or the antigen-binding protein constructs (e.g., bispecific antibodies) can comprises one, two, or three light chain variable region CDRs selected from FIGS. 14, 15, 17, and 18.

In some embodiments, the antibodies, the antigen-binding fragments thereof, or the antigen-binding protein constructs (e.g., bispecific antibodies) described herein can be conjugated to a therapeutic agent. The antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof can covalently or non-covalently bind to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxyanthracin, maytansinoids such as DM-1 and DM-4, dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs) .

Single-chain Fv or (scFv) antibody fragments comprise the VH and VL domains (or regions) of antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.

The Fab fragment contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CH1) of the heavy chain. F (ab') 2 antibody fragments comprise a pair of Fab fragments which are generally covalently linked near their carboxy termini by hinge cysteines between them. Other chemical couplings of antibody fragments are also known in the art.

Diabodies are small antibody fragments with two antigen-binding sites, which fragments comprise a VH connected to a VL in the same polypeptide chain (VH and VL) . By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

Linear antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

Multimerization of antibodies may be accomplished through natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, some percentage of purified antibody preparations (e.g., purified IgG1 molecules) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody multimers.

In some embodiments, the multi-specific antibody is a bi-specific antibody. Bi-specific antibodies can be made by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers that are recovered from recombinant cell culture. For example, the interface can contain at least a part of the CH3 domain of an 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) . Compensatory “cavities” of identical or similar size to the large side chain (s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine) . This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. This method is described, e.g., in WO 96/27011, which is incorporated by reference in its entirety.

Bi-specific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. Heteroconjugate antibodies can also be made using any convenient cross-linking methods. Suitable cross-linking agents and cross-linking techniques are well known in the art and are disclosed in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.

Methods for generating bi-specific antibodies from antibody fragments are also known in the art. For example, bi-specific antibodies can be prepared using chemical linkage. Brennan et al. (Science 229: 81, 1985) describes a procedure where intact antibodies are proteolytically cleaved to generate F (ab’) ₂ fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab’ fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab’ TNB derivatives is then reconverted to the Fab’ thiol by reduction with mercaptoethylamine, and is mixed with an equimolar amount of another Fab’ TNB derivative to form the bi-specific antibody.

Any of the antibodies, the antigen-binding fragments thereof, or the antigen-binding protein constructs (e.g., bispecific antibodies) described herein may 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 in solution) . Non-limiting examples of stabilizing molecules include: a polymer (e.g., a polyethylene glycol) or a protein (e.g., serum albumin, such as human serum albumin) . The conjugation of a stabilizing molecule can increase the half-life or extend the biological activity of an antibody or an antigen-binding fragmentin vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in a human) .

The antibodies, the antigen-binding fragments thereof, or the antigen-binding protein constructs (e.g., bispecific antibodies) can also have various forms. Many different formats of antigen binding constructs are known in the art, and are described e.g., in Suurs, et al. "A review of bispecific antibodies and antibody constructs in oncology and clinical challenges, " Pharmacology &therapeutics (2019) , which is incorporated herein by reference in the entirety.

In some embodiments, the antigen-binding protein construct is a BiTe, a (scFv) 2, a nanobody, a nanobody-HSA, a DART, a TandAb, a scDiabody, a scDiabody-CH3, scFv-CH-CL-scFv, a HSAbody, scDiabody-HAS, or a tandem-scFv. In some embodiments, the antigen- binding protein construct is a VHH-scAb, a VHH-Fab, a Dual scFab, a F (ab') 2, a diabody, a crossMab, a DAF (two-in-one) , a DAF (four-in-one) , a DutaMab, a DT-IgG, a knobs-in-holes common light chain, a knobs-in-holes assembly, a charge pair, a Fab-arm exchange, a SEEDbody, a LUZ-Y, a Fcab, a κλ-body, an orthogonal Fab, a DVD-IgG, a IgG (H) -scFv, a scFv- (H) IgG, IgG (L) -scFv, scFv- (L) IgG, IgG (L, H) -Fv, IgG (H) -V, V (H) -IgG, IgG (L) -V, V (L) -IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, Diabody-CH3, a triple body, a miniantibody, a minibody, a TriBiminibody, scFv-CH3 KIH, Fab-scFv, a F (ab') 2-scFv2, a scFv-KIH, a Fab-scFv-Fc, a tetravalent HCAb, a scDiabody-Fc, a Diabody-Fc, a tandem scFv-Fc, an Intrabody, a dock and lock, a lmmTAC, an IgG-IgG conjugate, a Cov-X-Body, or a scFv1-PEG-scFv2.

In some embodiments, the antigen-binding protein construct can be a TrioMab. In a TrioMab, the two heavy chains are from different species, wherein different sequences restrict the heavy-light chain pairing.

In some embodiments, the antigen-binding protein construct has two different heavy chains and one common light chain. Heterodimerization of heavy chains can be based on the knob-in-holes or some other heavy chain pairing technique.

In some embodiments, CrossMAb technique can be used produce bispecific antibodies. CrossMAb technique can be used enforce correct light chain association in bispecific heterodimeric IgG antibodies, this technique allows the generation of various bispecific antibody formats, including bi- (1+1) , tri- (2+1) and tetra- (2+2) valentbispecific antibodies, as well as non-Fc tandem antigen-binding fragment (Fab) -based antibodies. These formats can be derived from any existing antibody pair using domain crossover, without the need for the identification of common light chains, post-translational processing/in vitro chemical assembly or the introduction of a set of mutations enforcing correct light chain association. The method is described in Klein et al., "The use of CrossMAb technology for the generation of bi-and multispecific antibodies. " MAbs. Vol. 8. No. 6. Taylor &Francis, 2016, which is incorporated by reference in its entirety. In some embodiments, the CH1 in the heavy chain and the CL domain in the light chain are swapped.

In some embodiments, the antigen-binding protein construct can be a 2: 1 CrossMab. An additional Fab-fragment is added to the N-terminus of its VH domain of the CrossMab. The added Fab-fragment to the CrossMab increases the avidity by enabling bivalent binding.

In some embodiments, the antigen-binding protein construct can be a 2: 2 CrossMab. This tetravalent bispecific antibody generated by fusing a Fab-fragment to each C-terminus of a CrossMab. These Fab-fragments can be crossed: their CH1 is switched with their CL. VH is fused to their CL and the VL to the CH1. CrossMab technique in Fab-fragments ensure specific pairing. Avidity can be enhanced by double bivalent binding.

The antigen-binding protein construct can be a Duobody. The Fab-exchange mechanism naturally occurring in IgG4 antibodies is mimicked in a controlled matter in IgG1 antibodies, a mechanism called controlled Fab exchange. This format can ensure specific pairing between the heavy-light chains.

In Dual-variable-domain antibody (DVD-Ig) , additional VH and variable light chain (VL) domain are added to each N-terminus for bispecific targeting. This format resembles the IgG-scFv, but the added binding domains are bound individually to their respective N-termini instead of a scFv to each heavy chain N-terminus.

In scFv-IgG, the two scFv are connected to the C-terminus of the heavy chain (CH3) . The scFv-IgG format has two different bivalent binding sites and is consequently also called tetravalent. There are no heavy-chain and light-chain pairing problem in the scFv-IgG.

In some embodiments, the antigen-binding protein construct can be have a IgG-IgG format. Two intact IgG antibodies are conjugated by chemically linking the C-terminals of the heavy chains.

The antigen-binding protein construct can also have a Fab-scFv-Fc format. In Fab-scFv-Fc format, a light chain, heavy chain and a third chain containing the Fc region and the scFv are assembled. It can ensure efficient manufacturing and purification.

In some embodiments, antigen-binding protein construct can be a TF. Three Fab fragments are linked by disulfide bridges. Two fragments target the tumor associated antigen (TAA) and one fragment targets a hapten. The TF format does not have an Fc region.

ADAPTIR has two scFvs bound to each sides of an Fc region. It abandons the intact IgG as a basis for its construct, but conserves the Fc region to extend the half-life and facilitate purification.

Bispecific T cell Engager ( “BiTE” ) consists of two scFvs, VLA VHA and VHB VLB on one peptide chain. It has only binding domains, no Fc region.

In BiTE-Fc, an Fc region is fused to the BiTE construct. The addition of Fc region enhances half-life leading to longer effective concentrations, avoiding continuous IV.

Dual affinity retargeting (DART) has two peptide chains connecting the opposite fragments, thus VLA with VHB and VLB with VHA, and a sulfur bond at their C-termini fusing them together. In DART, the sulfur bond can improve stability over BiTEs.

In DART-Fc, an Fc region is attached to the DART structure. It can be generated by assembling three chains, two via a disulfide bond, as with the DART. One chain contains half of the Fc region which will dimerize with the third chain, only expressing the Fc region. The addition of Fc region enhances half-life leading to longer effective concentrations, avoiding continuous IV.

In tetravalent DART, four peptide chains are assembled. Basically, two DART molecules are created with half an Fc region and will dimerize. This format has bivalent binding to both targets, thus it is a tetravalent molecule.

Tandem diabody (TandAb) comprises two diabodies. Each diabody consists of an VHA and VLB fragment and a VHA and VLB fragment that are covalently associated. The two diabodies are linked with a peptide chain. It can improve stability over the diabody consisting of two scFvs. It has two bivalent binding sites.

The ScFv-scFv-toxin includes toxin and two scFv with a stabilizing linker. It can be used for specific delivery of payload.

In modular scFv-scFv-scFv, one scFv directed against the TAA is tagged with a short recognizable peptide is assembled to a bsAb consisting of two scFvs, one directed against CD3 and one against the recognizable peptide.

In ImmTAC, a stabilized and soluble T cell receptor is fused to a scFv recognizing CD3. By using a TCR, the ImmTAC is suitable to target processed, e.g. intracellular, proteins.

Tri-specific nanobody has two single variable domains (nanobodies) with an additional module for half-life extension. The extra module is added to enhance half-life.

In Trispecific Killer Engager (TriKE) , two scFvs are connected via polypeptide linkers incorporating human IL-15. The linker to IL-15 is added to increase survival and proliferation of NKs.

Methods of Making Antigen-Binding Protein Constructs

An isolated fragment of human protein can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be raised 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 can be conjugated to an agent that is immunogenic in the species to be immunized. Animals can be injected with the antigenic peptide or protein more than one time (e.g., twice, three times, or four times) .

The full-length polypeptide or protein can be used or, alternatively, antigenic peptide fragments thereof can be used as immunogens. The 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 encompasses an epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with the protein.

An immunogen typically is used to prepare antibodies by immunizing a suitable subject (e.g., human or transgenic animal expressing at least one human immunoglobulin locus) . An appropriate immunogenic preparation can contain, for example, a recombinantly-expressed or a chemically-synthesized polypeptide. The preparation can further include an adjuvant, such as Freund’s complete or incomplete adjuvant, or a similar immunostimulatory agent.

Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide, or an antigenic peptide thereof (e.g., part of the protein) as an immunogen. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme-linked immunosorbent assay (ELISA) using the immobilized polypeptide or peptide. If desired, the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A of protein G chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the specific antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al. (Nature 256: 495-497, 1975) , the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4: 72, 1983) , the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985) , or trioma techniques. The technology for producing hybridomas is well known (see, generally, Current Protocols in Immunology, 1994, Coligan et al. (Eds. ) , John Wiley &Sons, Inc., New York, NY) . Hybridoma cells producing a monoclonal antibody 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 described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding a human, humanized, or chimeric antibody, or antigen-binding fragment thereof described herein, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acids sequences that make-up the antigen-binding site of the antibody or an 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 arrive at an antibody or antigen-binding fragment thereof that has increased binding affinity for the target. The amino acid changes introduced into the antibody or antigen-binding fragment can also alter or introduce new post-translational modifications into the antibody or antigen-binding fragment, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence such that a different sugar is attached by enzymes present in a cell) , or introducing new glycosylation sites.

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

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

Human and humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequence as those derived from) human germline immunoglobulin sequences. Human antibodies may include amino acid residues 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) , for example in the CDRs.

A humanized antibody, typically has a human framework (FR) grafted with non-human CDRs. Thus, a humanized antibody has one or more amino acid sequence introduced into it from a source which is non-human. 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 e.g., substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. These methods are described in e.g., Jones et al., Nature, 321: 522-525 (1986) ; Riechmann et al., Nature, 332: 323-327 (1988) ; Verhoeyen et al., Science, 239: 1534-1536 (1988) ; each of which is incorporated by reference herein in its entirety. Accordingly, “humanized” antibodies are chimeric antibodies wherein substantially less than an intact human V domain has been substituted by 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 that antibodies be humanized with retention of high specificity and affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies can be prepared by a process of analysis of 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 are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen (s) , is achieved.

Identity or homology with respect to an original sequence is usually the percentage of amino acid residues present within the candidate sequence that are identical with a sequence present within the human, humanized, or chimeric antibody or fragment, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.

In some embodiments, a covalent modification can be made to the antibodies, the antigen-binding fragments thereof, or the antigen-binding protein constructs (e.g., bispecific antibodies) . These covalent modifications can be made by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of the antibody or antibody fragment are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with an organic derivatization agent that is capable of reacting with selected side chains or the 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 antibody may be from 1%to 80%, from 1%to 65%, from 5%to 65%or from 20%to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues; or position 314 in Kabat numbering) ; however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody can 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 antibodies was further engineered to replace the serine at position 228 (EU numbering) of IgG4 with proline (S228P) . A detailed description regarding S228 mutation is described, e.g., in Silva et al. "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation. " Journal of Biological Chemistry 290.9 (2015) : 5462-5469, which is incorporated by reference in its entirety.

In some embodiments, the methods described here are designed to make a bispecific antibody. Bispecific antibodies can be made by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers that are recovered from recombinant cell culture. For example, the interface can contain at least a part of the CH3 domain of an 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) . Compensatory “cavities” of identical or similar size to the large side chain (s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine) . This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. This method is described, e.g., in WO 96/27011, which is incorporated by reference in its entirety.

In some embodiments, knob-into-hole (KIH) technology can be used, which involves engineering CH3 domains to create either a “knob” or a “hole” in each heavy chain to promote heterodimerization. The KIH technique is described e.g., in Xu, Yiren, et al. "Production of bispecific antibodies in ‘knobs-into-holes’ using a cell-free expression system. " MAbs. Vol. 7. No. 1. Taylor &Francis, 2015, which is incorporated by reference in its entirety. In some embodiments, one heavy chain has a T366W, and/or S354C (knob) substitution (EU numbering) , and the other heavy chain has an Y349C, T366S, L368A, and/or Y407V (hole) substitution (EU numbering) . In some embodiments, one heavy chain has one or more of the following substitutions Y349C and T366W (EU numbering) . The other heavy chain can have one or more the following substitutions E356C, T366S, L368A, and Y407V (EU numbering) . Furthermore, a substitution (-ppcpScp-->-ppcpPcp-) can also be introduced at the hinge regions of both substituted IgG.

Furthermore, an anion-exchange chromatography can be used to purify bispecific antibodies. Anion-exchange chromatography is a process that separates substances based on their charges using an ion-exchange resin containing positively charged groups, such as diethyl-aminoethyl groups (DEAE) . In solution, the resin is coated with positively charged counter-ions (cations) . Anion exchange resins will bind to negatively charged molecules, displacing 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 a protein has no net charge. When the pH >pI, a protein has a net negative charge and when the pH <pI, a protein has a net positive charge. Thus, in some embodiments, different amino acid substitution can be introduced into two heavy chains, so that the pI for the homodimer comprising two Arm A and the pI for the homodimer comprising two Arm B is different. The pI for the bispecific antibody having Arm A and Arm B will be somewhere between the two pIs of the homodimers. Thus, the two homodimers and the bispecific antibody can be released at different pH conditions. The present disclosure shows that a few amino acid residue substitutions can be introduced to the heavy chains to adjust 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 residues at one or more of the

positions

1, 6, 43, 81, and 105 (Kabat numbering) is aspartic acid or glutamic acid. In some embodiments, the amino acid residues at one or more of the positions 13 and 105 (Kabat numbering) is aspartic acid or glutamic acid. In some embodiments, the amino acid residues at one or more of the positions 13 and 42 (Kabat numbering) is lysine, arginine, histidine, or glycine.

Bispecific antibodies can also include e.g., cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. Heteroconjugate antibodies can also be made using any convenient cross-linking methods. Suitable cross-linking agents and cross-linking techniques are well known in the art and are disclosed in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.

Methods for generating bispecific antibodies from antibody fragments are also known in the art. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al. (Science 229: 81, 1985) describes a procedure where intact antibodies are proteolytically cleaved to generate F (ab’) 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab’ fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab’ TNB derivatives is then reconverted to the Fab’ thiol by reduction with mercaptoethylamine, and is mixed with an equimolar amount of another Fab’ TNB derivative to form the bispecific antibody.

Recombinant Vectors

The present disclosure also provides recombinant vectors (e.g., expression vectors) that include an isolated polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein) , host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleotide) , and the production of recombinant antibody polypeptides or fragments thereof by recombinant techniques.

As used herein, a “vector” is any construct capable of delivering one or more polynucleotide (s) of interest to a host cell when the vector is introduced to the host cell. An “expression vector” is capable of delivering and expressing the one or more polynucleotide (s) of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and/or a poly-A tail, either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector.

A vector can be introduced into the host cell by methods known in the art, e.g., electroporation, chemical transfection (e.g., DEAE-dextran) , transformation, transfection, and infection and/or transduction (e.g., with recombinant virus) . Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant virus) , naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

In some implementations, a polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein) is introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus) , which may involve the use of a non-pathogenic (defective) , replication competent virus, or may use a replication defective virus. In the latter case, viral propagation generally will occur only in complementing virus packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86: 317-321; Flexner et al., 1989, Ann. N.Y. Acad Sci. 569: 86-103; Flexner et al., 1990, Vaccine, 8: 17-21; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. 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: 431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91: 215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90: 11498-11502; Guzman et al., 1993, Circulation, 88: 2838-2848; 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: 1745-1749, and Cohen, 1993, Science, 259: 1691-1692. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads that are efficiently transported into the cells.

For expression, the DNA insert comprising an antibody-encoding or polypeptide-encoding polynucleotide disclosed herein can be operatively linked to an appropriate 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 promoters of retroviral LTRs, to name a few. Other suitable promoters are known to the skilled artisan. The expression constructs can further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs may include a translation initiating at the beginning and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated.

As indicated, the expression vectors can include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate 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 such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells; and plant cells. Appropriate culture mediums and conditions for the host cells described herein are known in the art.

Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, 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 readily apparent to the skilled artisan.

Non-limiting bacterial promoters suitable for use include the E. colilacI 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 early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV) , and metallothionein promoters, such as the mouse metallothionein-I promoter.

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

Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many 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 an antibody of the present disclosure by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, 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, appropriate secretion signals may be incorporated into the expressed polypeptide. The signals may be endogenous to the polypeptide or they may be heterologous signals.

The polypeptide (e.g., antibody) can be expressed in a 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 additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to the polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.

The 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 nucleotide sequence as described herein, and 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 amino acid sequence as described herein.

The disclosure also provides a nucleic acid sequence that has a homology of 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%to any nucleotide sequence as described herein, and an amino acid sequence that has a homology of 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%to any amino acid sequence as described herein.

In some embodiments, the disclosure relates to nucleotide sequences encoding any peptides that are described herein, or any amino acid sequences that are encoded by any nucleotide sequences as 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 one of the sequences as described herein.

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

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) . The length of a reference sequence aligned for comparison purposes is at least 80%of the length of the reference sequence, and in some embodiments is at least 90%, 95%, or 100%. The amino acid residues or nucleotides at 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 amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology” ) . The percent identity between the 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 need to be introduced for optimal alignment of the two sequences. For purposes of the present invention, the comparison of 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 extend 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 percentage of sequence homology is known in the art. In some embodiments, amino acid residues conserved with similar physicochemical properties (percent homology) , e.g. leucine and isoleucine, can be used to measure sequence similarity. Families of amino acid residues having similar physicochemical properties have been defined in the art. These families include e.g., 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) . The homology percentage, in many cases, is higher than the identity percentage.

The disclosure provides one or more nucleic acid encoding any of the polypeptides as described herein. In some embodiments, the nucleic acid (e.g., cDNA) includes a polynucleotide encoding a polypeptide of a heavy chain as described herein. In some embodiments, the nucleic acid includes a polynucleotide encoding a polypeptide of a light chain as described herein. In some embodiments, the nucleic acid includes a polynucleotide encoding a scFv polypeptide as described herein.

In some embodiments, the vector can have two of the nucleic acids as described herein, wherein the vector encodes the VL region and the VH region that together bind to CTLA4. In some embodiments, a pair of vectors is provided, wherein each vector comprises one of the nucleic acids as described herein, wherein together the pair of vectors encodes the VL region and the VH region that together bind to CTLA4.

In some embodiments, the vector includes two of the nucleic acids as described herein, wherein the vector encodes the VL region and the VH region that together bind to OX40. In some embodiments, a pair of vectors is provided, wherein each vector comprises one of the nucleic acids as described herein, wherein together the pair of vectors encodes the VL region and the VH region that together bind to OX40.

Vectors can also be constructed to express specific antibodies or polypeptides. In some embodiments, a vector can be constructed to co-express anti-CTLA4 antibody light chain (CTLA4-K) and heavy chain (CTLA4-H) . In some embodiments, a vector can contain sequences of, from 5’ end to 3’ end, cytomegalovirus promotor (CMV) , CTLA4-K, polyadenylation (PolyA) , CMV, CTLA4-H, PolyA, simian vacuolating virus 40 terminator (SV40) and glutamine synthetase marker (GS) (FIG. 1A) . In some embodiments, a vector can be constructed to co-express anti-OX40 antibody light chain (OX40-K) and anti-OX40 antibody heavy chain (OX40-H) . In some embodiments, a vector can contain sequences of, from 5’ end to 3’ end, CMV, CTLA4-K, PolyA, CMV, CTLA4-H, SV40 and GS (FIG. 1B) . In some embodiments, a vector can be constructed to express anti-OX40 antibody scFv polypeptide chain.

Methods of Treatment

The methods described herein include methods for the treatment of disorders associated with cancer. Generally, the methods include administering a therapeutically effective amount of engineered antibodies, the antigen-binding fragments thereof, or the antigen-binding protein constructs (e.g., bispecific antibodies) as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment.

As used in this context, to “treat” means to ameliorate at least one symptom of the disorder associated with cancer. Often, cancer results in death; thus, a treatment can result in an increased life expectancy (e.g., by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years) . Administration of a therapeutically effective amount of an agent described herein for the treatment of a condition associated with cancer will result in decreased number of cancer cells and/or alleviated symptoms.

As used herein, the term “cancer” refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. The term “tumor” as used herein refers to cancerous cells, e.g., a mass of cancerous cells. Cancers that can be treated or diagnosed using the methods described herein include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. In some embodiments, the agents described herein are designed for treating or diagnosing a carcinoma in a subject. The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. In some embodiments, the cancer is renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.

In some embodiments, the cancer is a chemotherapy resistant cancer.

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

In one aspect, the disclosure features methods that include administering a therapeutically effective amount of antibodies, the antigen-binding fragments thereof, or the antigen-binding protein constructs (e.g., bispecific antibodies) , or an antibody drug conjugatesdisclosed herein to a subject in need thereof, e.g., a subject having, or identified or diagnosed as having, a cancer, e.g., breast cancer (e.g., triple-negative breast cancer) , carcinoid cancer, 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, urethral cancer, or hematologic malignancy.

As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present invention is provided. Veterinary and non-veterinary applications are contemplated by the present invention. Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old) . In addition to humans, patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, non-human primates (e.g., monkey, chimpanzee, gorilla, and the like) , rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits) , lagomorphs, swine (e.g., pig, miniature pig) , equine, canine, feline, bovine, and other domestic, farm, and zoo animals.

In some embodiments, the cancer isbladder cancer, colon cancer, or pancreas cancer. In some embodiments, the cancer is non-small cell lung cancer (NSCLC) , renal cell carcinoma (RCC) , urothelial carcinoma or prostate cancer. In some embodiments, the subject has solid tumor or neoplasms.

In some embodiments, the compositions and methods disclosed herein can be used for treatment of patients at risk for a cancer. Patients with cancer can be identified with various methods known in the art.

As used herein, by an “effective amount” is meant an amount or dosage sufficient to effect beneficial or desired results including halting, slowing, retarding, or inhibiting progression of a disease, e.g., a cancer. An effective amount will vary depending upon, e.g., an age and a body weight of a subject to which the antibody, antigen binding fragment, antibody-drug conjugates, antibody-encoding polynucleotide, vector comprising the polynucleotide, and/or compositions thereof is to be administered, a severity of symptoms and a route of administration, and thus administration can be determined on an individual basis.

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

Effective amounts and schedules for administering the antibodies, antibody-encoding polynucleotides, antibody-drug conjugates, and/or compositions disclosed herein may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal that will receive the antibodies, antibody-encoding polynucleotides, antibody-drug conjugates, and/or compositions disclosed herein, the route of administration, the particular type of antibodies, antibody-encoding polynucleotides, antigen binding fragments, antibody-drug conjugates, and/or compositions disclosed herein used and other drugs being administered to the mammal. Guidance in selecting appropriate doses for antibody or antigen binding fragment can be found in the literature on therapeutic uses of antibodies and antigen binding fragments, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., 1985, ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York, 1977, pp. 365-389.

A typical daily dosage of an effective amount of an antibody, the antigen-binding fragment thereof, or the antigen-binding protein construct (e.g., a bispecific antibody) is 0.01 mg/kg to 100 mg/kg. In some embodiments, the dosage can be less than 100 mg/kg, 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, or 0.1 mg/kg. In some embodiments, the dosage can be greater than 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, 0.1 mg/kg, 0.05 mg/kg, or 0.01 mg/kg. In some embodiments, the dosage is about or at least 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.9 mg/kg, 0.8 mg/kg, 0.7 mg/kg, 0.6 mg/kg, 0.5 mg/kg, 0.4 mg/kg, 0.3 mg/kg, 0.2 mg/kg, or 0.1 mg/kg.

In any of the methods described herein, the at least one antibody, the antigen-binding fragment thereof, or the antigen-binding protein construct (e.g., a bispecific antibody) , antibody-drug conjugates, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding fragments, antibody-drug conjugates, or pharmaceutical compositions described herein) and, optionally, at least one additional therapeutic agent can be administered to the subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a 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, at least one antibody, the antigen-binding fragment thereof, the antigen-binding protein construct (e.g., a bispecific antibody) , or antibody-drug conjugate, and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition) . In some embodiments, the at least one antibody or antigen-binding fragment and the at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition containing at least one antibody or antigen-binding fragment and a solid oral composition containing at least one additional therapeutic agent) . In some embodiments, the at least one additional therapeutic agent is administered as 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, the one or more additional therapeutic agents can be administered to the subject prior to, or after administering the 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) . In some embodiments, the one or more additional therapeutic agents and the 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) are administered to the subject such that there is an overlap in the bioactive period 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) in the subject.

In some embodiments, the subject can be administered the 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) over an extended period of time (e.g., over a period of 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) . A skilled medical professional may determine the length of the treatment period using any of the methods described herein for diagnosing or following the effectiveness of treatment (e.g., the observation of at least one symptom of cancer) . As described herein, a skilled medical professional can also change the identity and number (e.g., increase or decrease) of antibodies or antigen-binding antibody fragments, antibody-drug conjugates (and/or one or more additional therapeutic agents) administered to the subject and can also adjust (e.g., increase or decrease) the dosage or frequency of administration of at least one antibody or antigen-binding antibody fragment (and/or one or more additional therapeutic agents) to the subject based on an assessment of the effectiveness of the treatment (e.g., using any of the methods described herein and known in the art) .

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

In some embodiments, the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of HER3, an inhibitor of LSD1, an inhibitor of MDM2, an inhibitor of BCL2, an inhibitor of CHK1, an inhibitor of activated hedgehog signaling pathway, and an agent that selectively degrades the estrogen receptor.

In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, Pazopanib, IMA-901, AGS-003, cabozantinib, Vinflunine, an Hsp90 inhibitor, Ad-GM-CSF, Temazolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid, amrubicine, carfilzomib, pralatrexate, and enzastaurin.

In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, tumor necrosis factor (TNF) alpha, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a treatment targeting CX3CL1, a treatment targeting CXCL9, a treatment targeting CXCL10, a treatment targeting CCL5, an LFA-1 agonist, an ICAM1 agonist, and a Selectin agonist.

In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI are administered to the subject.

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

Pharmaceutical Compositions and Routes of Administration

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

Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) . The compositions can include a sterile diluent (e.g., sterile water or saline) , a fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvents, antibacterial or antifungal agents, such as benzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid, buffers, such as acetates, citrates, or phosphates, and isotonic agents, such as sugars (e.g., dextrose) , polyalcohols (e.g., mannitol or sorbitol) , or salts (e.g., sodium chloride) , or any combination thereof. Liposomal suspensions can also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Patent No. 4,522,811) . Preparations of the compositions can be formulated and enclosed in ampules, disposable syringes, or multiple dose vials. Where required (as in, for example, injectable formulations) , proper fluidity can be maintained by, for example, the use of a coating, such as lecithin, or a surfactant. Absorption of the antibody or antigen-binding fragment thereof can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin) . Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc. ) .

Compositions containing one or more of any of the antigen-binding protein constructs, 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 (i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage) .

Toxicity and therapeutic efficacy of compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys) . One can determine the LD50 (the dose lethal to 50%of the population) and the ED50 (the dose therapeutically effective in 50%of the population) : the therapeutic index being the ratio of LD50: ED50. Agents that exhibit high therapeutic indices are preferred. Where an agent exhibits an undesirable side effect, care should be taken to minimize potential damage (i.e., reduce unwanted side effects) . Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

Data obtained from cell culture assays and animal studies can be used in formulating an appropriate dosage of any given agent for use in a subject (e.g., a human) . A therapeutically effective amount of the one or more (e.g., one, two, three, or four) antigen-binding protein constructs, antibodies or antigen-binding fragments thereof (e.g., any of the antibodies or antibody fragments described herein) will be an amount that treats the disease in a subject (e.g., kills cancer cells ) in a subject (e.g., a human subject identified as having cancer) , or a subject identified as being at risk of developing the disease (e.g., a subject who has previously developed cancer but now has been cured) , decreases the severity, frequency, and/or duration of one or more symptoms of a disease in a subject (e.g., a human) . The effectiveness and dosing of any of the antigen-binding protein constructs, antibodies or antigen-binding fragments described herein can be determined by a health care professional or veterinary professional using methods known in the art, as well as by the observation of one or more symptoms of disease in a subject (e.g., a human) . Certain factors may influence the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and the presence of other diseases) .

Exemplary doses include milligram or microgram amounts of any of the antigen-binding protein constructs, antibodies or antigen-binding fragments, or antibody-drug conjugates described herein per kilogram of the subject’s weight (e.g., about 1 μg/kg to about 500 mg/kg; about 100 μg/kg to about 500 mg/kg; about 100 μg/kg to about 50 mg/kg; about 10 μg/kg to about 5 mg/kg; about 10 μg/kg to about 0.5 mg/kg; or about 0.1 mg/kg to about 0.5 mg/kg) . While these doses cover a broad range, one of ordinary skill in the art will understand that therapeutic agents, including antigen-binding protein constructs, antibodies and antigen-binding fragments thereof, vary in their potency, and effective amounts can be determined by methods known in the art. Typically, relatively low doses are administered at first, and the attending health care professional or veterinary professional (in the case of therapeutic application) or a researcher (when still working at the development stage) can subsequently and gradually increase the dose until an appropriate response is obtained. In addition, it is 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, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the half-life of the antibody or antibody fragmentin vivo.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. The disclosure also provides methods of manufacturing the antibodies or antigen binding fragments thereof, or antibody-drug conjugates for various uses as 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: Preparation of anti-CTLA4/OX40 bispecific antibody

Anti-CTLA4 and anti-OX40 antibody light chain and heavy chain were respectively constructed into a vector with two CMV (Cytomegalovirus) promoters (FIG. 1A-1B) , and the two vectors were co-transfected into CHO-Scells. After 14 days of culture, the cell supernatant was collected and purified by Protein A affinity chromatography, followed by size-exclusion chromatography (SEC) to obtain the a bispecific antibody (BCG-93412-1) (FIG. 2) . The BCG-93412-1 antibody has an anti-CTLA4 arm comprising a heavy chain and a light chain, and an anti-OX40 arm comprising a heavy chain and a light chain. Many methods can be used to reduce the chance of wrong pairing between the heavy chain and the light chain, and between the two heavy chains. In this example, the constant domain in the light chain (CL1) and the first constant domain (CH1) in the heavy chain in the anti-OX40 arm was exchanged between the heavy and light chain. Thus, only specific interaction can take place in the Fab region. The anti-OX40 arm heavy chain would not pair with the anti-CTLA4 arm light chain. In the Fc region, knobs-into-holes mutations were introduced to the anti-OX40 arm heavy chain and the anti-CTLA4 arm heavy chain. The full-length sequences of the heavy chain and the light chain are shown below.

Full length for CTLA4-K light chain of BCG-93412-1 (SEQ ID NO: 157) :

Full length for CTLA4-H heavy chain of BCG-93412-1 (SEQ ID NO: 158) :

Full length for OX40-K light chain of BCG-93412-1 (SEQ ID NO: 159) :

Full length for OX40-H heavy chain of BCG-93412-1 (SEQ ID NO: 160) :

In order to increase the binding affinity with FcγRIIIA, SI mutations S239D/I332E (EU numbering) were introduced to the heavy chains. The full-length sequences of the heavy chain and the light chain for BCG-93412-2 are shown below.

Full length for CTLA4-K light chain of BCG-93412-2 (SEQ ID NO: 161) :

Full length for CTLA4-H heavy chain of BCG-93412-2 (SEQ ID NO: 162) :

Full length for OX40-K light chain of BCG-93412-2 (SEQ ID NO: 163) :

Full length for OX40-H heavy chain of BCG-93412-2 (SEQ ID NO: 164) : QVQLVESGGGVVQPGRSLRISCAVSGFSLTSYGVLWVRQAPGKGLEWLGVIWSGGSTDYNAAFISRLTISRDNSKSTVYFQMNSLRAEDTAVYYCAREEFGYWGQGTLVTVSSASTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPA PELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The CTLA-4 x OX40 bispecific antibody ATOR-1015 (Kvarnhammar et al. "The CTLA-4 x OX40 bispecific antibody ATOR-1015 induces anti-tumor effects through tumor-directed immune activation. " Journal for immunotherapy of cancer 7.1 (2019) : 103) were also used for comparison purpose. ATOR-1015 was based on anti-OX40 antibody. The CTLA-4 binding domain (CD86) was fused to the C-terminal end of the κ light chain of the OX40 antibody with a S (GGGGS) 2 linker (S (GGGGS) (GGGGS) SEQ ID NO: 172) . The sequences for theATOR1015-K light chain and ATOR1015-H heavy chain were shown below.

Full length of ATOR1015-K light chain (SEQ ID NO: 165) :

Full length of ATOR1015-H heavy chain (SEQ ID NO: 166) :

Purified BCG-93412-2 were detected by a non-reducing SDS-PAGE (6%separation gel) as shown in FIG. 3. Each lane was loaded by proteins as described in Table 1 below.

Table 1

EXAMPLE 2: Antibody binding affinity

The binding affinity against human OX40 (hOX40) or human CTLA4 (hCTLA4) were determined by Biacore systems. Results are summarized in the table below.

Table 2

The result showedbispecific antibody BCG-93412-1had a binding affinity that is comparable to its parent monoclonal antibodies. As the additional mutations in BCG-93412-2 will not affect binding affinity, BCG-93412-2 should have a similar binding affinity as compared to the parent monoclonal antibodies.

EXAMPLE 3: Blocking effect of monoclonal antibody and bispecific antibody on CTLA4/B7-1 binding

Experiments were performed to test the blocking effects of three tested antibodies on CTLA4/B7-1 interaction.

His-tag labelled CTLA4 protein (CTLA4-His) was diluted to 1 μg/mL with phosphate-buffered saline (PBS) . 100 μLof the diluted solution was added to each well of aELISA (enzyme-linked immunosorbent assay) plate to coat the plate at 4 ℃ overnight. The liquid in the plate was then removed. 300 μL of 1×PBST (PBST (PBS with Tween 20) was added to each well, and the plate was washed by PBST for 4 times. PBS with 1.5%BSA was prepared and added to the plate at 200 μL per well, then incubated at 37 ℃ for 1 hour. The plate was then washed by PBST for 4 times.

Biotin-labeled B7-1/B7-2protein solution at a final concentration of 200 ng/mL with 1.5%BSA was prepared. The tested antibody wasserially diluted to final concentrations of 3000, 1500, 750, 375, 187.5, 93.75, 46.88, 23.44, 11.72, 5.86, 2.93, and 0 ng/mL by the Biotin-labeled B7-1/B7-2 protein solution. Diluted samples were added to the ELISA plate at 100 μL per well, then incubate at 37 ℃ for 1 hour. Next, the plate was washed by PBST for a total of 4 times. HRP-labeled Streptavidin was diluted at 1: 3000 in 1.5%BSA and was then added to the ELISA plate at 100 μL per well. The plate was incubated at 37 ℃ for 1 hour. Next, the plate was washed by PBST for a total of 4 times. 100 μLTMB chromogen solution was added to each well of the plate, and developed at 37 ℃ for 30 minutes in the dark. At the end of the development, 100 μL stop solution was added to each well. The ELISA plate was then placed in a microplate reader to read the absorbance values at OD450 and OD570. Sample absorbance was calculated based on OD450-OD570. The antibody concentration (Log ₁₀) was plotted on the X-axis and the OD450-OD570 value was plotted on the Y-axis (FIG. 4) . The analysis was performed using GraphPad Prism 7 to calculate the IC50 values. The IC50 and R ²values are shown in Table 3.

Table 3

Antibody Name	IC50 Value (ug/ml)	R ² value
4G12-H1K1-IgG1	0.0986	0.9957
BCG-93412-2	0.2532	0.9710

As shown in FIG. 4 and Table 3, the bispecific antibody BCG-93412-2 has a higher IC50as compared to 4G12-H1K1-IgG1. In other words, it has a decreased blocking effect. Themonoclonal antibody 9H3-H3K1-IgG1 did not bind to B7-1/B7-2, and was used as a negative control. The decreased blocking effect of the bispecific antibody can be a factor that contributes to its enhanced safety.

EXAMPLE 4: Jurkat-luc-hOX40 reporter cell activation assay

The experiment was performed to test whether the bispecific antibody can activate the OX40 pathway.

Adherent CHO-K1-FcγRIIB cells (Promega, Cat#CS1979A09) were plated in a 96-well flat bottom plate (cell density 4×10 ⁴ cells/well) . On the next day, 25 μL of Jurkat-Luc-hOX40 (Promega, Cat#CS197703) suspension cells were added to each well in the 96-well plate (cell density 5 × 10 ⁴ cells/well) . 25 μL of antibody solution was added to each well following a 3-fold serial dilution. The above 96-well plate was incubated in a 37 ℃ incubator for 6 hours. After the incubation, the plate was taken out, and 25 μl of the chromogensolution was added to incubate at room temperature for 5 minutes in the dark. The place was then placed in a luminescence detector to detect the fluorescence signal. The analysis was performed using GraphPad software.

As shown in FIG. 5A-5B, due to the monovalent structure, the activity induced by bispecific antibody in OX40 reporter cell was smaller than that of monoclonal antibody, but this may not be the primary mechanism of action for this bispecific antibody.

EXAMPLE 5: Total binding avidity test

The experiment was performed to determine avidity. CHO-K1-hOX40-hCTLA4 cells co-express hOX40 and hCTLA4 antigens. 5 × 10 ⁵ cells were added in each well on a 96-well round bottom plate with a volume of 25 μl. The antibody was diluted to a final concentration of 100 nM, then a 3-fold serial dilution was performed. The diluted antibody with a volume of 25 μl was added and incubated for 30 minutes at 4 ℃. Next, the cells were washed by PBS and spun down at 1200 rpm for 3 minutes. The supernatant was removed. 50 μl of 1: 500 diluted goat anti-human antibody (labeled by FITC) secondary antibody was added to the cells and incubated at 4 ℃ for 30 minutes. The cells were washed by PBS and spun down at 1200 rpm for 3 minutes. The pelleted cells were resuspended in 30 μl PBS, and the average fluorescence intensity was calculated by flow cytometry.

As shown in FIG. 6, bispecific antibodies BCG-93412-1 and BCG-93412-2 were determined to have a higher avidity for cells that co-express both antigens than the remaining testedmonoclonal and bispecific antibodies.

EXAMPLE 6: ADCC activity assay

The experiment was performed to test ADCC activity. CHO-K1-hOX40-hCTLA4 cells were prepared at 37 ℃ and plated in a 96-well plates (cell density 4 × 10 ⁴ cells /well) . 25 μL of Jurkat-Luc-NFAT-hCD16A-158F (a reporter cell line expressing the luciferase reporter gene under the control of an promoter fused to NFAT response elements) suspension cells were added to each well in the 96-well plate (cell density 5 × 10 ⁴ cells/well) . 25 μL of Assay Buffer diluted antibody at a final concentration of 100 nM (15 μg/ml) was added to each well following a 3-fold serial dilution. The 96-well plate was incubated in a 37 ℃ incubator for 6 hours. After the incubation, the plate was taken out, and the substrate solution was added to incubate at room temperature for 5 minutes in the dark. Then the platewas placed in a luminescence detector to detect the fluorescence signal. The analysis was performed using GraphPad software.

As shown in FIG. 7, BCG-93412-2 (with SI mutations) had stronger ADCC activity than BCG-93412-1 and the tested monoclonal antibodies. ATOR-1015 also had strong ADCC effects, particularly at higher concentration.

EXAMPLE 7: Inhibitory effect on tumor growth

Mouse colon cancer cell expressing PD-L1 (MC38-PDL1) was subcutaneously injected to mice. When the tumor volume grew to about 100-150 mm ³, the mice were divided into a control group and several treatment groups. For treatment groups G2-G6, either monoclonal or bispecific antibodies were injected with a treatment dosage of 0.3 mg/kg. For treatment group G8, ATOR-1015 was injected at a dosage of 0.4 mg/kg. The control group G1 was injected with an equal volume of saline solution. Intraperitoneal injection was performed and the frequency of administration was once a week (3 administrations in total) . The administrations were indicated in the figure.

Table 4

The weight of the mice was monitored during the entire treatment period (FIGS. 8A and 8B) . Not much difference in weight was observed among these groups. The results showed that the antibodies were well tolerated and not toxic to the mice.

As shown in FIG. 8C, ATOR-1015 did not exhibit good antitumor effects. In contrast, tumor unexpectedly disappeared in mice treated with BCG-93412-2bispecific antibodies. The tumor inhibitory effects of BCG-93412-2 is much stronger than all other tested antibody.

Table 5

The same experiment was repeated to confirm the results. The weight of the mice was monitored during the entire treatment period (FIGS. 9A and 9B) . Similarly, not much difference in weight was observed among these groups. The results showed that the antibodies were well tolerated and not toxic to the mice.

Table 6

As shown in FIG. 9C, the experiment confirmed that BCG-93412-2 had very strong tumor inhibitory effects. The tumor almost disappeared in the mice.

Table 7

EXAMPLE 9: Bispecific antibody with Fab-ScFv-Fc structure

Based on the sequences in the previous Examples, a bispecific antibodyBCG-93412-3 with Fab-ScFv-Fc structure was developed (FIG. 10) . The purified protein was detected by a non-reducing SDS-PAGE (6%separation gel) as shown in FIG. 11.

Three vectors expressing CTLA4-K, CTLA4-H and OX40-ScFv were co-transfected into CHO-Scells. After 14 days of culture, the cell supernatant was harvested and subjected to Protein A affinity chromatography, followed by size-exclusion chromatography (SEC) to obtain the bispecific antibody protein. The full-length sequence of the heavy chain and the light chain are shown below:

Full length of CTLA4-K light chain of BCG-93412-3 (SEQ ID NO: 167) :

Full length of CTLA4-H heavy chainof BCG-93412-3 (SEQ ID NO: 168) :

Full length of OX40-ScFv chain of BCG-93412-3 (SEQ ID NO: 169) :

In order to increase the stability, the VH and VL sequences in the OX40-ScFv chain had additional modifications. The VH in the OX40-ScFv chain is shown below:

The VL in the OX40-ScFv chain is shown below:

EXAMPLE 10: Antibody binding affinity

The binding affinity to human OX40 (hOX40) or human CTLA4 (hCTLA4) of monoclonal antibodies 9H3-H3K1-IgG1, 4G12-H1K1-IgG1 and bispecific antibodies BCG-93412-1, BCG-93412-3 were determined by Biacore systems. Results are shown in the table below.

Table 4

The results show that BCG-93412-1 and BCG-93412-3 have similar binding affinity against hOX40 and hCTLA4.

EXAMPLE 11: Blocking effect of monoclonal antibody and bispecificantibody on CTLA4/B7-1 binding

Experiments were performed to determine the blocking effect on CTLA4/B7-1 binding. Methods were described in Example 3.

As shown in FIG. 12, the two bispecific antibodies with two distinct structures have comparable blocking effect on CTLA4/B7-1 binding. The effects were weaker than the effect of the monoclonal antibody. Calculated IC50 and R ² values are shown in the table below.

Table 5

Antibody Name	IC50 Value	R ² value
4G12-H1K1-IgG1	0.0463	0.9804
BCG-93412-1	0.2647	0.9843
BCG-93412-3	0.4006	0.9677

EXAMPLE 11: Total binding avidity test

The experiment was performed to determine avidity against CHO-K1-hOX40-hCTLA4 cells co-expressing hOX40 and hCTLA4 antigens. Methods for determining avidity was described in Example 5.

Results are shown in FIG. 13. As shown in the figure, BCG-93412-3 has a higher avidity than the tested monoclonal antibodies.

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 following claims.

Claims

An antigen-binding protein construct, comprising

a first heavy chain variable region and a first 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 specifically binds to CTLA4; and

a second heavy chain variable region and a second light chain variable region, wherein 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 to OX40.
The antigen-binding protein construct of claim 1, wherein the antigen-binding protein construct comprises

a first polypeptide comprising the first heavy chain variable region, a first heavy chain constant region 2 (CH2) , and a first heavy chain constant region 3 (CH3) ; and

a second polypeptide comprising the second heavy chain variable region, a second heavy chain constant region 2 (CH2) , and a second heavy chain constant region 3 (CH3) .
The antigen-binding protein construct of claim 2, wherein either one or both of the first polypeptide and the second polypeptide comprises one or both of the following:

(i) Asp at position 239 (EU numbering) ; and

(ii) Glu at position 332 (EU numbering) .
The antigen-binding protein construct ofclaim 2 or 3, wherein the antigen-binding protein construct comprises

a third polypeptide comprising the first light chain variable region; and

a fourth polypeptide comprising the second light chain variable region.
The antigen-binding protein construct of claim 4, wherein the amino acid sequence of the first light chain variable region and the amino acid sequence of the second light chain variable region are at least or about 90%, 95%, or 100%identical.
The antigen-binding protein construct ofclaim 2 or 3, wherein the second polypeptide further comprises the second light chain variable region.
The antigen-binding protein construct of any one of claims 2, 3, and 6, wherein the first polypeptide further comprises the first light chain variable region.
The antigen-binding protein construct of any one of claims 2-7, wherein the first polypeptide and the second polypeptide associate with each other by knobs into holes.
The antigen-binding protein construct ofclaim 8, wherein the first polypeptide has one or more of the following:

(i) Cys at position 354 (EU numbering) ;

(ii) Trp at position 366 (EU numbering) ,

wherein the second polypeptide has one or more of the following:

(i) Cys at position 349 (EU numbering) ;

(ii) Ser at position 366 (EU numbering) ;

(iii) Ala at position 368 (EU numbering) ;

(iv) Val at position 407 (EU numbering) .
The antigen-binding protein construct ofclaim 8, wherein the second polypeptide has one or more of the following:

(i) Cys at position 354 (EU numbering) ;

(ii) Trp at position 366 (EU numbering) ;

wherein the first polypeptide has one or more of the following:

(i) Cys at position 349 (EU numbering) ;

(ii) Ser at position 366 (EU numbering) ;

(iii) Ala at position 368 (EU numbering) ; and

(iv) Val at position 407 (EU numbering) .
The antigen-binding protein construct of any one of claim 1-10, wherein

the first heavy chain variable region (VH1) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR3 amino acid sequence; and

the first light chain variable region (VL1) comprising CDRs 1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL1 CDR3 amino acid sequence,

wherein the selected VH1 CDRs 1, 2, and 3 amino acid sequences, the selected VL1 CDRs 1, 2, and 3 amino acid sequences are one of the following:

(1) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 85, 86, 87, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 88, 89, 90, respectively, according to the Kabat numbering scheme;

(2) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 109, 110, 111, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 112, 113, 114, respectively, according to the Chothia numbering scheme;

(3) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 91, 92, 93, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 94, 95, 96, respectively, according to the Kabat numbering scheme;

(4) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 115, 116, 117, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 118, 119, 120, respectively, according to the Chothia numbering scheme;

(5) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 97, 98, 99, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 100, 101, 102, respectively, according to the Kabat numbering scheme;

(6) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 121, 122, 123, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 124, 125, 126, respectively, according to the Chothia numbering scheme;

(7) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 103, 104, 105, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 106, 107, 108, respectively, according to the Kabat numbering scheme; and

(8) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 127, 128, 129, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 130, 131, 132, respectively, according to the Chothia numbering scheme.
The antigen-binding protein construct of any one of claim 1-11, wherein

the second heavy chain variable region (VH2) comprising CDRs 1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH2 CDR3 amino acid sequence; and

the second light chain variable region (VL2) comprising CDRs 1, 2, and 3, wherein the VL2 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL2 CDR3 amino acid sequence,

wherein the selected VH2 CDRs 1, 2, and 3 amino acid sequences, and the selected VL2 CDRs 1, 2, and 3 amino acid sequences are one of the following:

(1) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 2, 3, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, 6, respectively, according to the Kabat numbering scheme;

(2) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 26, 27, 28, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 29, 30, 31, respectively, according to the Chothia numbering scheme;

(3) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 7, 8, 9, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10, 11, 12, respectively, according to the Kabat numbering scheme;

(4) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 32, 33, 34, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 35, 36, 37, respectively, according to the Chothia numbering scheme;

(5) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 13, 14, 15, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, 18, respectively, according to the Kabat numbering scheme;

(6) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 38, 39, 40, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 41, 42, 43, respectively, according to the Chothia numbering scheme;

(7) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 19, 20, 21, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22, 23, 24, respectively, according to the Kabat numbering scheme;

(8) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 44, 45, 46, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 47, 48, 49, respectively, according to the Chothia numbering scheme;

(9) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 2, 3, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 25, 6, respectively, according to the Kabat numbering scheme; and

(10) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 26, 27, 28, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 29, 50, 31, respectively, according to the Chothia numbering scheme.
The antigen-binding protein construct of claim11 or 12, wherein

(1) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 91, 92, 93, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 94, 95, 96, respectively, the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 2, 3, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 25, 6, respectively, according to the Kabat numbering scheme; or

(2) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 115, 116, 117, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 118, 119, 120, respectively, the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 26, 27, 28, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 29, 50, 31, respectively, according to the Chothia numbering scheme.
The antigen-binding protein construct of any one of claims 1-13, wherein the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 133, 134, 135, 136, 137, or 149, and the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 138, 139, 140, or 150.
The antigen-binding protein construct of any one of claims 1-13, wherein the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 141, 142, 143, 144, or 151, and the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 145, 146, 147, 148, or 152.
The antigen-binding protein construct of any one of claims 1-13, wherein the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 153, and the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 154.
The antigen-binding protein construct of any one of claims 1-13, wherein the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 155, and the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 156.
The antigen-binding protein construct of any one of claims 1-13, wherein the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 51, 52, 53, 77, or 170, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 54, 55, 56, 78, or 171.
The antigen-binding protein construct of any one of claims 1-18, wherein the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 57, 58, 59, or 79, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 60, 61, 62, 63, or 80.
The antigen-binding protein construct of any one of claims 1-18, wherein the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 64, 65, 66, or 81, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 67, 68, 69, 70, or 82.
The antigen-binding protein construct of any one of claims 1-18, wherein the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 71, 72, 73, or 83, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 74, 75, 76, or 84.
The antigen-binding protein construct of any one of claims 1-13, wherein the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 141, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 145, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 53, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 54.
The antigen-binding protein construct of any one of claims 4, 5, and 8-22, wherein the first polypeptide comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 158, and the third polypeptide comprise a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 157.
The antigen-binding protein construct of any one of claims 4, 5, and 8-23, wherein the second polypeptide comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 160, and the fourth polypeptide comprise a sequence that is at least80%, 85%, 90%, or 95%identical to SEQ ID NO: 159.
The antigen-binding protein construct of any one of claims 4, 5, and 8-22, wherein the first polypeptide comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 162, and the third polypeptide comprise a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 161.
The antigen-binding protein construct of any one of claims 4, 5, 8-22, and 25, wherein the second polypeptide comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 164, and the fourth polypeptide comprise a sequence that is at least80%, 85%, 90%, or 95%identical to SEQ ID NO: 163.
The antigen-binding protein construct of any one of claims 2, 3, and 6-22, wherein the second polypeptide comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 169.
The antigen-binding protein construct of any one of claims 1-27, wherein the antigen-binding protein construct is a bispecific antibody.
The antigen-binding protein construct of claim 28, wherein the bispecific antibody has a common light chain.
The antigen-binding protein construct of any one of claims 1-27, wherein the antigen-binding protein construct is a Fab-scFv-Fc.
The antigen-binding protein construct of any one of claims 1-27, wherein the antigen-binding protein construct is a TrioMab, a bispecific antibody with a common light chain, a CrossMab, a 2: 1 CrossMab, a 2: 2 CrossMab, a Duobody, a Dual-variable-domain antibody (DVD-Ig) , a scFv-IgG, a IgG-IgG format antibody, a Fab-scFv-Fc format antibody, a TF, an ADAPTIR, a Bispecific T cell Engager (BiTE) , a BiTE-Fc, a Dual affinity retargeting (DART) , a DART-Fc, a tetravalent DART, a Tandem diabody (TandAb) , a scFv-scFv-scFv, an ImmTAC, a Tri-specific nanobody, or a Trispecific Killer Engager (TriKE) .
A bispecificantibody or antigen-binding fragment thereof, comprising

a first heavy chain polypeptide comprising a first heavy chain variable region;

a first light chain polypeptide comprising a first light chain variable region;

a second heavy chain polypeptide comprising a second heavy chain variable region; and

a second light chain polypeptide, 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, forming a first antigen binding region that specifically binds to CTLA4, 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 to OX40.
The bispecificantibody or antigen-binding fragment thereof of claim 32, comprising the first heavy chain variable region (VH1) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR3 amino acid sequence;

the first light chain variable region (VL1) comprising CDRs 1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL1 CDR3 amino acid sequence;

the second heavy chain variable region (VH2) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH2 CDR3 amino acid sequence; and

the second light chain variable region (VL2) comprising CDRs 1, 2, and 3, wherein the VL2 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL2 CDR3 amino acid sequence;

wherein the selected VH1 CDRs 1, 2, and 3 amino acid sequences, the selected VL1 CDRs 1, 2, and 3 amino acid sequences, the selected VH2 CDRs 1, 2, and 3 amino acid sequences, and the selected VL2 CDRs 1, 2, and 3 amino acid sequences are one of the following:

(1) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 91, 92, 93, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 94, 95, 96, respectively, the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 2, 3, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 25, 6, respectively, according to the Kabat numbering scheme; and

(2) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 115, 116, 117, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 118, 119, 120, respectively, the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 26, 27, 28, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 29, 50, 31, respectively, according to the Chothia numbering scheme.
The bispecificantibody or antigen-binding fragment thereof of claim 32or 33, wherein the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 141, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 145, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 53, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 54.
Thebispecificantibody or antigen-binding fragment thereof of any one of claims 32-34, wherein

(1) the first heavy chain polypeptide comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 158, and the first light chain polypeptide comprise a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 157; and

(2) the second heavy chain polypeptide comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 160, and the second light chain polypeptide comprise a sequence that is at least80%, 85%, 90%, or 95%identical to SEQ ID NO: 159.
The bispecific antibody or antigen-binding fragment thereof of any one of claims 32-34, wherein

(1) the first heavy chain polypeptide comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 162, and the first light chain polypeptide comprise a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 161; and

(2) the second heavy chain polypeptide comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 164, and the second light chain polypeptide comprise a sequence that is at least80%, 85%, 90%, or 95%identical to SEQ ID NO: 163.
A bispecificantibody or antigen-binding fragment thereof, comprising

a heavy chain polypeptide comprising the first heavy chain variable region;

a light chain polypeptide comprising the first light chain variable region; and

a single-chain variable fragment polypeptide comprising a second heavy 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 specifically binds to CTLA4, 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 to OX40.
The bispecific antibody or antigen-binding fragment thereof of claim 37, wherein either one or both of the heavy chain polypeptide and the single-chain variable fragment polypeptide comprises one or both of the following:

(iii) Asp at position 239 (EU numbering) ; and

(iv) Glu at position 332 (EU numbering) .
The bispecific antibody or antigen-binding fragment thereof of claim 37or 38, comprising the first heavy chain variable region (VH1) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR3 amino acid sequence;

the first light chain variable region (VL1) comprising CDRs 1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL1 CDR3 amino acid sequence;

the second heavy chain variable region (VH2) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH2 CDR3 amino acid sequence; and

the second light chain variable region (VL2) comprising CDRs 1, 2, and 3, wherein the VL2 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL2 CDR3 amino acid sequence;

wherein the selected VH1 CDRs 1, 2, and 3 amino acid sequences, the selected VL1 CDRs 1, 2, and 3 amino acid sequences, the selected VH2 CDRs 1, 2, and 3 amino acid sequences, and the selected VL2 CDRs 1, 2, and 3 amino acid sequences are one of the following:

(1) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 91, 92, 93, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 94, 95, 96, respectively, the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 2, 3, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 25, 6, respectively, according to the Kabat numbering scheme; and

(2) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 115, 116, 117, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 118, 119, 120, respectively, the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 26, 27, 28, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 29, 50, 31, respectively, according to the Chothia numbering scheme.
The bispecificantibody or antigen-binding fragment thereof of any one of claims 37-39, wherein the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 141, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 145, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 53 or 170, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 54 or 171.
The bispecific antibody or antigen-binding fragment thereof of any one of claims 37-40, wherein

(1) the heavy chain polypeptide comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 168, and the light chain polypeptide comprise a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 167; and

(2) the single-chain variable fragment polypeptide comprises a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 169.
An antibody or antigen-binding fragment thereof that binds to OX40 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 that is at least 80%identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is 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 that is at least 80%identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR3 amino acid sequence, wherein

(1) the selected VH CDRs 1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1, 2, 3, respectively, and the selected VL CDRs, 1, 2, and 3 amino acid sequences are set forth in SEQ IN NOs: 4, 25, 6, respectively, according to the Kabat numbering scheme; and

(2) the selected VH CDRs 1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 26, 27, 28, respectively, and the selected VL CDRs, 1, 2, and 3 amino acid sequences are set forth in SEQ IN NOs: 29, 50, 31, respectively, according to the Chothia numbering scheme.
The antibody or antigen-binding fragment thereof of claim 42, wherein the antibody or antigen-binding fragment specifically binds to human OX40.
The antibody or antigen-binding fragment thereof of claim 42or 43, wherein the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof.
The antibody or antigen-binding fragment thereof of any one of claims 42-44, wherein the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) .
The antibody or antigen-binding fragment thereof of any one of claims 42-45, wherein the heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%identical to a selected VH sequence, and the light chain variable region (VL) comprising an amino acid sequence that is at least 90%identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

the selected VH sequence is SEQ ID NOs: 51, 52, 53, 77, or 170, and the selected VL sequence is SEQ ID NOs: 54 or 171.
An antibody-drug conjugate comprising the antigen-binding protein construct of any one of claims 1-31, the bispecific antibody or antigen-binding fragment thereof of any one of claims 32-41, or the antibody or antigen-binding fragment thereof of any one of claims 42-46, that covalently bound to a therapeutic agent.
The antibody drug conjugate of claim 47, wherein the therapeutic agent is a cytotoxic or cytostatic agent.
A method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the antigen-binding protein construct of any one of claims 1-31, the bispecific antibody or antigen-binding fragment thereof of any one of claims 32-41, the antibody or antigen-binding fragment thereof of any one of claims 42-46, or the antibody-drug conjugate of claims 47or 48, to the subject.
The method of claim 49, wherein the subject has a solid tumor.
The method of claim 49, wherein the cancer is breast cancer, oropharyngeal cancer, ovarian cancer, bladder cancer, colon cancer, pancreas cancer, B cell lymphoma, or Non-Hodgkin's lymphoma.
The method of any one of claims 49-51, wherein the method further comprises administering an anti-PD1 antibody to the subject.
The method of any one of claims 49-52, wherein the method further comprises administering a chemotherapy to the subject.
A method of decreasing the rate of tumor growth, the method comprising administering to a subject in need thereof an effective amount of a composition comprising the antigen-binding protein construct of any one of claims 1-31, the bispecific antibody or antigen-binding fragment thereof of any one of claims 32-41, the antibody or antigen-binding fragment thereof of any one of claims 42-46, or the antibody-drug conjugate of claims 47 or 48, to the subject.
A method of killing a tumor cell, the method comprising administering to a subject in need thereofan effective amount of a composition comprising the antigen-binding protein construct of any one of claims 1-31, the bispecific antibody or antigen-binding fragment thereof of any one of claims 32-41, the antibody or antigen-binding fragment thereof of any one of claims 42-46, or the antibody-drug conjugate of claims 47 or 48, to the subject.
A pharmaceutical composition comprising the antigen-binding protein construct of any one of claims 1-31, the bispecific antibody or antigen-binding fragment thereof of any one of claims 32-41, or the antibody or antigen-binding fragment thereof of any one of claims 42-46, and a pharmaceutically acceptable carrier.
A pharmaceutical composition comprising the antibody drug conjugate of claim 47or 48, and a pharmaceutically acceptable carrier.