WO2022156773A1

WO2022156773A1 - Protein complexes targeting il12 pathway

Info

Publication number: WO2022156773A1
Application number: PCT/CN2022/073224
Authority: WO
Inventors: Yuan GUO; Yi Yang; Yuelei SHEN
Original assignee: Biocytogen Pharmaceuticals (Beijing) Co., Ltd
Priority date: 2021-01-21
Filing date: 2022-01-21
Publication date: 2022-07-28

Abstract

This disclosure provides protein complexes targeting IL12 pathway and methods of use thereof.

Description

PROTEIN COMPLEXES TARGETING IL12 PATHWAY

CLAIM OF PRIORITY

This application claims the benefit of PCT Application No. PCT/CN2021/073089, filed on January 21, 2021. The entire contents of the foregoing are incorporated herein by reference.

TECHNICAL FIELD

This disclosure relates to protein complexes targeting IL12 pathway and methods of use thereof.

BACKGROUND

According to World Health Organization, cancer is the second leading cause of death globally, accounting for an estimated 9.6 million deaths in 2018. Lung, prostate, colorectal, stomach and liver cancer are the most common types of cancer in men, while breast, colorectal, lung, cervical and thyroid cancer are the most common among women.

Recent clinical and commercial success of checkpoint inhibitors has created great interest in immunotherapies. These checkpoint inhibitors (e.g., anti-PD-1 antibodies) have changed the treatment landscape of many cancers. However, response rate remains relatively low in many cases. There is a need to develop immunotherapies that can further overcome tumor-induced immune suppression while minimizing toxicity of these therapies.

SUMMARY

The present disclosure provides protein complexes targeting IL12R.

In one aspect, the disclosure is related to a protein complex comprising a targeting moiety fused with an immunomodulatory moiety. In some embodiments, (a) the targeting moiety specifically binds to a PD-1 ligand; and (b) the immunomodulatory moiety specifically binds to interleukin-12 receptor (IL12R) .

In some embodiments, the immunomodulatory moiety comprises an antibody or antigen-binding fragment, a single chain variable fragment (scFv) , a Fc-containing polypeptide, or a fusion protein that specifically binds IL12R.

In some embodiments, the immunomodulatory moiety is an IL12R agonist.

In some embodiments, the immunomodulatory moiety comprises IL12. In some embodiments, the IL12 comprises a IL12a (e.g., a human IL12a) , a IL12b (e.g., a human IL12b) , and/or a variant thereof.

In some embodiments, the targeting moiety comprises an antibody or antigen-binding fragment, a single chain variable fragment (scFv) , a Fc-containing polypeptide, or a fusion protein that specifically binds to the PD-1 ligand.

In some embodiments, the targeting moiety comprises a full-length antibody.

In some embodiments, the targeting moiety comprises an extracellular domain of PD-1.

In some embodiments, the PD-1 ligand is PD-L1 or PD-L2.

In some embodiments, the targeting moiety blocks or does not block the interaction between PD-1 and the PD-1 ligand. In some embodiments, the targeting moiety interferes the interaction between PD-1 and the PD-1 ligand. In some embodiments, the interference described herein can reduce the toxicity of the protein complex.

In some embodiments, targeting moiety has a KD of less than 1 x 10 ^-7 M, less than 1 x 10 ^-8 M or less than 1 x 10 ^-9 M with the PD-1 ligand.

In some embodiments, the targeting moiety comprises a polypeptide. In some embodiments, the immunomodulatory moiety is fused to the N-terminus or the C-terminus of the polypeptide.

In some embodiments, the immunomodulatory moiety comprises a polypeptide. In some embodiments, the targeting moiety is fused to the N-terminus or the C-terminus of the polypeptide.

In some embodiments, the targeting moiety comprises a polypeptide comprising a CH3 domain. In some embodiments, the immunomodulatory moiety is fused to the CH3 domain.

In some embodiments, the targeting moiety and the immunomodulatory moiety are fused to a scaffold protein (e.g., an albumin) .

In some embodiments, the protein complex comprises a bispecific antibody. In some embodiments, the bispecific antibody binds to the PD-1 ligand and the IL12R.

In some embodiments, the protein complex comprises two or more immunomodulatory moieties that specifically bind to IL12R.

In some embodiments, the protein complex comprises two or more targeting moieties that specifically bind to a PD-1 ligand.

In some embodiments, the protein complex comprises an Fc comprising two CH3 domains.

In some embodiments, the immunomodulatory moiety is linked to a CH3 domain of the two CH3 domains in the Fc.

In some embodiments, the immunomodulatory moiety is linked to the C-terminus of a CH3 domain of the two CH3 domains.

In some embodiments, the immunomodulatory moiety is fused to a CH3 domain of the two CH3 domains in the Fc at a region from position 344 to position 382 of the CH3 domain according to EU numbering.

In some embodiments, the immunomodulatory moiety comprises IL12a and IL12b. In some embodiments, IL12a is linked to the Fc.

In some embodiments, the immunomodulatory moiety comprises IL12a and IL12b. In some embodiments, IL12b is linked to the Fc.

In some embodiments, the targeting moiety is linked to a CH3 domain of the two CH3 domains in the Fc.

In some embodiments, the targeting moiety is linked to the C-terminus of a CH3 domain of the two CH3 domains in the Fc.

In some embodiments, the targeting moiety is fused to a CH3 domain of the two CH3 domains in the Fc at a region from position 344 to position 382 of the CH3 domain according to EU numbering.

In some embodiments, the targeting moiety comprises a scFv (e.g., a scFv targeting the PD-1 ligand) .

In some embodiments, the protein complex comprises two light chains.

In some embodiments, the immunomodulatory moiety is linked to one of the two light chains.

In some embodiments, the targeting moiety is linked to one of the two light chains.

In some embodiments, the protein complex further comprises an interferon (e.g., IFNa1, IFNa2, IFNa3, IFNa4, and/or IFNγ) .

In some embodiments, the protein complex further comprises a cytokine (e.g., IL2, IL7, IL15, IL18, and/or IL21) .

In some embodiments, the interferon or the cytokine is linked to the protein complex by a linker sequence.

In some embodiments, the protein complex comprises a linker sequence (GGGGS) n. In some embodiments, n can be 1, 2, 3, 4, 5, 6, 7 or 8.

In some embodiments, the targeting moiety comprises an anti-PD-L1 antibody or antigen-binding fragment thereof.

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof is an IgG (e.g., IgG1, IgG2 or IgG4) .

In some embodiments, the targeting moiety comprises a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, and a light chain variable region (VL) comprising

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. In some embodiments, the selected

VH CDRs

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

(1) the selected

VH CDRs

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

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, and 6, respectively;

(2) the selected

VH CDRs

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

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10, 11, and 12, respectively;

(3) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 104, 105, and 106, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 107, 108, and 109 respectively;

(4) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 134, 135, and 136, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 137, 138, and 139, respectively;

(5) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 110, 111, and 112, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 113, 114, and 115, respectively;

(6) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 140, 141, and 142, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 143, 144, and 145, respectively;

(7) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 116, 117, and 118, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 119, 120, and 121, respectively;

(8) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 146, 147, and 148, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 149, 150, and 151, respectively;

(9) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 122, 123, and 124, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 125, 126, and 127, respectively;

(10) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 152, 153, and 154, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 155, 156, and 157, respectively;

(11) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 128, 129, and 130, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 131, 132, and 133, respectively; and

(12) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 158, 159, and 160, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 161, 162, and 163, respectively.

In some embodiments, the targeting moiety comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%identical to SEQ ID NO: 13, 14, 15, or 16, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90%identical to SEQ ID NO: 17, 18, or 19.

In some embodiments, the immunomodulatory moiety comprises a sequence that is at least 80%identical to SEQ ID NO: 28 and a sequence that is at least 80%identical to SEQ ID NO: 29.

In some embodiments, the targeting moiety comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%identical to SEQ ID NO: 66, 68, 70, 78, 80, or 98, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90%identical to SEQ ID NO: 67, 69, 71, 79, 81, or 99.

In one aspect, the disclosure is related to an antibody or antigen-binding fragment thereof that binds to PD-L1, comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence 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, in some embodiments, the selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are one of the following:

(1) the selected

VH CDRs

VL CDRs

(2) the selected

VH CDRs

VL CDRs

(3) the selected

VH CDRs

VL CDRs

(4) the selected

VH CDRs

VL CDRs

(5) the selected

VH CDRs

VL CDRs

(6) the selected

VH CDRs

VL CDRs

(7) the selected

VH CDRs

VL CDRs

(8) the selected

VH CDRs

VL CDRs

(9) the selected

VH CDRs

VL CDRs

(10) the selected

VH CDRs

VL CDRs

In some embodiments, the VH comprises

CDRs

1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 104, 105, and 106 respectively, and the VL comprises

CDRs

1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 107, 108, and 109, respectively.

In some embodiments, the VH comprises

CDRs

1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 110, 111, and 112, respectively, and the VL comprises

CDRs

1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 113, 114, and 115, respectively.

In some embodiments, the VH comprises

CDRs

1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 116, 117, and 118, respectively, and the VL comprises

CDRs

1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 119, 120, and 121, respectively.

In some embodiments, the VH comprises

CDRs

1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 122, 123, and 124, respectively, and the VL comprises

CDRs

1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 125, 126, and 127, respectively.

In some embodiments, the VH comprises

CDRs

1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 128, 129, and 130, respectively, and the VL comprises

CDRs

1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 131, 132, and 133, respectively.

In some embodiments, the antibody or antigen-binding fragment specifically binds to human PD-L1.

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

In some embodiments, the antibody or antigen-binding fragment is a single-chain variable fragment (scFV) , or a multi-specific antibody (e.g., a bispecific antibody) .

In one aspect, the disclosure is related to a nucleic acid comprising a polynucleotide encoding a polypeptide comprising:

(1) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 104, 105 and 106, respectively, and in some embodiments, the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 81, binds to PD-L1;

(2) an immunoglobulin light chain or a fragment thereof comprising a

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 107, 108, and 109, respectively, and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 80, binds to PD-L1;

(3) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 110, 111, and 112, respectively, and in some embodiments, the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 79, binds to PD-L1;

(4) an immunoglobulin light chain or a fragment thereof comprising a

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 113, 114, and 115, respectively, and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 78, binds to PD-L1;

(5) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 116, 117, and 118, respectively, and in some embodiments, the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 71, binds to PD-L1;

(6) an immunoglobulin light chain or a fragment thereof comprising a

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 119, 120, and 121, respectively, and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 70, binds to PD-L1;

(7) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 122, 123, and 124, respectively, and in some embodiments, the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 67, binds to PD-L1;

(8) an immunoglobulin light chain or a fragment thereof comprising a

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 125, 126, and 127, respectively, and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 66, binds to PD-L1;

(9) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 128, 129, and 130, respectively, and in some embodiments, the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 99, binds to PD-L1; or

(10) an immunoglobulin light chain or a fragment thereof comprising a

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 131, 132, and 133, respectively, and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 98, binds to PD-L1.

In some embodiments, the VH when paired with a VL specifically binds to human PD-L1, or the VL when paired with a VH specifically binds to human PD-L1.

In some embodiments, the immunoglobulin heavy chain or the fragment thereof is a human or humanized immunoglobulin heavy chain or a fragment thereof, and the immunoglobulin light chain or the fragment thereof is a human or humanized immunoglobulin light chain or a fragment thereof.

In some embodiments, the nucleic acid encodes a single-chain variable fragment (scFv) .

In some embodiments, the nucleic acid is cDNA.

In one aspect, the disclosure is related to a vector comprising one or more of the nucleic acids as described herein.

In one aspect, the disclosure is related to a vector comprising two of the nucleic acids as described herein, in some embodiments, the vector encodes the VL region and the VH region that together bind to PD-L1.

In one aspect, the disclosure is related to a pair of vectors, wherein each vector comprises one of the nucleic acids as described herein, in some embodiments, together the pair of vectors encodes the VL region and the VH region that together bind to PD-L1.

In one aspect, the disclosure is related to a cell comprising the vector, or the pair of vectors as described herein. In some embodiments, the cell is a CHO cell.

In one aspect, the disclosure is related to a cell comprising one or more of the nucleic acids as described herein.

In one aspect, the disclosure is related to a cell comprising two of the nucleic acids as described herein. In some embodiments, the two nucleic acids together encode the VL region and the VH region that together bind to PD-L1.

In one aspect, the disclosure is related to a method of producing an antibody or an antigen-binding fragment thereof, the method comprising (a) culturing the cell as described herein under conditions sufficient for the cell to produce the antibody or the antigen-binding fragment; and (b) collecting the antibody or the antigen-binding fragment produced by the cell.

In one aspect, the disclosure is related to an antibody or antigen-binding fragment thereof that binds to PD-L1 comprising a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%identical to a selected VH sequence, and a 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 is SEQ ID NO: 66, 70, 78, 80, or 98, and the selected VL sequence is SEQ ID NO: 67, 71, 79, 81, or 99. In some embodiments, the VH comprises the sequence of SEQ ID NO: 66 and the VL comprises the sequence of SEQ ID NO: 67. In some embodiments, the VH comprises the sequence of SEQ ID NO: 70 and the VL comprises the sequence of SEQ ID NO: 71. In some embodiments, the VH comprises the sequence of SEQ ID NO: 78 and the VL comprises the sequence of SEQ ID NO: 79. In some embodiments, the VH comprises the sequence of SEQ ID NO: 80 and the VL comprises the sequence of SEQ ID NO: 81. In some embodiments, the VH comprises the sequence of SEQ ID NO: 98 and the VL comprises the sequence of SEQ ID NO: 99.

In one aspect, the disclosure is related to a method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the protein complex or the antibody or antigen-binding fragment thereof as described herein, to the subject.

In some embodiments, the subject has a cancer that expresses PD-L1. In some embodiments, the subject has a cancer that does not express PD-L1.

In some embodiments, the cancer is resistant to anti-PD-1 antibody or anti-PD-L1 antibody treatment.

In some embodiments, the method described herein further comprises administering an effective amount of an anti-PD-1 antibody or an anti-PD-L1 antibody to the subject.

In one aspect, the disclosure is related to a method of increasing immune response (e.g., in tumor microenvironment, spleen, lymph nodes, and/or lymphatic system) in a subject, the method comprising administering a therapeutically effective amount of a composition comprising the protein complex or the antibody or antigen-binding fragment thereof as described herein to the subject.

In one aspect, the disclosure is related to a method of activating T cells (e.g., in tumor microenvironment, spleen, lymph nodes, and/or lymphatic system) of a subject, the method comprising administering a therapeutically effective amount of a composition comprising the protein complex as described herein to the subject.

In one aspect, the disclosure is related to a method of reducing IL12 toxicity when delivering IL12 into a subject, the method comprising administering a therapeutically effective amount of a composition comprising the protein complex as described herein, to the subject.

In one aspect, the disclosure is related to an isolated molecule comprising the protein complex or the antibody or antigen-binding fragment thereof as described herein; covalently bound to a therapeutic agent.

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

In one aspect, the disclosure is related to a pharmaceutical composition comprising the protein complex or the antibody or antigen-binding fragment thereof as described herein; and a pharmaceutically acceptor carrier.

In one aspect, the disclosure is related to a nucleic acid encoding the protein complex or the antigen-binding fragment thereof as described herein.

In one aspect, the disclosure is related to a vector comprising the nucleic acid as described herein.

In one aspect, the disclosure is related to a host cell comprising the nucleic acid as described herein.

In one aspect, the disclosure is related to a method for producing a protein complex, the method comprising culturing the host cell as described herein under conditions suitable to produce the protein complex.

In one aspect, the disclosure is related to a method of reducing IL12 toxicity when delivering an agent comprising IL12 into a subject, in some embodiments, the IL12 is conjugated (e.g., fused) to an PD-L1 targeting moiety. In some embodiments, the PD-L1 targeting moiety is an anti-PD-L1 antibody or antigen-binding fragment thereof (e.g., any one of the anti-PD-L1 antibody or antigen-binding fragment thereof as described herein) . In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof can block or interfere the interaction between PD-1 (e.g., human PD-1) and PD-L1 (e.g., human PD-L1) .

As used herein, the term “protein complex” refers to a group of associated polypeptides. These polypeptides may have different or the same functions. They can associate with each other by non-covalent interactions or covalent bonds (e.g., peptide bonds or disulfide bonds) . A protein complex can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 polypeptide chains.

As used herein, the term “antibody” or “immunoglobulin” refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementary determining region (CDR) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope. Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bi-specific antibodies) , single-chain antibodies, single domain antibodies, nanobodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, an antibody contains an Fc region of a human antibody. The term antibody also includes derivatives, e.g., bi-specific antibodies, single-chain antibodies, scFv, diabodies, linear antibodies, and multi-specific antibodies formed from antibody fragments.

As used herein, the term “IgG-like” refers to a molecule that is largely similar to an IgG antibody. Non-limiting examples of IgG-like molecules include an IgG antibody with one or more non-native polypeptides that are added to the IgG antibody. In some embodiments, the protein complex as described herein is an IgG-like molecule.

As used herein, the term “antigen-binding fragment” refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of a heavy chain or a variable domain of light chain) . Non-limiting examples of antibody fragments include, e.g., Fab, Fab’, F (ab’) ₂, Fv fragments, and VHH (single domain antibody or the variable domain of a heavy chain antibody) .

As used herein, the term "full length antibody" refers to an antibody that has an intact structure as compared to a wildtype antibody. In some embodiments, a full length antibody is an antibody having two full-length heavy chains and two full length light chains.

As used herein, the term “human antibody” refers to an antibody that is encoded by an endogenous nucleic acid (e.g., rearranged human immunoglobulin heavy or light chain locus) present in a human. In some embodiments, a human antibody is collected from a human or produced in a human cell culture (e.g., human hybridoma cells) . In some embodiments, a human antibody is produced in a non-human cell (e.g., a mouse or hamster cell line) .

As used herein, the term “chimeric antibody” refers to an antibody that contains a sequence present in at least two different antibodies (e.g., antibodies from two different mammalian species such as a human and a mouse antibody) . A non-limiting example of a chimeric antibody is an antibody containing the variable domain sequences (e.g., all or part of a light chain and/or heavy chain variable domain sequence) of a non-human (e.g., mouse) antibody and the constant domains of a human antibody.

As used herein, the term “humanized antibody” refers to a non-human antibody which contains minimal sequence derived from a non-human (e.g., mouse) immunoglobulin and contains sequences derived from a human immunoglobulin. In non-limiting examples, humanized antibodies are human antibodies (recipient antibody) in which hypervariable (e.g., CDR) region residues of the recipient antibody are replaced by hypervariable (e.g., CDR) region residues from a non-human antibody (e.g., a donor antibody) , e.g., a mouse, rat, or rabbit antibody, having the desired specificity, affinity, and capacity. In some embodiments, the Fv framework residues of the human immunoglobulin are replaced by corresponding non-human (e.g., mouse) immunoglobulin residues. In some embodiments, humanized antibodies may contain residues which are not found in the recipient antibody or in the donor antibody. These modifications can be made to further refine antibody performance.

As used herein, the term “single-chain antibody” refers to a single polypeptide that contains at least two immunoglobulin variable domains (e.g., a variable domain of a mammalian immunoglobulin heavy chain or light chain) that is capable of specifically binding to an antigen. Non-limiting examples of single-chain antibodies are described herein.

As used herein, the term “multispecific antibody” refers to an antibody that can specifically bind to two or more different antigens or epitopes. In some embodiments, the multispecific antibody is able to crosslink one target molecule (e.g., PD-L1) to at least one second target molecule (e.g., IL12R) on the surface of a mammalian cell (e.g., a human T-cell) . In some embodiments, a multispecific antibody is a bispecific antibody.

As used herein, when referring to an antibody, the phrases “specifically binding” and “specifically binds” mean that the antibody interacts with its target molecule (e.g., PD-L1) preferably to other molecules, because the interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) on the target molecule; in other words, the reagent is recognizing and binding to molecules that include a specific structure rather than to all molecules in general. An antibody that specifically binds to the target molecule may be referred to as a target-specific antibody. For example, an antibody that specifically binds to a PD-L1 molecule may be referred to as a PD-L1-specific antibody or an anti-PD-L1 antibody.

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.

As used herein, the terms “polypeptide, ” “peptide, ” and “protein” are used interchangeably to refer to polymers of amino acids of any length of at least two amino acids.

As used herein, the terms “polynucleotide, ” “nucleic acid molecule, ” and “nucleic acid sequence” are used interchangeably herein to refer to polymers of nucleotides of any length of at least two nucleotides, and include, without limitation, DNA, RNA, DNA/RNA hybrids, and modifications thereof.

As used herein, the term “fusion” or “fused” when used with respect to amino acid sequences (e.g. peptide, polypeptide or protein) refers to combination of two or more amino acid sequences, for example by chemical bonding or recombinant means, into a single amino acid sequence. A fusion amino acid sequence can be produced by genetic recombination of two encoding polynucleotide sequences, and can be expressed by a method of introducing a construct containing the recombinant polynucleotides into a host cell.

As used herein, the term “linked” when used with respect to amino acid sequences (e.g. peptide, polypeptide or protein) refers to combination of two or more amino acid sequences by chemical bonds (e.g., a peptide bond, a disulfide bond, bis-sulfone linker) .

As used herein, the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1%to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In some embodiments, the terms “about” or “approximately” when preceding a numerical value or a level indicates the value plus or minus a range of 15%, 10%, 5%, or 1%. The term “about the same level” indicates the level plus or minus a range of 15%, 10%, 5%, or 1%.

As used herein, the term “operably link” or “operably linked” refers to a juxtaposition, with or without a spacer or linker sequence, of two or more biological sequences of interest in such a way that they are in a relationship permitting them to function in an intended manner. When used with respect to polypeptides, it is intended to mean that the polypeptide sequences are linked in such a way that permits the linked product to have the intended biological function. For example, an antibody variable region may be operably linked to a constant region so as to provide for a stable product with antigen-binding activity. The term can also be used with respect to polynucleotides. For one instance, when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., promoter, enhancer, silencer sequence, etc. ) , it is intended to mean that the polynucleotide sequences are linked in such a way that permits regulated expression of the polypeptide from the polynucleotide.

As used herein, the term “binding partner” refers to a member of a pair of molecules capable of recognizing a specific structural aspect of another molecule, wherein the binding partners interact with each other by means of a specific, noncovalent or covalent interaction. Examples of such binding partners and corresponding molecules or compositions include, but are not limited to, any of the class of immune-type binding pairs, such as antigen/antibody; and also any of the class of nonimmune-type binding pairs, such as ligand/receptor, biotin/avidin, biotin/streptavidin, digoxigenin/anti-digoxigenin F (ab’) 2, folic acid/folate binding protein, complementary nucleic acid segments, protein A or G/immunoglobulins, lectin/carbohydrate, substrate/enzyme, inhibitor/enzyme, or virus/cellular receptor.

As used herein, a “targeting moiety” refers to a molecule that has the ability to localize and bind to a specific molecule or cellular component. The targeting moiety can be an antibody, antibody fragment, scFv, Fc-containing polypeptide, fusion antibody, polypeptide, peptide, aptamer, ligand, nucleic acid, or any combination thereof. In some embodiments, a targeting moiety can bind to a molecule present in a cell or tissue. In some embodiments, the targeting moiety can bind to a molecule in a diseased cell or tissue, e.g., a cancer cell or tumor. In some embodiments, the targeting molecule can bind to a normal cell or tissue, e.g., an immune cell such as T cell. In some embodiments, the targeting moiety can bind to a cellular or extracellular molecule that modulates the immune response. In some embodiments, the targeting moiety binds to a PD-1 ligand (e.g., PD-L1) .

As used herein, an “immunomodulatory moiety” refers to a ligand, peptide, polypeptide, or Fc-containing polypeptide that binds a specific component of an immune cell (e.g., T cell, regulatory T cell, myeloid suppressor cell, or dendritic cell) and modulates the number or function of immune cells. In some embodiments, the “immunomodulatory moiety” specifically binds a cytokine, cytokine receptor, co-stimulatory molecule, or co-inhibitory molecule that modulates the immune system. In some embodiments, the immunomodulatory moiety specifically binds to IL-12R. In some embodiments, the immunomodulatory moiety is an agonist that increases the function of the bound molecule.

As used herein, the term “IL12” refers to interleukin 12, which can be a wildtype IL12 (e.g., a human IL12, a mouse IL12, a feline IL12, a monkey IL12, or a canine IL12) , a polypeptide derived from a wildtype IL12, a variant thereof (e.g., with mutations) , or a portion thereof (e.g., all or a portion of IL12a and/or all or a portion of IL12b) . In some embodiments, IL12a can be a wildtype IL12a (e.g., a human IL12a, a mouse IL12a, a feline IL12a, a monkey IL12a, or a canine IL12a) , a polypeptide derived from a wildtype IL12a (e.g., a domain with mutations) , a variant thereof (e.g., with mutations) , or a portion thereof. In some embodiments, IL12b can be a wildtype IL12b (e.g., a human Il12b, a mouse IL12b, a feline IL12b, a monkey IL12b, or a canine IL12b) , a polypeptide derived from a wildtype IL12b (e.g., a domain with mutations) , a variant thereof (e.g., with mutations) , or a portion thereof.

In some embodiments, the immunomodulatory moiety described herein can specifically bind to IL12R (e.g., either one or both of IL12Rb1 and IL12Rb2) . The IL12R can be a wildtype IL12R (e.g., a human IL12R, a mouse IL12R, a feline IL12R, a monkey IL12R, or a canine IL12R) , a polypeptide derived from a wildtype IL12R, a variant thereof (e.g., with mutations) , or a portion thereof (e.g., all or a portion of IL12Rb1 and/or all or a portion of IL12Rb2) . In some embodiments, IL12Rb1 can be a wildtype IL12Rb1 (e.g., a human IL12Rb1, a mouse IL12Rb1, a feline IL12Rb1, a monkey IL12Rb1, or a canine IL12Rb1) , a polypeptide derived from a wildtype IL12Rb1 (e.g., a domain with mutations) , a variant thereof (e.g., with mutations) , or a portion thereof. In some embodiments, IL12Rb2 can be a wildtype IL12Rb2 (e.g., a human IL12Rb2, a mouse IL12Rb2, a feline IL12Rb2, a monkey IL12Rb2, or a canine IL12Rb2) , a polypeptide derived from a wildtype IL12Rb2 (e.g., a domain with mutations) , a variant thereof (e.g., with mutations) , or a portion thereof.

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. 1A shows a schematic structure of 3F2-IL12-3A.

FIG. 1B shows schematic diagrams of plasmids encoding the modified heavy chain, light chain and IL12b of 3F2-mIL12-3A.

FIG. 2A shows a schematic structure of 3F2-IL12-3A-IFNa4.

FIG. 2B shows schematic diagrams of plasmids encoding the modified heavy chain, light chain and IL12b of 3F2-mIL12-3A-mIFNa4. L indicates a linker sequence.

FIG. 3 shows non-reducing SDS-PAGE results of 3F2-mIL12-3A and 3F2-mIL12-3A-mIFNa4.

FIG. 4A shows SEC result of 3F2-mIL12-3A.

FIG. 4B shows SEC result of 3F2-mIL12-3A-mIFNa4.

FIG. 5 is a graph showing body weight over time of double humanized hPD-1/hPD-L1 mice that were injected with mouse colon cancer cells MC38, and treated with PDL1-3F2 IgG1 (PDL1-3F2) , 3F2-mIL12-3A, or 3F2-mIL12-3A-mIFNa4. Saline solution was injected as a control.

FIG. 6 is a graph showing body weight change over time of double humanized hPD-1/hPD-L1 mice that were injected with mouse colon cancer cells MC38, and treated with PDL1-3F2 IgG1 (PDL1-3F2) , 3F2-mIL12-3A, or 3F2-mIL12-3A-mIFNa4. Saline solution was injected as a control.

FIG. 7 is a graph showing average tumor volume in different groups of double humanized hPD-1/hPD-L1 mice that were injected with mouse colon cancer cells MC38, and treated with PDL1-3F2 IgG1 (PDL1-3F2) , 3F2-mIL12-3A, or 3F2-mIL12-3A-mIFNa4. Saline solution was injected as a control.

FIG. 8 shows the average tumor volume in different groups of double humanized hPD-1/hPD-L1 mice that were injected with MC38-hPD-L1, and treated with PDL1-3F2 IgG1 or 3F2-mIL12-3A. PBS solution was injected as a control.

FIG. 9 shows survival curves of double humanized hPD-1/hPD-L1 mice that were injected with MC38-hPD-L1, and treated with PDL1-3F2 IgG1 or 3F2-mIL12-3A. PBS solution was injected as a control.

FIG. 10 shows the average tumor volume in different groups of double humanized hPD-1/hPD-L1 mice that were injected with B16F10-hPDL1, and treated with PDL1-3F2 IgG1 or 3F2-mIL12-3A. PBS solution was injected as a control.

FIG. 11 shows survival curves of double humanized hPD-1/hPD-L1 mice that were injected with B16F10-hPDL1, and treated with PDL1-3F2 IgG1 or 3F2-mIL12-3A. PBS solution was injected as a control.

FIG. 12 shows the average tumor volume in different groups of double humanized hPD-1/hPD-L1 mice that were injected with MC38-hPD-L1, and treated with PDL1-3F2 IgG1 (G2) , Avelumab IgG1 (G3) , Atezolizumab IgG1 (G4) , 3F2-mIL12-3A (G5) , Avelumab-mIL12-3A IgG1 (G6) , or Atezolizumab-mIL12-3A IgG1 (G7) . PBS solution was injected as a control (G1) .

FIG. 13 shows survival curves of double humanized hPD-1/hPD-L1 mice that were injected with MC38-hPD-L1, and treated with PDL1-3F2 IgG1 (G2) , Avelumab IgG1 (G3) , Atezolizumab IgG1 (G4) , 3F2-mIL12-3A (G5) , Avelumab-mIL12-3A IgG1 (G6) , or Atezolizumab-mIL12-3A IgG1 (G7) . PBS solution was injected as a control (G1) .

FIG. 14 shows the average tumor volume in different groups of double humanized hPD-1/hPD-L1 mice that were injected with MC38-hPD-L1, and treated with PDL1-3F2 IgG1 (G2) , 30-2C12-IgG1 (G3) , 23-2A6-IgG1 (G4) , 23-10A11-IgG1 (G5) , 3F2-mIL12-3A (G6) , PL1-2C12-mIL12-3A-IgG1 (G7) , PL1-2A6-mIL12-3A-IgG1 (G8) , or PL1-10A11-mIL12-3A-IgG1 (G9) . PBS solution was injected as a control (G1) .

FIG. 15 shows survival curves of double humanized hPD-1/hPD-L1 mice that were injected with MC38-hPD-L1, and treated with PDL1-3F2 IgG1 (G2) , 30-2C12-IgG1 (G3) , 23-2A6-IgG1 (G4) , 23-10A11-IgG1 (G5) , 3F2-mIL12-3A (G6) , PL1-2C12-mIL12-3A-IgG1 (G7) , PL1-2A6-mIL12-3A-IgG1 (G8) , or PL1-10A11-mIL12-3A-IgG1 (G9) . PBS solution was injected as a control (G1) .

FIG. 16 shows the competitive binding assay results of anti-PD-L1 antibodies with different antigen-binding epitopes.

FIG. 17 is a schematic diagram showing different classes of anti-PD-L1 antibodies based on the competitive binding assay results.

FIG. 18 shows the average tumor volume in different groups of double humanized hPD-1/hPD-L1 mice that were injected with MC38-hPD-L1, and treated with PDL1-3F2 IgG1 (PDL1-3F2; G2) , 30-2C12-IgG1 (G3) , 23-2A5-IgG1 (G4) , 17-1C9-IgG1 (G5) , 3F2-mIL12-3A (G6) , PL1-2C12-mIL12-3A-IgG1 (G7) , PL1-2A5-mIL12-3A-IgG1 (G8) , or PL1-1C9-mIL12-3A-IgG1 (G9) . PBS solution was injected as a control (G1) .

FIG. 19 shows survival curves of double humanized hPD-1/hPD-L1 mice that were injected with MC38-hPD-L1, and treated with PDL1-3F2 IgG1 (G2) , 30-2C12-IgG1 (G3) , 23-2A5-IgG1 (G4) , 17-1C9-IgG1 (G5) , 3F2-mIL12-3A (G6) , PL1-2C12-mIL12-3A-IgG1 (G7) , PL1-2A5-mIL12-3A-IgG1 (G8) , or PL1-1C9-mIL12-3A-IgG1 (G9) . PBS solution was injected as a control (G1) .

FIG. 20 shows the body weight over time of double humanized hPD-1/hPD-L1 mice that were injected with MC38-hPD-L1, and treated with 0.1-30 mg/kg 3F2-mIL12-3A. PBS solution was injected as a control.

FIG. 21 shows the IFN-γ concentration in mouse serum samples.

FIG. 22 shows the average tumor volume in different groups of double humanized hPD-1/hPD-L1 mice that were injected with MC38-hPD-L1, and treated with 0.1-30 mg/kg 3F2-mIL12-3A. PBS solution was injected as a control (G1) .

FIG. 23 shows survival curves of double humanized hPD-1/hPD-L1 mice that were injected with MC38-hPD-L1, and treated with 0.1-30 mg/kg 3F2-mIL12-3A. PBS solution was injected as a control (G1) .

FIG. 24 shows the average tumor volume in double humanized hPD-1/hPD-L1 mice (G1) or tumor-regression hPD-1/hPD-L1 mice (G2) that were injected with MC38-hPD-L1. Tumor size was measured for 25 days.

FIG. 25A shows a schematic structure of 3F2-IL12-HC-IgG1.

FIG. 25B shows a schematic structure of 3F2-IL12-CK-IgG1.

FIG. 25C shows a schematic structure of IL12-3F2-IgG1.

FIG. 25D shows a schematic structure of IL12-NK-3F2-IgG1.

FIGS. 26A-26C are native gel electrophoresis results showing purified fusion proteins.

FIG. 26D is a table showing the lane representing each fusion protein in FIGS. 26A-26C. M is a protein marker.

FIG. 27 shows the body weight over time of double humanized hPD-1/hPD-L1 mice that were injected with MC38-hPD-L1, and treated with PDL1-3F2 IgG1 (G2) , 3F2-mIL12-3A (G3) , mIL12-3F2-IgG1 (G4) , mIL12-NK-3F2-IgG1 (G5) , 3F2-mIL12-HC-IgG1 (G6) , 3F2-mIL12-CK-IgG1 (G7) , or MOCK-mIL12-3A-IgG1 (G8) . PBS solution was injected as a control (G1) .

FIG. 28 shows the average tumor volume in different groups of double humanized hPD-1/hPD-L1 mice that were injected with MC38-hPD-L1, and treated with PDL1-3F2 IgG1 (G2) , 3F2-mIL12-3A (G3) , mIL12-3F2-IgG1 (G4) , mIL12-NK-3F2-IgG1 (G5) , 3F2-mIL12-HC-IgG1 (G6) , 3F2-mIL12-CK-IgG1 (G7) , or MOCK-mIL12-3A-IgG1 (G8) . PBS solution was injected as a control (G1) .

FIG. 29 shows survival curves of double humanized hPD-1/hPD-L1 mice that were injected with MC38-hPD-L1, and treated with PDL1-3F2 IgG1 (G2) , 3F2-mIL12-3A (G3) , mIL12-3F2-IgG1 (G4) , mIL12-NK-3F2-IgG1 (G5) , 3F2-mIL12-HC-IgG1 (G6) , 3F2-mIL12-CK-IgG1 (G7) , or MOCK-mIL12-3A-IgG1 (PL1-MOCK-mIL12-3A-IgG1; G8) . PBS solution was injected as a control (G1) .

FIG. 30 shows the body weight over time of double humanized hPD-1/hPD-L1 mice that were injected with MC38-hPD-L1, and treated with PD1-25-1A7-IgG4-S228P (G2) , MOCK-mIL12-3A-IgG4-S228P (G3) , a combination of PD1-25-1A7-IgG4-S228P and MOCK-mIL12-3A-IgG4-S228P (G4) , or PD1-1A7-mIL12-3A-IgG4 (G5) . PBS solution was injected as a control (G1) .

FIG. 31 shows the average tumor volume in different groups of double humanized hPD-1/hPD-L1 mice that were injected with MC38-hPD-L1, and treated with PD1-25-1A7-IgG4-S228P (G2) , MOCK-mIL12-3A-IgG4-S228P (G3) , a combination of PD1-25-1A7-IgG4-S228P and MOCK-mIL12-3A-IgG4-S228P (G4) , or PD1-1A7-mIL12-3A-IgG4 (G5) . PBS solution was injected as a control (G1) . “*” indicates p < 0.05 relative to G1 data.

FIG. 32 shows the body weight over time of double humanized hPD-1/hPD-L1 mice that were injected with MC38-hPD-L1, and treated with PDL1-3F2 IgG1 (PDL1-3F2; G2) , Fc-mIL12-HC (G3) , a combination of PDL1-3F2 IgG1 and Fc-mIL12-HC (PDL1-3F2 + Fc-mIL12- HC; G4) , 3F2-mIL12-3A (G5) , 3F2-mIL12-3A-IgG1-N297A (G6) , or 3F2-mIL12-3A-IgG1-knob (G7) . PBS solution was injected as a control (G1) .

FIG. 33 shows the average tumor volume in different groups of double humanized hPD-1/hPD-L1 mice that were injected with MC38-hPD-L1, and treated with PDL1-3F2 IgG1 (PDL1-3F2; G2) , Fc-mIL12-HC (G3) , a combination of PDL1-3F2 IgG1 and Fc-mIL12-HC (PDL1-3F2 + Fc-mIL12-HC; G4) , or 3F2-mIL12-3A (G5) . PBS solution was injected as a control (G1) .

FIG. 34 shows the average tumor volume in different groups of double humanized hPD-1/hPD-L1 mice that were injected with MC38-hPD-L1, and treated with PDL1-3F2 IgG1 (PDL1-3F2; G2) , 3F2-mIL12-3A (G5) , 3F2-mIL12-3A-IgG1-N297A (G6) , or 3F2-mIL12-3A-IgG1-knob (G7) . PBS solution was injected as a control (G1) .

FIG. 35 shows the IFN-γ concentration in mouse serum samples.

FIG. 36 shows survival curves of double humanized hPD-1/hPD-L1 mice that were injected with MC38-hPD-L1, and treated with PDL1-3F2 IgG1 (G2) , 3F2-mIL12-3A (; G5) , 3F2-mIL12-3A-IgG1-N297A (G6) , or 3F2-mIL12-3A-IgG1-knob (G7) . PBS solution was injected as a control (G1) .

FIG. 37 shows the body weight over time of B-NDG mice that were injected with MC38-hPD-L1, and treated with PDL1-3F2 IgG1 (G2) , Atezolizumab IgG1 (G3) , 3F2-mIL12-3A (3F2-mIL12-3A IgG1; G4) , or Atezolizumab-mIL12-3A IgG1 (G5) . PBS solution was injected as a control (G1) .

FIG. 38 shows the average tumor volume in different groups of B-NDG mice that were injected with MC38-hPD-L1, and treated with PDL1-3F2 IgG1 (G2) , Atezolizumab IgG1 (G3) , 3F2-mIL12-3A (3F2-mIL12-3A IgG1; G4) , or Atezolizumab-mIL12-3A IgG1 (G5) . PBS solution was injected as a control (G1) .

FIG. 39 shows the body weight over time of C57BL/6 wild-type mice that were injected with MC38-hPD-L1, and treated with PDL1-3F2 IgG1 (G2) , Atezolizumab IgG1 (G3) , 3F2-mIL12-3A (3F2-mIL12-3A IgG1; G4) , or Atezolizumab-mIL12-3A IgG1 (G5) . PBS solution was injected as a control (G1) .

FIG. 40 shows the average tumor volume in different groups of C57BL/6 wild-type mice that were injected with MC38-hPD-L1, and treated with PDL1-3F2 IgG1 (G2) , Atezolizumab IgG1 (G3) , 3F2-mIL12-3A (3F2-mIL12-3A IgG1; G4) , or Atezolizumab-mIL12-3A IgG1 (G5) . PBS solution was injected as a control (G1) .

FIG. 41 shows the body weight over time of double humanized hPD-1/hPD-L1 mice that were injected with wild-type MC38 cells, and treated with PDL1-3F2 IgG1 (G2) , Atezolizumab IgG1 (G3) , 3F2-mIL12-3A (3F2-mIL12-3A IgG1; G4) , or Atezolizumab-mIL12-3A IgG1 (G5) . PBS solution was injected as a control (G1) .

FIG. 42 shows the average tumor volume in different groups of double humanized hPD-1/hPD-L1 mice that were injected with wild-type MC38 cells, and treated with PDL1-3F2 IgG1 (G2) , Atezolizumab IgG1 (G3) , 3F2-mIL12-3A (3F2-mIL12-3A IgG1; G4) , or Atezolizumab-mIL12-3A IgG1 (G5) . PBS solution was injected as a control (G1) .

FIG. 43 shows the body weight over time of double humanized hPD-1/hPD-L1 mice that were injected with MC38-hPD-L1, and treated with MOCK-mIL12-3A-IgG1 (G2) , PL1-2H9-mIL12-3A-IgG1 (G3) , PL1-2H9-mIL12-3A-IgG1-N297A (G4) , PD1-1A7-mIL12-3A-IgG4 (G5) , Fc-mIL12-HC (G6) , or BC-2H9-IgG1 (G7) . PBS solution was injected as a control (G1) .

FIGS. 44A-44B shows the blood biochemical test results. Both alanine Aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured in different groups.

FIG. 45 shows the average tumor volume in different groups of double humanized hPD-1/hPD-L1 mice that were injected with MC38-hPD-L1, and treated with MOCK-mIL12-3A-IgG1 (G2) , PL1-2H9-mIL12-3A-IgG1 (G3) , PL1-2H9-mIL12-3A-IgG1-N297A (G4) , PD1-1A7-mIL12-3A-IgG4 (G5) , Fc-mIL12-HC (G6) , or BC-2H9-IgG1 (G7) . PBS solution was injected as a control (G1) .

FIG. 46 shows the body weight over time of double humanized hPD-1/hPD-L1 mice that were injected with B16F10-hPDL1, and treated with MOCK-mIL12-3A-IgG1 (G2) , PL1-2H9-mIL12-3A-IgG1 (G3) , PL1-2H9-mIL12-3A-IgG1-N297A (G4) , PD1-1A7-mIL12-3A-IgG4 (G5) , Fc-mIL12-HC (G6) , or BC-2H9-IgG1 (G7) . PBS solution was injected as a control (G1) .

FIG. 47 shows the average tumor volume in different groups of double humanized hPD-1/hPD-L1 mice that were injected with B16F10-hPDL1, and treated with MOCK-mIL12-3A-IgG1 (G2) , PL1-2H9-mIL12-3A-IgG1 (G3) , PL1-2H9-mIL12-3A-IgG1-N297A (G4) , PD1-1A7-mIL12-3A-IgG4 (G5) , Fc-mIL12-HC (G6) , or BC-2H9-IgG1 (G7) . PBS solution was injected as a control (G1) .

FIGS. 48A-48B shows the blood biochemical test results. Both ALT and AST levels were measured in different groups.

FIGS. 49-53 are tables showing additional affinity data of the antibodies or fusion proteins used in the present disclosure.

FIG. 54 lists CDR sequences of anti-PD-L1 antibody 08-3F2 as defined by Kabat numbering.

FIG. 55 lists CDR sequences of anti-PD-L1 antibody 08-3F2 as defined by Chothia numbering.

FIG. 56 lists amino acid sequences of heavy chain variable regions and light chain variable regions of humanized anti-PD-L1 antibody 08-3F2.

FIG. 57 lists amino acid sequences of human, mouse, and chimeric PD-1.

FIG. 58 lists amino acid sequences of human, mouse, and chimeric PD-L1.

FIGS. 59A-59B list amino acid sequences discussed in the disclosure.

DETAILED DESCRIPTION

Tumor-induced immune suppression is recognized as an important mechanism by which tumors evade immune-mediated detection and destruction. A number of strategies to overcome this suppression have been evaluated. Among them, IL-12 appears to be a potent agent for anti-tumor immunotherapy due to its central role in T-and NK-cell-mediated inflammatory responses. Unfortunately, it has been repeatedly established that clinical application of IL-12-based therapies remains problematic due to rapid development of lethal inflammatory syndrome. This potentially lethal toxicity associated with systemic administration of IL-12 precludes almost all of its clinical applications.

This disclosure is based on, in part, the unexpected discovery that IL12 can be fused to an antibody that targets a PD-1 ligand. The protein complex can greatly reduce the toxicity of IL12 so that IL12 can be delivered to a subject with a dosage that is much higher than the typical tolerated amount. As such, the protein complex can greatly increase the immune response in the tumor microenvironment, and further increase the therapeutic efficacy of the antibody that targets the PD-1 ligand.

Protein complex targeting IL12 pathway

Interleukin 12 (IL-12) is a pleiotropic cytokine. It is naturally produced by dendritic cells, macrophages, neutrophils, and human B-lymphoblastoid cells (NC-37) in response to antigenic stimulation, and creates an interconnection between the innate and adaptive immunity. IL-12 is a heterodimeric cytokine encoded by two separate genes, IL-12A (p35) and IL-12B (p40) . It specifically binds to the IL-12 receptor (IL-12R) , which is a heterodimeric receptor formed by IL-12Rβ1 and IL-12Rβ2. Upon binding, IL-12R-β2 becomes tyrosine phosphorylated and provides binding sites for kinases, Tyk2 and Jak2. IL12 is important in activating critical transcription factor proteins such as STAT4 that are implicated in IL-12 signaling in T cells and NK cells, and activating the JAK-STAT pathway.

IL-12 can stimulate the growth and function of T cells. It stimulates the production of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) from T cells and natural killer (NK) cells, and reduces IL-4 mediated suppression of IFN-γ. IL-12 also mediates enhancement of the cytotoxic activity of NK cells and CD8+ cytotoxic T lymphocytes. Due to its ability of bridging the innate and adaptive immunity and potently stimulating the production of IFN-γ, IL-12 appears to be an ideal candidate for tumor immunotherapy in humans. However, severe side effects were associated with systemic administration of IL-12 in clinical investigations. The very narrow therapeutic index of this cytokine markedly tempered enthusiasm for the use of this cytokine in cancer patients. Some early phase I clinical trials have determined that the maximal tolerated dose of 500 ng/kg/day. This dose was found toxic in phase II trial and severe side effects of the treatment developed in 12 of 17 enrolled patients leading to death of two patients (Jenks, Susan. "After initial setback, IL-12 regaining popularity. " (1996) : 576-577) . As such, the maximal tolerated doses in escalating dose protocols ranged, in relation to the treatment schedule, usually between 250 and 500 ng/kg. Despite of high expectations, these early clinical studies with IL-12 did not yield satisfactory therapeutic results. The function of IL-12, and the details of some of these clinical trials are described, e.g., in Lasek et al., "Interleukin 12: still a promising candidate for tumor immunotherapy? " Cancer Immunology, Immunotherapy 63.5 (2014) : 419-435, which is incorporated herein by reference in its entirety.

This disclosure relates to protein complexes targeting IL12R. The disclosure demonstrates that the protein complex as described herein can activate T cells in a tumor microenvironment, spleen, lymph nodes, and/or lymphatic system. Particularly, the present disclosure demonstrates that by targeting the tumor microenvironment, the protein complex as described herein can unexpectedly reduce IL12 toxicity when it is administered to a subject, thereby greatly increasing maximal tolerated doses for IL12. It has also been shown that the protein complex can effectively activate IL12 signaling pathway in T cells that are in close proximity of tumor cells. In some embodiments, the protein complex binds to PD-L1 on immune cells and activates T cells in that microenvironment. In one aspect, the protein complex can further block the interaction between PD-1 and PD-L1, thereby further increasing the immune response in the tumor microenvironment.

In one aspect, the disclosure provides a protein complex comprising (a) a targeting moiety; and (b) an immunomodulatory moiety specifically binds to interleukin-12 receptor (IL12R) . In some embodiments, the targeting moiety specifically binds to a PD-1 ligand.

In one aspect, the disclosure also provides a protein complex that comprises (a) a first portion (e.g., a functional domain, a functional unit, or an immunomodulatory moiety) targeting Interleukin-12 receptor (IL12R) ; and (b) a second portion (e.g., a functional domain, a functional unit, or a targeting moiety) targeting PD-1 pathway. As used herein, a “functional domain” refers to a distinct functional and structural unit in a protein. A functional domain can vary in length, but it generally has between about 50 amino acids to about 250 amino acids in length. In some embodiments, a functional domain is a segment of a polypeptide. In some embodiments, a functional domain can be formed by multiple segments of one polypeptide (e.g., a VH-VL pair in scFv) or multiple segments from different polypeptides (e.g., a VH-VL pair in Fab) . In some embodiments, a targeting moiety comprises a functional domain. In some embodiments, an immunomodulatory moiety comprises a functional domain.

The protein complex can have 1, 2, 3, 4, 5, 6 or more than 6 functional domains or targeting moieties that target PD-1 pathway. In some embodiments, the protein complex can have 1, 2, 3, 4, 5, 6 or more than 6 functional domains or immunomodulatory moieties that target IL12R. In some embodiments, the ratio of the portions that target PD-1 pathway to the portions that target PD-1 pathway is 4: 1, 2: 1, 1: 1, 1: 2, or 1: 4.

In some embodiments, the functional domains or moieties targeting PD-1 pathway can block the interaction between PD-1 and PD-L1. In some embodiments, the functional domains or moieties targeting PD-1 pathway cannot block the interaction between PD-1 and PD-L1 (e.g., a non-blocking anti-PD1 antibody or a non-blocking anti-PD-L1 antibody) . In some embodiments, the functional domain or moiety targeting PD-1 pathway is a full length antibody or antigen binding fragment. In some embodiments, the functional domain or moiety targeting PD-1 pathway is an anti-PD-L1 antibody or an anti-PD-L2 antibody.

In some embodiments, the functional domain or the immunomodulatory moiety targeting IL-12R is IL-12 (e.g., human IL-12) . In some embodiments, the IL12 comprises IL12a (e.g., human IL12a) . In some embodiments, the IL12 comprises IL12b (e.g., human IL12b) . In some embodiments, the IL12 comprises a variant (e.g., a fusion protein or a truncated protein thereof) of IL12a and/or IL12b. In some embodiments, the IL12 does not comprise a signal peptide (e.g., a signal peptide of IL12a or IL12b) . In some embodiments, the functional domain or the immunomodulatory moiety targeting IL-12R is an anti-IL12R antibody or antigen binding fragment thereof (e.g., scFV or VHH) . In some embodiments, the anti-IL12R antibody is an agonist anti-IL12R antibody.

In some embodiments, the immunomodulatory moiety binds to IL12 receptor (e.g., human IL12 receptor) with a KD value less than 7 × 10 ^-8 M, less than 6.5 × 10 ^-8 M, less than 6 ×10 ^-8 M, less than 5 × 10 ^-8 M, less than 4× 10 ^-8 M, less than 3 × 10 ^-8 M, less than 2 × 10 ^-8 M, or less than 1 × 10 ^-8 M.

In some embodiments, the immunomodulatory moiety and the targeting moiety are linked (e.g., fused) together, optionally through a linker sequence. In some embodiments, the immunomodulatory moiety and the targeting moiety are linked to a third functional domain or moiety, such as a protein scaffold (e.g., Fc, albumin) . In some embodiments, they can be linked to the third functional domain or moiety through a linker sequence. In some embodiments, the immunomodulatory moiety and the targeting moiety are linked to a nanoparticle.

In some embodiments, the functional domain or moiety targeting IL-12R is fused to the heavy chain at a region from position 344 to position 382 of the heavy chain CH3 domain according to EU numbering, or is linked (e.g., fused) to the N-terminus or C-terminus of a heavy chain or a light chain.

In some embodiments, the protein complex comprises (a) a first portion (e.g., an immunomodulatory moiety) targeting Interleukin-12 receptor (IL12R) ; and (b) a second portion (e.g., a targeting moiety) targeting PD-1 pathway. The first portion (e.g., an immunomodulatory moiety) can be linked or fused to a heavy chain or a portion thereof (e.g., CH3, or Fc) by various ways as described herein. In some embodiments, the first portion (e.g., an immunomodulatory moiety) targeting IL12R comprises or consists of IL12a and IL12b subunits. In some embodiments, IL12a and IL12b are linked together. In some embodiments, IL12a is fused to the heavy chain at a region from position 344 to position 382 of the heavy chain CH3 domain according to EU numbering, or is linked to the N-terminus or C-terminus of a heavy chain or a light chain, and IL12a and IL12b associate with each other, e.g., by non-covalent interaction, forming a functional domain that target IL12R. In some embodiments, IL12b is fused to the heavy chain at a region from position 344 to position 382 of the heavy chain CH3 domain according to EU numbering, or is linked to the N-terminus or C-terminus of a heavy chain or a light chain, and IL12a and IL12b associate with each other, e.g., by non-covalent interaction, forming a functional domain that target IL12R.

In some embodiments, the protein complex can further include a cytokine (e.g., IL2, IL7, IL15, IL18, and/or IL21) and/or an interferon (e.g., IFNa1, IFNa2, IFNa3, IFNa4, and/or IFNγ) . The cytokine or interferon can be linked or fused to the protein complex, e.g., linked or fused to the targeting moiety, the immunomodulatory moiety, or the protein scaffold, directly or indirectly through a linker sequence.

As systematic administration of IL12 is toxic, the present disclosure also provides a protein complex comprising IL12 that can be enriched in a tumor microenvironment. The effects of IL12 is largely limited to the tumor microenvironment, thereby reducing the toxicity of IL12 on the entire immune system. In one aspect, the present disclosure provides a fusion protein comprising IL12, wherein the fusion protein is engineered to target a tumor associated antigen.

In some embodiments, the binding affinity for the tumor antigen needs to be higher than the binding affinity of IL12 to IL12R. In some embodiments, the binding affinity to the tumor antigen (e.g., PD-L1) has a KD value of less than 1 × 10 ^-7 M, less than 1 × 10 ^-8 M, less than 1 ×10 ^-9 M, less than 5 × 10 ^-10 M, less than 4.5 × 10 ^-10 M, or less than 4 × 10 ^-10 M.

In some embodiments, the fusion protein comprises an Fc region or a full length antibody, e.g., anti-PD-L1 antibody. In some embodiments, either IL12a or IL12b is linked to a region from position 344 to position 382 of a CH3 domain of the Fc region according to EU numbering, wherein IL12a associates with IL12b. In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a VHH.

Without wishing to be bound by theory, it is also hypothesized that the full-fledged activation of antitumor T cells by PD-1 pathway blockers (e.g., anti-PD-L1 antibody) further requires T cell: dendritic cell (DC) crosstalk, which involves IFN-γ and IL-12. The IL-12 is required for stimulating antitumor T cell immunity. In the absence of IL-12 released from dendritic cells in the tumor microenvironment, it can be difficult to activate antitumor T cells. Thus, in one aspect, the present disclosure provides a method of improving the efficacy of PD-1 pathway blockers (e.g., anti-PD-1 antibody or anti-PD-L1 antibody) , and the modified antibody that can effectively increase the immune response.

In one aspect, the disclosure provides a protein complex comprising or consisting of an anti-PD-L1 antibody or antigen-binding fragment thereof, and an IL12. In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof is an immunoglobulin (e.g., IgM, IgA, IgE, IgD, or IgG) . In some embodiments, the protein complex comprises 1, 2, 3, 4, 5, 6 or more than 6 IL12 functional domains or immunomodulatory moieties. These IL12 functional domains or immunomodulatory moieties can be linked to N terminal or C terminal of a heavy chain and/or a light chain. In some embodiments, the IL12 functional domains or immunomodulatory moieties can be linked to the CH3 domain in the anti-PD-L1 antibody or antigen-binding fragment thereof.

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises or consists of: a first heavy chain polypeptide comprising a first heavy chain CH3 domain, a second heavy chain polypeptide comprising a second heavy chain CH3 domain, a first light chain polypeptide, and a second light chain polypeptide; wherein the first heavy chain polypeptide and the first light chain polypeptide associate with each other, forming a first antigen-binding region targeting PD-L1; wherein the second heavy chain polypeptide and the second light chain polypeptide associate with each other, forming a second antigen-binding region targeting PD-L1.

In some embodiments, IL12a is fused to the CH3 domain of the first heavy chain polypeptide and/or the second heavy chain polypeptide. In some embodiments, IL12a is linked to the C terminal of the first heavy chain polypeptide and/or the second heavy chain polypeptide. In some embodiments, IL12a is linked to the C terminal of the first light chain polypeptide and/or the second light chain polypeptide.

In some embodiments, IL12b is fused to the CH3 domain of the first heavy chain polypeptide and/or the second heavy chain polypeptide. In some embodiments, IL12b is linked to the C terminal of the first heavy chain polypeptide and/or the second heavy chain polypeptide. In some embodiments, IL12b is linked to the C terminal of the first light chain polypeptide and/or the second light chain polypeptide.

In some embodiments, IL12 (e.g., IL12a or IL12b) is linked to the anti-PD-L1 antibody or antigen-binding fragment thereof with a linker sequence as described herein.

In some embodiments, IL12a has a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to SEQ ID NO: 28. In some embodiments, IL12b has a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to SEQ ID NO: 29.

In some embodiments, at least two functional domains or immunomodulatory moieties (e.g., 2, 3, 4, 5, or 6 IL12 functional subunits) are linked to the anti-PD-L1 antibody or antigen-binding fragment thereof.

In some embodiments, one or more additional cytokine functional domains (e.g., IL2, IL7, IL15, IL18, and/or IL21) and/or one or more interferon functional domains (e.g., IFNa1, IFNa2, IFNa3, IFNa4, and/or IFNγ) are linked to the anti-PD-L1 antibody or antigen-binding fragment thereof.

In one aspect, the disclosure provides a modified polypeptide comprising a CH3 domain. In some embodiments, the polypeptide comprises a first portion of CH3; a region comprising an interleukin 12 polypeptide (e.g., IL12a, IL12b, or both) ; and a second portion of CH3. In some embodiments, the first portion of CH3 comprises amino acid residues 341-343 of a CH3 domain according to EU numbering, and the second portion of CH3 comprises amino acid residues 383-447 of a CH3 domain according to EU numbering. In some embodiments, the modified polypeptide further comprises a CH2 domain. In some embodiments, two of these modified polypeptides associate with each other, forming an Fc-like structure. In some embodiments, the Fc-like structure only has one IL12 functional domain.

In one aspect, the disclosure further provides a fusion heavy chain polypeptide, wherein the fusion heavy chain polypeptide comprises, e.g., from N-terminus to C-terminus: a first region comprising VH, CH1, CH2, and CH3; and a second region comprising an interleukin 12 polypeptide; wherein the first region is linked to the second region through a linker sequence. In one aspect, the disclosure further provides a fusion light chain polypeptide, wherein the fusion light chain polypeptide comprises, e.g., from N-terminus to C-terminus: a first region comprising VL and CL; and a second region comprising an interleukin 12 polypeptide; wherein the first region is linked to the second region through a linker sequence. In some embodiments, the interleukin 12 polypeptide is IL12a or IL12b. In some embodiments, the interleukin 12 polypeptide is an IL12a-IL12b fusion protein.

Modified immunoglobulins

In one aspect, the disclosure provides a protein complex that comprises (a) a first portion (e.g., a functional domain, a functional unit, or an immunomodulatory moiety) targeting IL12 pathway; and (b) a second portion (e.g., a functional domain, a functional unit, or a targeting moiety) targeting PD-1 pathway. In some embodiments, the protein complex can comprise an antibody or a portion thereof (e.g., Fc) . The first portion (e.g., an immunomodulatory moiety) and the second portion (e.g., a targeting moiety) can be linked to the antibody or a portion thereof (e.g., Fc) by various ways.

In general, antibodies (also called immunoglobulins) are made up of two classes of polypeptide chains, light chains and heavy chains. A non-limiting examples of antibody of the present disclosure can be an intact, four immunoglobulin chain antibody comprising two heavy chains and two light chains. The heavy chains, which each contain one variable domain (or variable region, V _H) 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, V _L) 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 antigen binding surface of the antibody. The four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting the beta-sheet structure, 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.

The heavy chain has four to five domains, depending on the isotype, including a variable (VH) domain and several constant (CH) domains: three CH domains (CH1, CH2, CH3) in IgG, IgA and IgD and four CH domains (CH1, CH2, CH3, CH4) in IgM and IgE. The antigen-binding fragment (Fab) is formed by the light chain (VL and CL) and the first two domains of the heavy chain (VH and CH1) and is specifically involved in antigen binding. The Ig Fc (fragment crystallizable) portion is formed by the CH2 and CH3 constant domains, and optionally with CH4 constant domain, from each heavy chain. The Fc region ensures that each antibody generates an appropriate immune response for a given antigen, by binding to a specific class of Fc receptors, and other immune molecules, such as complement proteins. By doing this, it mediates different physiological effects, including recognition of opsonized particles (binding to FcγR) , lysis of cells (binding to complement) , and degranulation of mast cells, basophils, and eosinophils (binding to FcεR) .

In some embodiments, the heavy chain described herein can be a heavy chain variant, e.g., a heavy chain variant without the CH1 domain; or a heavy chain variant with at least the CH3 domain (e.g., CH2 and CH3 domains) .

All domains in immunoglobulins have a similar structure and are constructed from two βsheets. The sheets are linked by a disulfide bridge and together form a roughly barrel-shaped structure, known as a β barrel. The distinctive folded structure of the immunoglobulin protein domain is known as the immunoglobulin fold. The constant domains are built up from seven βstrands arranged such that four strands form one β sheet and three strands form a second sheet. The loops connecting the β strands are relatively short and, as a result, a majority of the residues of the domain are contained in the two β sheets. These strands include A-strand, B-strand, C-strand, D-strand, E-strand, F-Strand, and G-strand. The sequence connecting the β strands include AB-turn, BC-loop, CD-strand, DE-turn, and EF-turn. A detailed description of the structure of the constant domain can be found e.g., in Lefranc et al., "

and 30 years of Immunoinformatics Insight in antibody V and C domain structure and function. " Antibodies 8.2 (2019) : 29, which is incorporated herein by reference in its entirety.

In some embodiments, the protein complex can comprise an antibody (IgM, IgA, IgE, IgD, IgG, IgG1, IgG2a, IgG2b, IgG3) or a portion thereof (e.g., Fc) . The functional domains, immunomodulatory moieties, and/or targeting moieties can be linked to the antibody or a portion thereof (e.g., Fc) by various ways. In some embodiments, the functional domains, immunomodulatory moieties, and/or targeting moieties can comprise an antibody or a portion thereof (e.g., Fc, Fab, scFv, VHH) .

In some embodiments, the functional domains, immunomodulatory moieties, and/or targeting moieties can be linked to the C terminal of an Fc. In some embodiments, the functional domains, immunomodulatory moieties, and/or targeting moieties can be linked to the N terminal of an Fc, e.g., they can be linked to CH2 domain through an optional hinge region or a linker sequence. In some embodiments, the functional domains, immunomodulatory moieties, and/or targeting moieties can be linked to the C terminal of an Fc, e.g., through a linker sequence. In some embodiments, the functional domains, immunomodulatory moieties, and/or targeting moieties can be linked to the N terminal a light chain in the protein complex or the C terminal a light chain in the protein complex, e.g., through a linker sequence.

In some embodiments, the functional domains, immunomodulatory moieties, and/or targeting moieties can be fused to a particular region in the Fc region. In some embodiments, a non-native polypeptide is fused to a particular region in the Fc region in the protein complex. As used herein, a “non-native” polypeptide refers to a polypeptide which cannot be found in the Fc region of a wildtype immunoglobulin. This particular region in the present disclosure is referred as the “3A site. ” The 3A site is located in the CH3 domain, and starts from position 344 to position 382 (EU numbering) . The fusion of a non-native polypeptide can provide superior results. As compared to some other modified immunoglobulins, the immunoglobulins with this modification is very stable, and the immunoglobulins with a non-native polypeptide fused at this site can be expressed at a high level and they do not form aggregates. The property of this fusion site is also unexpected, as the 3A site is located in the A-strand and B-strand, which seems to be important for the function and stability of the CH3 domain.

In some embodiments, the modification is made to IgG, IgM, IgD, IgE, or IgA. Thus, in one aspect, the disclosure provides a modified Fc region or a polypeptide complex comprising a first polypeptide comprising a first heavy chain CH3 domain, a second polypeptide comprising a second heavy chain CH3 domain. The two polypeptides interact with each other and can form a homodimer or a heterodimer. One or two non-native polypeptides can be fused to the heavy chain CH3 domains of the one or two polypeptides at the 3A site. The 3A site starts from position 344 to position 382 (EU numbering) . In some embodiments, the functional domains, immunomodulatory moieties, or targeting moieties replaces 1, 2, 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 amino acids at the fusion site or is inserted between any of the two amino acids at this fusion site. In some embodiments, when a non-native polypeptide is inserted between two non-consecutive amino acids at the fusion site, it also replaces all amino acids between the two non-consecutive amino acids. In some embodiments, the non-native polypeptide is linked to two amino acid residues of the heavy chain CH3 domain of the modified immunoglobulin. The two amino acid residues can be consecutive or non-consecutive.

In some embodiments, the functional domains, immunomodulatory moieties, and/or targeting moieties are linked to two amino acid residues of the heavy chain CH3 domain of a modified immunoglobulin. In some embodiments, the two residues are selected from any two of positions 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, and 383 of the heavy chain CH3 domain according to EU numbering. The functional domains, immunomodulatory moieties, and/or targeting moieties are linked to a starting amino acid and an ending amino acid in the heavy chain CH3 domain. The present disclosure also provides all different combinations of the starting amino acid and the ending amino acid at the 3A site. For example, if the functional domains, immunomodulatory moieties, and/or targeting moieties are linked to the amino acid residues located at position 343 and 383, the entire 3A site (positions 344-382) is replaced by the functional domains, immunomodulatory moieties, and/or targeting moieties. If the functional domains, immunomodulatory moieties, and/or targeting moieties are linked to two consecutive amino acid residues, e.g., located at position 357 and 358, the functional domains, immunomodulatory moieties, and/or targeting moieties are inserted between the two consecutive amino acid residues. The specific combinations of any two amino residues at positions 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, and 383 of the heavy chain CH3 domain (EU numbering) are provided.

In some embodiments, the starting amino acid is selected from 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, or 356. In some embodiments, the starting amino acid is selected from 357, 358, 359, 360, 361, or 362. In some embodiments, the ending amino acid is selected from 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, or 383. In some embodiments, the ending amino acid is selected from 358, 359, 360, 361, 362, or 363.

In some embodiments, the fusion site is located at a region from position 351 to 362 (EU numbering) . In some embodiments, the functional domains, immunomodulatory moieties, and/or targeting moieties replace 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all amino acids (e.g., 351-362) at the fusion site, or are inserted between any of the two amino acids at this fusion site, e.g., inserted at the position 351-352, 352-353, 353-354, 354-355, 355-356, 356-357, 357-358, 358-359, 359-360, 360-361, or 361-362.

In some embodiments, the two residues are selected from any two of positions 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, and 363 of the heavy chain CH3 domain according to EU numbering. The combinations of these starting amino acids and the ending amino acids are provided in the table below. For example, if the functional domains, immunomodulatory moieties, and/or targeting moieties are linked to the amino acid residues located at position 350 and 363, the amino acids in this fusion site (positions 351-362) are replaced by the functional domains, immunomodulatory moieties, and/or targeting moieties. If the functional domains, immunomodulatory moieties, and/or targeting moieties are linked to two consecutive amino acid residues, e.g., located at position 350 and 351, the functional domains, immunomodulatory moieties, and/or targeting moieties are inserted between the two consecutive amino acid residues.

Table 1

Start	End	Comment	Start	End	Comment	Start	End	Comment
350	351	Insert	352	359	Insert/delete	355	363	Insert/delete
350	352	Insert/delete	352	360	Insert/delete	356	357	Insertion
350	353	Insert/delete	352	361	Insert/delete	356	358	Insert/delete
350	354	Insert/delete	352	362	Insert/delete	356	359	Insert/delete
350	355	Insert/delete	352	363	Insert/delete	356	360	Insert/delete
350	356	Insert/delete	353	354	Insertion	356	361	Insert/delete
350	357	Insert/delete	353	355	Insert/delete	356	362	Insert/delete
350	358	Insert/delete	353	356	Insert/delete	356	363	Insert/delete
350	359	Insert/delete	353	357	Insert/delete	357	358	Insertion
350	360	Insert/delete	353	358	Insert/delete	357	359	Insert/delete
350	361	Insert/delete	353	359	Insert/delete	357	360	Insert/delete
350	362	Insert/delete	353	360	Insert/delete	357	361	Insert/delete
350	363	Insert/delete	353	361	Insert/delete	357	362	Insert/delete
351	352	Insertion	353	362	Insert/delete	357	363	Insert/delete
351	353	Insert/delete	353	363	Insert/delete	358	359	Insertion

351	354	Insert/delete	354	355	Insertion	358	360	Insert/delete
351	355	Insert/delete	354	356	Insert/delete	358	361	Insert/delete
351	356	Insert/delete	354	357	Insert/delete	358	362	Insert/delete
351	357	Insert/delete	354	358	Insert/delete	358	363	Insert/delete
351	358	Insert/delete	354	359	Insert/delete	359	360	Insertion
351	359	Insert/delete	354	360	Insert/delete	359	361	Insert/delete
351	360	Insert/delete	354	361	Insert/delete	359	362	Insert/delete
351	361	Insert/delete	354	362	Insert/delete	359	363	Insert/delete
351	362	Insert/delete	354	363	Insert/delete	360	361	Insertion
351	363	Insert/delete	355	356	Insertion	360	362	Insert/delete
352	353	Insertion	355	357	Insert/delete	360	363	Insert/delete
352	354	Insert/delete	355	358	Insert/delete	361	362	Insertion
352	355	Insert/delete	355	359	Insert/delete	361	363	Insert/delete
352	356	Insert/delete	355	360	Insert/delete	362	363	Insert
352	357	Insert/delete	355	361	Insert/delete
352	358	Insert/delete	355	362	Insert/delete

In some embodiments, the fusion site is located at a region from 358 to 362 (EU numbering) . In some embodiments, the functional domains or the moieties can replace 1, 2, 3, 4, 5 or all amino acids at the fusion site or is inserted between any of the two amino acids at this fusion site, e.g., inserted at the position 358-359, 359-360, 360-361, or 361-362.

In some embodiments, the two residues are selected from any two of positions 357, 358, 359, 360, 361, 362, and 363 of the heavy chain CH3 domain according to EU numbering. The combinations of these starting amino acids and the ending amino acids are provided in the table below.

Table 2

Starting	Ending	Comment	Starting	Ending	Comment
357	358	Insert	359	360	Insert
357	359	Insert/delete	359	361	Insert/delete
357	360	Insert/delete	359	362	Insert/delete
357	361	Insert/delete	359	363	Insert/delete
357	362	Insert/delete	360	361	Insert
357	363	Insert/delete	360	362	Insert/delete
358	359	Insert	360	363	Insert/delete
358	360	Insert/delete	361	362	Insert
358	361	Insert/delete	361	363	Insert/delete
358	362	Insert/delete	362	363	Insert

358

363

Insert/delete

Thus, in some embodiments, the two residues are positions 357 and 358 of the heavy chain CH3 domain according to EU numbering. In some embodiments, the two residues are positions 357 and 359 of the heavy chain CH3 domain according to EU numbering. In some embodiments, the two residues are positions 357 and 360 of the heavy chain CH3 domain according to EU numbering. In some embodiments, the two residues are positions 357 and 361 of the heavy chain CH3 domain according to EU numbering. In some embodiments, the two residues are positions 357 and 362 of the heavy chain CH3 domain according to EU numbering. In some embodiments, the two residues are positions 357 and 363 of the heavy chain CH3 domain according to EU numbering. In some embodiments, the two residues are positions 358 and 359 of the heavy chain CH3 domain according to EU numbering. In some embodiments, the two residues are positions 358 and 360 of the heavy chain CH3 domain according to EU numbering. In some embodiments, the two residues are positions 358 and 361 of the heavy chain CH3 domain according to EU numbering. In some embodiments, the two residues are positions 358 and 362 of the heavy chain CH3 domain according to EU numbering. In some embodiments, the two residues are positions 358 and 363 of the heavy chain CH3 domain according to EU numbering. In some embodiments, the two residues are positions 359 and 360 of the heavy chain CH3 domain according to EU numbering. In some embodiments, the two residues are positions 359 and 361 of the heavy chain CH3 domain according to EU numbering. In some embodiments, the two residues are positions 359 and 362 of the heavy chain CH3 domain according to EU numbering. In some embodiments, the two residues are positions 359 and 363 of the heavy chain CH3 domain according to EU numbering. In some embodiments, the two residues are positions 360 and 361 of the heavy chain CH3 domain according to EU numbering. In some embodiments, the two residues are positions 360 and 362 of the heavy chain CH3 domain according to EU numbering. In some embodiments, the two residues are positions 360 and 363 of the heavy chain CH3 domain according to EU numbering. In some embodiments, the two residues are positions 361 and 362 of the heavy chain CH3 domain according to EU numbering. In some embodiments, the two residues are positions 361 and 363 of the heavy chain CH3 domain according to EU numbering. In some embodiments, the two residues are positions 362 and 363 of the heavy chain CH3 domain according to EU numbering.

The fusion of the functional domains, immunomodulatory moieties, and/or targeting moieties at the 3A site does not interfere with the antigen-binding site of the immunoglobulins. In addition, the fused functional domains, immunomodulatory moieties, or targeting moieties can maintain its bioactivity. Moreover, the modification at the 3A site does not significantly affect the binding affinity of Fc to FcγRIIA, FcγRIIIA, FcγRIIIB, or FcRn receptors. In some embodiments, the binding affinities of the modified Fc to FcγRIIA, FcγRIIIA, FcγRIIIB, or FcRn receptors are about the same as compared to the same immunoglobulins before any modifications. In some embodiments, the binding affinities of the modified Fc to FcγRIIA, FcγRIIIA, FcγRIIIB, or FcRn receptors are higher (e.g., at least 10%, 20%, 30%, 40%, or 50%) as compared to the same immunoglobulins before any modifications. In some embodiments, the binding affinities of the modified Fc to FcγRIIA, FcγRIIIA, FcγRIIIB, or FcRn receptors are lower (e.g., no more than 10%, 20%, 30%, 40%, or 50%lower) as compared to the same immunoglobulins before any modifications.

In some embodiments, the protein complex has at least one of antibody-dependent cell cytotoxicity (ADCC) , antibody-dependent cellular phagocytosis (ADCP) , complement dependent cytotoxicity (CDC) or apoptotic activity. In some embodiments, when the fused polypeptide at the 3A site interacts with a target protein, the Fc will not bind FcγRIIA, FcγRIIIA, or FcγRIIIB receptors because of steric effects. Thus, when the fused polypeptide at the 3A site interacts with a target protein, the ADCC, ADCP, and CDC effects are reduced.

Moreover, the fusion of the functional domains, immunomodulatory moieties, or targeting moieties at the 3A site does not interfere with the function of the functional domains, immunomodulatory moieties, or targeting moieties. Particularly, the functional domains, immunomodulatory moieties, or targeting moieties in the modified immunoglobulins can have about the same or even a higher level of biological activity as compared to an isolated functional domains, immunomodulatory moieties, or targeting moieties. In some embodiments, the biological activity of the functional domains, immunomodulatory moieties, or targeting moieties in the modified immunoglobulins can be at least or about 85%, 90%, 95%, or 100%of the isolated functional domains, immunomodulatory moieties, or targeting moieties.

The fusion of the functional domains, immunomodulatory moieties, or targeting moieties at the 3A site does not interfere with the binding affinity of the modified immunoglobulins. Particularly, the modified immunoglobulins can have about the same or even a higher level of binding affinity as compared to the parent immunoglobulins. As used herein, the term “parent” molecule refers to a molecule before any non-native peptides as described herein is fused to the molecule or any other modifications are made to the molecule. In some embodiments, the binding affinity can be at least or about 85%, 90%, 95%, or 100%of the parent immunoglobulins.

The fusion of the functional domains, immunomodulatory moieties, or targeting moieties at the 3A site does not interfere with the expression level and does not reduce expression yield. Particularly, the protein complex has a very high expression level. In some embodiments, the expression level can be at least or about 50%, 60%, 70%, 80%, 90%, or 100%higher than the expression level of a similar bispecific antibody (e.g., an antibody that binds to the same targets) or an immunocytokine (e.g., an antibody with a cytokine that is linked at the C-terminal of the Fc) . In some embodiments, the modified immunoglobulins do not form aggregates easily. In some embodiments, the percentage of aggregates in the purified form (e.g., after purified by Protein A chromatography) is less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%or 1%.

The protein complex can have various forms or formats. In some embodiments, the protein complex or the targeting moiety can comprise 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 EU numbering of these immunoglobulins, 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; Lefranc et al., "

and 30 years of Immunoinformatics Insight in antibody V and C domain structure and function. " Antibodies 8.2 (2019) : 29; each of which is incorporated herein by reference in its entirety.

The protein complex can comprise an immunoglobulin molecule that is derived from any species (e.g., human, rodent, rat, mouse, camelid, dog, horn shark, Xenopus laevis, rhesus monkey, cat, rabbit) . 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, VHH, 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 VHH, a bispecific antibody, a bispecific scFv, 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.

Fragments of antibodies are suitable for use in the protein complex described herein are also provided. 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.

In some embodiments, 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 two identical copies of a heavy chain.

In some embodiments, the protein complex can have a format as shown in the table below. In some embodiments, a functional domain, an immunomodulatory moiety, or a targeting moiety is linked to a format as shown in the table below. Detailed descriptions of these antibody formats can be found, e.g., in Brinkmann, et al., "The making of bispecific antibodies. " MAbs. Vol. 9. No. 2. Taylor &Francis, 2017, which is incorporated herein by reference in the entirety.

Table 3

In some embodiments, the protein complex comprises 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. In some embodiments, the protein complex targets both PD-L1 and IL12R. 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. While the modification is made to Fc region, the present disclosure also shows that the modification is compatible with knobs-in-holes. The "knobs into holes" approach introduces a mutation for an amino acid with a large sidechain in one heavy chain, and a mutation for an amino acid with a small sidechain in the other heavy chain. Thus, the same heavy chains are less likely to associate with each other and the two different heavy chains have a higher chance to associate with each other. The “knobs into holes” approaches are described, e.g., in Ridgway, John BB, Leonard G. Presta, and Paul Carter. " ‘Knobs-into-holes’ engineering of antibody CH3 domains for heavy chain heterodimerization. " Protein Engineering, Design and Selection 9.7 (1996) , which is incorporated herein by reference in its entirety. In some embodiments, one or more amino acid residues in the CH3 domain of the IgG are substituted. In some embodiments, one heavy chain has one or more of the following substitutions Y349C and T366W. The other heavy chain can have one or more the following substitutions E356C, T366S, L368A, and Y407V. In some embodiments, one heavy chain has a T366Y (knob) substitution, and the other heavy chain has a Y407T (hole) substitution. In some embodiments, one heavy chain has a T366Y (knob) substitution, and the other heavy chain has one, two, or three of these substitutions T366S, L368A, Y407V (hole) .

In some embodiments, the protein complex comprises an antibody with various formats, such as DVD-Ig, CrossMab, BiTE etc. In some embodiments, the protein complex comprises a targeting moiety and an immunomodulatory moiety. The targeting moiety and the immunomodulatory moiety, together with some optional moieties, can form an antibody with various formats. In some embodiments, the targeting moiety and the immunomodulatory moiety are linked to an antibody with various formats.

These formats are described in e.g., Spiess et al., "Alternative molecular formats and therapeutic applications for bispecific antibodies. " Molecular immunology 67.2 (2015) : 95-106, which is incorporated herein by reference in its entirety. In certain embodiments, the bispecific polypeptide complex as provided herein is based on a bispecific format selected from Triomabs; hybrid hybridoma (quadroma) ; Multispecific anticalin platform (Pieris) ; Diabodies; Single chain diabodies; Tandem single chain Fv fragments; TandAbs, Trispecific Abs; Darts (dual affinity retargeting; Macrogenics) ; Bispecific Xmabs (Xencor) ; Bispecific T cell engagers (Bites; Amgen; 55 kDa) ; Triplebodies; Tribody (Fab-scFv) Fusion Protein (CreativeBiolabs) multifunctional recombinant antibody derivates; Duobody platform (Genmab) ; Dock and lock platform; Humanized bispecific IgG antibody (REGN1979) (Regeneron) ; Mab2 bispecific antibodies (F-Star) ; DVD-Ig (dual variable domain immunoglobulin) (Abbvie) ; kappa-lambda bodies; TBTI (tetravalent bispecific tandem Ig) ; and CrossMab. In certain embodiments, the bispecific polypeptide complex as provided herein is based on the format of a “whole” antibody, such as whole IgG or IgG-like molecules, and small recombinant formats, such as tandem single chain variable fragment molecules (taFvs) , diabodies (Dbs) , single chain diabodies (scDbs) and various other derivatives of these, and BiTE (bispecific T cell engager) .

“MAb-Fv” or “IgG-Fv” refers to a fusion protein formed by fusion of VH to the C-terminus of one Fc chain and the VL domain either expressed separately or fused to the C-terminus of the other resulted in a bispecific, trivalent IgG-Fv (mAb-Fv) fusion protein, with the Fv stabilized by an interdomain disulphide bond.

“ScFab-Fc-scFv2” and “ScFab-Fc-scFv” refer to a fusion protein formed by fusion of a single-chain Fab with Fc and disulphide-stabilized Fv domains.

“Appended IgG” refers to a fusion protein with a Fab arm fused to an IgG to form the format of bispecific (Fab) 2-Fc. It can form a “IgG-Fab” or a “Fab-IgG” , with a Fab fused to the C-terminus or N-terminus of an IgG molecule with or without a connector.

“DVD-Ig” refers to a dual-variable-domain antibody that is formed by fusion of an additional VH domain and VL domain of a second specificity to an IgG heavy chain and light chain. “CODV-Ig” refers to a related format where the two VH and two VL domains are linked in a way that allows crossover pairing of the variable VH-VL domains, which are arranged either (from N-to C-terminus) in the order VH A-VH B and VL B-VL A, or in the order HV B-HV A and VL A-VL B.

A “CrossMab” refers to a technology of pairing of unmodified light chain with the corresponding unmodified heavy chain and pairing of the modified light chain with the corresponding modified heavy chain, thus resulting an antibody with reduced mispairing in the light chain.

A “BiTE” is a bispecific T-cell engager molecule, comprising a first scFv with a first antigen specificity in the VL-VL orientation linked to a second scFv with a second specificity in the VH-VL orientation.

In some embodiments, the protein complex comprises an Fc. The Fc region can be modified to provide desired effector functions or serum half-life.

Any of the protein complex described herein can be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the modified immunoglobulin 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 fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in a human) .

In some embodiments, the protein complex described herein can be conjugated 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, dihydroxy anthracin, maytansinoids such as DM-1 and DM-4, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs) .

In some embodiments, the therapeutic agent can be linked to the functional domains, immunomodulatory moieties, or targeting moieties as described herein. For example, one or more therapeutic agents can be covalently linked to one or more amino acids (e.g., side chains) of the functional domains, immunomodulatory moieties, or targeting moieties (e.g., fused to different fusion sites as described herein) .

In some embodiments, the protein complex comprises a modified immunoglobulin. Various polypeptides can be fused to modified immunoglobulins. The polypeptides can be fused to any sites described herein (e.g., the 3A site) , the N-terminal, and/or the C-terminal of the heavy chain or light chain. As shown in the present disclosure, after the polypeptide is fused to the Fc region, the fused polypeptide can adopt a proper conformation and maintain its bioactivity. In some embodiments, at least 1, 2, 3, 4, 5, or 6 polypeptides can be fused to the modified immunoglobulin. In some embodiments, a polypeptide is fused to a heavy chain CH3 domain of a modified immunoglobulin from one or more amino acid residues. In some embodiments, the polypeptide is fused to one amino acid residue of the heavy chain CH3 domain. In some embodiments, the polypeptide is fused to the C-terminal amino acid residue of the heavy chain CH3 domain. In some embodiments, the polypeptide is fused to the N-terminal amino acid residue of the heavy chain VH. In some embodiments, the polypeptide is fused to the C-terminal amino acid residue of the light chain CL domain. In some embodiments, the polypeptide is fused to the N-terminal amino acid residue of the light chain VL.

The disclosure further provides fusion proteins comprising a targeting moiety and an immunomodulatory moiety as described herein. As used herein, the term “fusion protein” in the present disclosure refers to a molecule comprising two or more proteins or the fragments thereof which are linked by the covalent bond via their respective main chains of the peptides, and more preferably, the fusion protein is generated by the genetic expression of the polynucleotide molecules encoding these proteins. In some embodiments, the fusion protein comprises an immunoglobulin domain. In some embodiments, the fusion protein is an Fc-fusion protein.

In one aspect, the protein complex comprises or is a modified antibody. In some embodiments, the modified antibody comprises or consists of two fusion heavy chain polypeptides and two light chain polypeptides. In some embodiments, the two fusion heavy chain polypeptides are identical. In some embodiments, the two fusion heavy chain polypeptides are different. In some embodiments, the modified antibody comprises or consists of a fusion heavy chain polypeptide, a heavy chain polypeptide, and two light chain polypeptides.

In some embodiments, the fusion heavy chain polypeptide comprises or consists of, e.g., preferably from N-terminus to C-terminus: a heavy chain variable region (VH) , a CH1 domain, a CH2 domain, a first portion of a CH3 domain, an optional first linker sequence, an interleukin 12 polypeptide, an optional second linker sequence, and a second portion of a CH3 domain. In some embodiments, the first portion of the CH3 domain comprises amino acid residues 341-343, 341-350, or 341-357 of the CH3 domain according to EU numbering. In some embodiments, the second portion of the CH3 domain comprises amino acid residues 383-447 or 363-447 of the CH3 domain according to EU numbering. In some embodiments, the optional first linker sequence and/or the optional second linker sequence are identical to any of the linker sequences described herein.

In some embodiments, the fusion heavy chain polypeptide and/or the heavy chain polypeptide comprise an IgG1 constant region. In some embodiments, the IgG1 constant region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 34. In some embodiments, the last lysine residue in SEQ ID NO: 31 is mutated (e.g., to alanine) in order to reduce hydrolysis rate (e.g., by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%) . In some embodiments, position 447 of the fusion heavy chain polypeptide and/or the heavy chain polypeptide is an amino acid with a hydrophobic side chain (e.g., alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan) according to EU numbering. In some embodiments, position 358 of the fusion heavy chain polypeptide and/or the heavy chain polypeptide is an amino acid with a hydrophobic side chain (e.g., alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan) according to EU numbering. In some embodiments, position 297 of the fusion heavy chain polypeptide and/or the heavy chain polypeptide is alanine according to EU numbering. In some embodiments, the mutation at position 297 can reduce the glycosylation level of Fc (e.g., to less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%) . In some embodiments, the light chain polypeptide comprises a light chain constant region (CL) . In some embodiments, the CL comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 33. In some embodiments, the antibody or antigen-binding fragment thereof, or the protein complex as described herein have an Fc region without effector function. In some embodiments, the Fc is a human IgG4 Fc. In some embodiments, the Fc does not have a functional Fc region. For example, the Fc region has LALA mutations (L234A and L235A mutations in EU numbering) , or LALA-PG mutations (L234A, L235A, P329G mutations in EU numbering) .

In some embodiments, the fusion heavy chain polypeptide and/or the heavy chain polypeptide comprise an IgG4 constant region. In some embodiments, the IgG4 constant region described herein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 166, 167, or 168. In some embodiments, the fusion heavy chain polypeptide and/or the heavy chain polypeptide comprise an IgG2 constant region. In some embodiments, the IgG2 constant region described herein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 165.

In some embodiments, the fusion heavy chain polypeptide comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 39 or 50. In some embodiments, the light chain polypeptide comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 40 or 51. In some embodiments, the modified antibody comprises or consists of a structure as shown in FIG. 1A.

In some embodiments, the interleukin 12 polypeptide is IL12a. In some embodiments, the IL12a is a mouse IL12a (mIL12a) . In some embodiments, the mIL12a comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 26. In some embodiments, the IL12a is a human IL12a (hIL12a) . In some embodiments, the hIL12a comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 28. In some embodiments, the interleukin 12 polypeptide is IL12b. In some embodiments, the IL12b is a mouse IL12b (mIL12b) . In some embodiments, the mIL12b comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 27. In some embodiments, the IL12b is a human IL12b (hIL12b) . In some embodiments, the hIL12b comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 29. In some embodiments, the interleukin 12 polypeptide is an IL12a and IL12b fusion protein, optionally linked with a linker sequence described herein.

In some embodiments, the fusion heavy chain polypeptide comprises or consists of, e.g., preferably from N-terminus to C-terminus: a VH, a CH1 domain, a CH2 domain, a first portion of a CH3 domain, an optional first linker sequence, an interleukin 12 polypeptide, an optional second linker sequence, a second portion of a CH3 domain, an optional third linker sequence, and a cytokine.

In some embodiments, the cytokine is IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 , IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, or IL-36. In some embodiments, the fusion heavy chain polypeptide comprises or consists of, e.g., preferably from N-terminus to C-terminus: a VH, a CH1 domain, a CH2 domain, a first portion of a CH3 domain, an optional first linker sequence, an interleukin 12 polypeptide, an optional second linker sequence, a second portion of a CH3 domain, an optional third linker sequence, and an interferon (IFN) . In some embodiments, the optional third linker sequence is identical to any of the linker sequences described herein. In some embodiments, the interferon is an IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-τ, IFN-δ, IFN-ζ, IFN-ω, or IFN-γ. In some embodiments, the interferon is IFNa1, IFNa2, IFNa4, IFNa5, IFNa6, IFNa7, IFNa8, IFNa10, IFNa13, IFNa14, IFNa16, IFNa17, or IFNa21. In some embodiments, the interferon is mouse IFNa4 (mIFNa4) . In some embodiments, the mIFNa4 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 30. In some embodiments, the interferon is human IFNa4 (hIFNa4) . In some embodiments, the hIFNa4 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 54. In some embodiments, the interferon is human IFNa2 (hIFNa2) . In some embodiments, the hIFNa2 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 57.

In some embodiments, the fusion heavy chain polypeptide comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 41, 52, or 55.In some embodiments, the light chain polypeptide comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 42, 53, or 56. In some embodiments, the modified antibody comprises or consists of a structure as shown in FIG. 2A.

In some embodiments, the modified antibody comprises one or more knobs-into-holes (KIH) modifications. The KIH modifications include mutations at position S354C, T366W, Y349C, T366S, L368A, Y407V according EU numbering.

In some embodiments, the modified antibody comprises or consists of a fusion heavy chain polypeptide, a heavy chain polypeptide, and two light chain polypeptides. In some embodiments, the fusion heavy chain polypeptide comprises one or more hole mutations and the heavy chain polypeptide comprises one or more corresponding knob mutations. In some embodiments, the fusion heavy chain polypeptide comprises one or more knob mutations and the heavy chain polypeptide comprises one or more corresponding hole mutations. In some embodiments, the fusion heavy chain polypeptide comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 43 or 88. In some embodiments, the heavy chain polypeptide comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 44 or 164. In some embodiments, the light chain polypeptide comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 45 or 89.

In some embodiments, the modified antibody comprises or consists of a first fusion heavy chain polypeptide, a second fusion heavy chain polypeptide, and two light chain polypeptides. In some embodiments, the first fusion heavy chain polypeptide comprises one or more hole mutations and the second fusion heavy chain polypeptide comprises one or more corresponding knob mutations.

In some embodiments, the fusion heavy chain polypeptide comprises or consists of, e.g., preferably from N-terminus to C-terminus, a VH, a CH1 domain, a CH2 domain, a CH3 domain, an optional linker sequence, and an interleukin 12 polypeptide. In some embodiments, the optional linker sequence is identical to any of the linker sequences described herein. In some embodiments, the fusion heavy chain polypeptide comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 46. In some embodiments, the light chain polypeptide comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 47. In some embodiments, the modified antibody comprises or consists of a structure as shown in FIG. 25A.

In one aspect, the disclosure is related to a modified antibody. In some embodiments, the modified antibody comprises or consists of two heavy chain polypeptides and two fusion light chain polypeptides. In some embodiments, the two fusion light chain polypeptides are identical. In some embodiments, the two fusion light chain polypeptides are different. In some embodiments, the modified antibody comprises or consists of two heavy chain polypeptides, a fusion light chain polypeptide, and a light chain polypeptide.

In some embodiments, the fusion light chain polypeptide comprises or consists of, e.g., preferably from N-terminus to C-terminus: a light chain variable region (VL) , a light chain constant region (CL) , an optional linker sequence, and an interleukin 12 polypeptide. In some embodiments, the optional linker sequence is identical to any of the linker sequences described herein. In some embodiments, the fusion light chain polypeptide comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 49. In some embodiments, the heavy chain polypeptide comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 48. In some embodiments, the modified antibody comprises or consists of a structure as shown in FIG. 25B.

In some embodiments, the fusion light chain polypeptide comprises or consists of, e.g., preferably from N-terminus to C-terminus: an interleukin 12 polypeptide, an optional linker sequence, a light chain variable region (VL) , and a light chain constant region (CL) . In some embodiments, the optional linker sequence is identical to any of the linker sequences described herein. In some embodiments, the fusion light chain polypeptide comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 93. In some embodiments, the heavy chain polypeptide comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 92. In some embodiments, the modified antibody comprises or consists of a structure as shown in FIG. 25D.

In one aspect, the disclosure is related to a modified antibody that comprises or consists of two fusion heavy chain polypeptides and two fusion light chain polypeptides. In some embodiments, the two fusion heavy chain polypeptides are identical. In some embodiments, the two fusion heavy chain polypeptides are different. In some embodiments, the two fusion light chain polypeptides are identical. In some embodiments, the two fusion light chain polypeptides are different. In some embodiments, the modified antibody comprises or consists of a fusionheavy chain polypeptides, a heavy chain polypeptide, a fusion light chain polypeptide, and a light chain polypeptide. In some embodiments, the fusion heavy chain polypeptide comprises or consists of, e.g., preferably from N-terminus to C-terminus: a first interleukin 12 polypeptide (e.g., IL12a or IL12b) , an optional linker sequence, a heavy chain variable region (VH) , a CH1 domain, a CH2 domain, and a CH3 domain. In some embodiments, the fusion light chain polypeptide comprises or consists of, e.g., preferably from N-terminus to C-terminus: a second interleukin 12 polypeptide (e.g., IL12a or IL12b) , an optional linker sequence, a light chain variable region (VL) , and a light chain constant region (CL) . In some embodiments, the optional linker sequence is identical to any of the linker sequences described herein. In some embodiments, the first and the second interleukin 12 polypeptides can associate with each other forming a functional IL12 complex. In some embodiments, the fusion heavy chain polypeptide comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 90. In some embodiments, the heavy chain polypeptide comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 91. In some embodiments, the modified antibody comprises or consists of a structure as shown in FIG. 25C.

In some embodiments, IL12 fusion of an anti-PD-L1 antibody (e.g., any one of the anti-PD-L1 antibody described herein) can improve the tumor growth inhibition efficacy as compared to an unmodified anti-PD-L1 antibody, e.g., by increasing the Tumor Growth Inhibition value (TGI _TV) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 5-fold, or at least 10-fold. In some embodiments, IL12 fusion of an anti-PD-L1 antibody does not increase the toxicity of the anti-PD-L1 antibody (e.g., as determined using any of the methods described herein) .

In some embodiments, treatment using an anti-PD-L1 antibody (e.g., any one of the anti-PD-L1 antibody described herein) that is fused with IL12 (e.g., having any one of the schematic structures described herein) can increase the survival period of a subject (e.g., a tumor-bearing mouse) by at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least a week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, or at least 10 weeks. In some embodiments, more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or 100%of the treated subjects are tumor-free after the treatment.

In some embodiments, the protein complex described herein (e.g., 3F2-mIL12-3A) can be administered at a dose level of less than 10 mg/kg, less than 9 mg/kg, less than 8 mg/kg, less than 7 mg/kg, less than 6 mg/kg, less than 5 mg/kg, less than 3 mg/kg, less than 2 mg/kg, or less than 1 mg/kg without causing toxicity to the subject for cancer treatment. In some embodiments, the toxicity of the protein complex is determined by measuring the serum IFN-γ concentration of the subject (e.g., a significantly high IFN-γ indicates toxicity) . In some embodiments, the toxicity of the protein complex is determined by measuring the ALT and/or AST level of the subject (e.g., a significantly high ALT and/or AST level indicate toxicity) . In some embodiments, the protein complex described herein (e.g., 3F2-mIL12-3A) can be administered at a dose level that is greater than 0.3 mg/kg, greater than 0.5 mg/kg, or greater than 1 mg/kg, such that an obvious tumor inhibition and/or survival period improvement can be observed.

In some embodiments, treatment of the protein complex described herein (e.g., 3F2-mIL12-3A) can have a long-term protective effect against tumor growth. For example, after tumor regression using the protein complex, new tumor cannot grow after at least 5 days, at least 10 days, at least 15 days, at least 20 days, at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 45 days, at least 50 days, at least 100 days, at least 150 days, at least 200 days, at least 250 days, at least 300 days, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years post injection of tumor cells.

In some embodiments, fusion to an anti-PD-L1 antibody or antigen-binding fragment thereof can decrease the toxicity of IL12. However, fusion to an Fc, or an anti-PD-1 antibody or antigen-binding fragment does not decrease the toxicity of IL12.

In some embodiments, a weaker Fc effector function (e.g., including an N297A mutation in the Fc region) of the protein complex described herein can improve the tumor growth inhibition efficacy and/or subject survival period as compared to an unmodified protein complex. In some embodiments, fusion of a single IL12 polypeptide to an anti-PD-L1 antibody or antigen-binding fragment thereof can improve the tumor growth inhibition efficacy and/or subject survival period as compared to a protein complex having two or more IL12 moieties fused to an anti-PD-L1 antibody or antigen-binding fragment thereof.

In some embodiments, fusion to anti-PD-L1 antibody or antigen-binding fragment can increase the activity of IL12, e.g., by decreasing the EC50 value to less than 0.2 nmol/L, less than 0.15 nmol/L, or less than 0.1 nmol/L using the methods described herein.

Targeting moieties

The targeting moiety can specifically bind to various antigens. In some embodiments, the antigen is a tumor-associated antigen. As used herein, the term “tumor associated antigen” refers to an antigen that is or can be presented on a tumor cell surface. In some embodiments, the tumor associated antigens can be exclusively expressed on tumor cells or may represent a tumor specific mutation compared to non-tumor cells. In some embodiments, the tumor associated antigens can be found in both tumor cells and non-tumor cells, but is overexpressed on tumor cells when compared to non-tumor cells or are accessible for antibody binding in tumor cells due to the less compact structure of the tumor tissue compared to non-tumor tissue. In some embodiments the tumor associated antigen is located on the vasculature of a tumor. Illustrative examples of a tumor associated surface antigen are CD10, CD19, CD20, CD22, CD21, CD22, CD25, CD30, CD33, CD34, CD37, CD44v6, CD45, CD133, Fms-like tyrosine kinase 3 (FLT-3, CD135) , chondroitin sulfate proteoglycan 4 (CSPG4, melanoma-associated chondroitin sulfate proteoglycan) , Epidermal growth factor receptor (EGFR) , Her2, Her2neu, Her3, IGFR, IL3R, fibroblast activating protein (FAP) , CDCP1, Derlin1, Tenascin, frizzled 1-10, the vascular antigens VEGFR2 (KDR/FLK1) , VEGFR3 (FLT4, CD309) , PDGFR-alpha (CD140a) , PDGFR-beta (CD140b) Endoglin, CLEC14, Tem1-8, and Tie2. Further examples may include A33, CAMPATH-1 (CDw52) , Carcinoembryonic antigen (CEA) , Carboanhydrase IX (MN/CA IX) , de2-7 EGFR, EGFRvIII, EpCAM, Ep-CAM, Folate-binding protein, G250, Fms-like tyrosine kinase 3 (FLT-3, CD135) , c-Kit (CD117) , CSF1R (CD115) , HLA-DR, IGFR, IL-2 receptor, IL3R, MCSP (Melanoma-associated cell surface chondroitin sulfate proteoglycane) , Muc-1, Prostate-specific membrane antigen (PSMA) , Prostate stem cell antigen (PSCA) , Prostate specific antigen (PSA) , and TAG-72. In some embodiments, the tumor associated antigen is PD-L1.

In some embodiments, the functional domain, the targeting moiety, and/or the immunomodulatory moiety comprises an antibody or antigen binding fragment thereof. The binding affinity of an antibody or antigen binding fragment to the antigen is determined by CDRs. They are part of the variable chains in immunoglobulins (antibodies) and T cell receptors. 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.

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 addition, the functional domains, immunomodulatory moieties, or targeting moieties can be fused to various modified immunoglobulins, antibodies, antibody-like, or IgG-like molecules.

In some embodiments, the protein complex or the targeting moiety comprises an antibody and antigen-binding fragment thereof that specifically binds to PD-L1. In some embodiments, the antibody and antigen-binding fragment described herein can block PD-1/PD-L1 interaction, thereby increasing immune response. Furthermore, it is contemplated that blocking (or interfering) the PD-1/PD-L1 interaction can activate the PD-1-expressing T cells, which may increase the expression of IL12R. These cells can be further activated by IL12. However, if the antibody or antigen-binding fragment thereof does not block the PD-1/PD-L1 interaction, the PD-1-expressing T cells are not activated and the off-target IL12 can exhibit toxicity.

In some embodiments, the antibody or antigen-binding fragment described herein is capable of binding to PD-L1 without blocking PD-1/PD-L1 interaction.

In some embodiments, the antibody or antigen-binding fragment thereof is an agonist. In some embodiments, the antibody or antigen-binding fragment thereof is an antagonist. The disclosure provides, e.g., mouse anti-PD-L1 antibody 08-3F2 ( “3F2” ) and the humanized antibodies thereof.

The CDR sequences for 3F2 ( “08-3F2” ) , and 3F2 derived 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. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 7-9, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 10-12.

The CDR sequences for 1C9 ( “17-1C9” ) , and 1C9 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 104-106, and CDRs of the light chain variable domain, SEQ ID NOs: 107-109, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 134-136, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 137-139.

The CDR sequences for 2A5 ( “23-2A5” ) , and 2A5 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 110-112, and CDRs of the light chain variable domain, SEQ ID NOs: 113-115, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 140-142, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 143-145.

The CDR sequences for 10A11 ( “23-10A11” ) , and 10A11 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 116-118, and CDRs of the light chain variable domain, SEQ ID NOs: 119-121, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 146-148, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 149-151.

The CDR sequences for 2C12 ( “30-2C12” ) , and 2C12 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 122-124, and CDRs of the light chain variable domain, SEQ ID NOs: 125-127, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 152-154, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 155-157.

The CDR sequences for 2H9 ( “BC-2H9” ) , and 2H9 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 128-130, and CDRs of the light chain variable domain, SEQ ID NOs: 131-133, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 158-160, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 161-163.

The amino acid sequence for heavy chain variable region and light variable region of humanized antibodies are also provided.

The amino acid sequence for the heavy chain variable region (VH) of 1C9 antibody is set forth in SEQ ID NO: 80, and the amino acid sequence for the light chain variable region (VL) of 1C9 antibody is set forth in SEQ ID NO: 81. The amino acid sequence for the VH of 2A5 antibody is set forth in SEQ ID NO: 78, and the amino acid sequence for the VL of 2A5 antibody is set forth in SEQ ID NO: 79. The amino acid sequence for the VH of 10A11 antibody is set forth in SEQ ID NO: 70, and the amino acid sequence for the VL of 10A11 antibody is set forth in SEQ ID NO: 71. The amino acid sequence for the VH of 2C12 antibody is set forth in SEQ ID NO: 66, and the amino acid sequence for the VL of 2C12 antibody is set forth in SEQ ID NO: 67. The amino acid sequence for the VH of 2H9 antibody is set forth in SEQ ID NO: 98, and the amino acid sequence for the VL of 2H9 antibody is set forth in SEQ ID NO: 99. The amino acid sequence for the VH of 2A6 antibody is set forth in SEQ ID NO: 68, and the amino acid sequence for the VL of 2A6 antibody is set forth in SEQ ID NO: 69.

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 version of humanized sequences. The amino acid sequences for the heavy chain variable region of humanized 3F2 antibody are set forth in SEQ ID NOs: 13-16. The amino acid sequences for the light chain variable region of humanized 9H3 antibody are set forth in SEQ ID NOs: 17-19. Any of these heavy chain variable region sequences (SEQ ID NO: 13-16) can be paired with any of these light chain variable region sequences (SEQ ID NO: 17-19) .

In some embodiments, the amino acid sequence for the heavy chain variable region of humanized 3F2 antibody is set forth in SEQ ID NO: 13. The amino acid sequence for the light chain variable region of humanized 3F2 antibody is set forth in SEQ ID NO: 17.

Furthermore, in some embodiments, the antibody or antigen-binding fragment 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: 104-106, SEQ ID NOs: 110-112; SEQ ID NOs: 116-118, SEQ ID NOs: 122-124; SEQ ID NOs: 128-130; SEQ ID NOs: 7-9, SEQ ID NOs: 134-136, SEQ ID NOs: 140-142, SEQ ID NOs: 146-148, SEQ ID NOs: 152-154, and SEQ ID NOs: 158-160; and/or one, two, or three light chain variable region CDRs selected from the group of SEQ ID NOs: 4-6, SEQ ID NOs: 107-109, SEQ ID NOs: 113-115, SEQ ID NOs: 119-121, SEQ ID NOs: 125-127, SEQ ID NOs: 131-133, SEQ ID NOs 10-12, SEQ ID NOs: 137-139, SEQ ID NOs: 143-145, SEQ ID NOs: 149-151, SEQ ID NOs: 155-157, and SEQ ID NOs: 161-163.

In some embodiments, the antibody or antigen-binding fragment thereof 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 of an 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. 54 (Kabat CDR) and FIG. 55 (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 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 antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the VH CDRs 1-3 described in FIG. 54 or FIG. 55 with zero, one or two amino acid insertions, deletions, or substitutions. In some embodiments, the antibody or antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the VL CDRs 1-3 described in FIG. 54 or FIG. 55 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 PD-L1. 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 SEQ ID NO: 13-16, 66, 68, 70, 78, 80, or 98; 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 SEQ ID NO: 17-19, 67, 69, 71, 79, 81, or 99.

In some embodiments, the antibody or antigen-binding fragment thereof described herein contains 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 SEQ ID NO: 58, 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 SEQ ID NO: 59. In some embodiments, the antibody or antigen-binding fragment thereof described herein contains 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 SEQ ID NO: 60, 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 SEQ ID NO: 61. In some embodiments, the antibody or antigen-binding fragment thereof described herein contains 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 the VH of durvalumab, atezolizumab or avelumab; 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 the VL of durvalumab, atezolizumab or avelumab.

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. 54 or FIG. 55, or have sequences as shown in FIG. 56. 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 PD-L1 (e.g., human PD-L1) .

In some embodiments, the 3F2, or 3F2 derived antibodies (e.g., humanized antibodies) ; the 2C12, or 2C12 derived antibodies (e.g., humanized antibodies) ; the 2A6, or 2A6 derived antibodies (e.g., humanized antibodies) ; the 10A11, or 10A11 derived antibodies (e.g., humanized antibodies) ; the 2A5, or 2A5 derived antibodies (e.g., humanized antibodies) ; the 1C9, or 1C9 derived antibodies (e.g., humanized antibodies) ; and the 2H9, or 2H9 derived antibodies (e.g., human antibodies) have an IgG1 or IgG4 subtype.

Details of the PD-L1 antibody 3F2 and 23-2A6 can be found, e.g., in PCT/CN2020/075983 and U.S. Patent Application No. 17/430,396, each of which is incorporated herein by reference in its entirety.

In some embodiments, the antibody and antigen-binding fragment thereof that specifically binds to PD-L1 is selected from the group consisting of Atezolizumab, Avelumab, or Durvalumab. The anti-PD-L1 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, multimeric, multispecific (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. In some embodiments, the antigen-binding fragment is a scFv or a Fab. Generally, the scFv 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.

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof described herein have a KD value against PD-L1 of less than 1 × 10 ^-7 M, less than 1 × 10 ^-8 M, less than 1 × 10 ^-9 M, or less than 5 × 10 ^-10 M. In some embodiments, the binding epitopes of anti-PD-L1 antibody or antigen-binding fragment thereof described herein are identical, partially overlapped, or completely different.

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof described herein comprises a heavy chain variable region (VH) comprising

VH CDRs

1, 2, 3, and a light chain variable region (VL) comprising

VL CDRs

1, 2, 3, wherein the

VH CDRs

1, 2, 3 and the

VL CDRs

1, 2, 3 are identical to any of the

VH CDRs

1, 2, 3 and any of the

VL CDRs

1, 2, 3 of the antibody or antigen-binding fragment thereof described herein.

In some embodiments, provided therein are anti-PD-L1 antibodies or antigen-binding fragments thereof that can cross-compete with any of the anti-PD-L1 antibodies or antigen-binding fragments thereof described herein. In some embodiments, the cross-competing effect can be achieved between two anti-PD-L1 antibodies by having identical or overlapping binding epitopes, e.g., between Class I (e.g., Avelumab, or Atezolizumab) , Class II (e.g., 3F2 or 3F2 derived antibodies) , Class III (e.g., 2A5 or 2A5 derived antibodies) , and Class IV (e.g., 2C12 or 2C12 derived antibodies) anti-PD-L1 antibodies.

Linker sequence

The functional domain, the immunomodulatory moiety (e.g., IL12) , and/or the targeting moiety can be fused to any protein complex as described herein or fused with each other (e.g., the N terminal or the C terminal of a heavy chain, or the N terminal or the C terminal of a light chain, or the 3A site in the CH3 domain) with or without a linker sequence. In some embodiments, the linker peptide is optional, i.e., the two regions that are linked together can be directly linked by a peptide bond.

In some embodiments, the linker sequence comprises at least or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 amino acid residues. In some embodiments, the linker sequence comprises at least or about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 25, 30, or 40 glycine residues. In some embodiments, the linker sequence comprises at least or about 1, 2, 3, 4, 5, 6, 7, or 8 serine residues. In some embodiments, the linker sequence comprises or consists of both glycine and serine residues. In some embodiments, the linker sequence comprises or consists of a sequence that is at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, or 100%identical to any SEQ ID NO: 35, 36, 37, or 38. In some embodiments, the linker sequence comprises at least 1, 2, 3, 4, 5, or 6 repeats of GGGGS (SEQ ID NO: 35) . In some embodiments, the linker sequence has no more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 amino acid residues.

In one aspect, a polypeptide is fused to a heavy chain CH3 domain. In some embodiments, the inserted polypeptide includes an N-terminal linker sequence and a C-terminal linker sequence. As used herein, the term “N-terminal linker sequence” refers to a linker sequence that is located at the N-terminal of the inserted or fused polypeptide. As used herein, the term “C-terminal linker sequence” refers to a linker sequence that is located at the C-terminal of the inserted or fused polypeptide. The N-terminal linker sequence and the C-terminal linker sequence can be the same or different, and can comprise or consist of any linker sequences as described herein.

Methods of making protein complexes

The present disclosure also provides recombinant vectors (e.g., an 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 protein complex 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.

The expression vectors can include at least one selectable marker. Such markers include e.g., 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.

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.

In some embodiments, a covalent modification can be made to the protein complex. These covalent modifications can be made by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications can be introduced into the molecule by reacting targeted amino acid residues in the protein complex with an organic derivatization agent that is capable of reacting with selected side chains or the N-or C-terminal residues.

In some embodiments, the protein complex can comprises an antibody having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody composition 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 one aspect, the disclosure provides a vector that comprises a sequence encoding a heavy chain, wherein IL12a is fused to the heavy chain, and/or a vector that comprises a sequence encoding a light chain and IL12b. In some embodiments, IL12b and the light chain have different promoters. In some embodiments, IFNa4 is further fused to the heavy chain.

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.

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, 400 or 500 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 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 example, 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.

Methods of treatment

The protein complex as described herein can be used for various therapeutic purposes. In one aspect, the disclosure 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.

As used herein, the term “cancer” refers to cells having the capacity for autonomous growth. Examples of such cells include cells having an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include cancerous growths, e.g., tumors; oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. Also included are malignancies of the various organ systems, such as respiratory, cardiovascular, renal, reproductive, hematological, neurological, hepatic, gastrointestinal, and endocrine systems; 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, and cancer of the small intestine. Cancer that is “naturally arising” includes any cancer that is not experimentally induced by implantation of cancer cells into a subject, and includes, for example, spontaneously arising cancer, cancer caused by exposure of a patient to a carcinogen (s) , cancer resulting from insertion of a transgenic oncogene or knockout of a tumor suppressor gene, and cancer caused by infections, e.g., viral infections. The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues. The term also includes carcinosarcomas, 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. The term “hematopoietic neoplastic disorders” includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin. A hematopoietic neoplastic disorder can arise from myeloid, lymphoid or erythroid lineages, or precursor cells thereof.

In one aspect, the disclosure features methods that include administering a therapeutically effective amount of a protein complex disclosed 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. In some embodiments, the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung carcinoma (NSCLC) , small cell lung cancer (SCLC) , bladder cancer, or metastatic hormone-refractory prostate cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is squamous cell carcinoma of the head and neck (SCCHN) , renal cell carcinoma (RCC) , triple-negative breast cancer (TNBC) , or colorectal carcinoma. In some embodiments, the subject has Hodgkin's lymphoma. In some embodiments, the subject has triple-negative breast cancer (TNBC) , gastric cancer, urothelial cancer, Merkel-cell carcinoma, or head and neck cancer. In some embodiments, the cancer is melanoma, pancreatic carcinoma, mesothelioma, hematological malignancies, especially Non-Hodgkin's lymphoma, lymphoma, chronic lymphocytic leukemia, or advanced solid tumors.

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.

In some embodiments, the cancer expresses PD-L1. In some embodiments, the cancer is resistant to the anti-PD-1 antibody treatment or the anti-PD-L1 antibody treatment. In some embodiments, the IL12 in the protein complex can activate tumor-infiltrating lymphocyte effector functions.

In some embodiments, the subject is not responsive to the immunotherapy. The immune-desert and immune-excluded phenotypes are known as cold tumors (non-inflamed) , and the density of CD8+ T cells in the tumors is low. As the protein complex can increase immune response in the tumor microenvironment, the protein complex can be used to treat patients who are not responsive to the immunotherapy.

Thus, in one aspect, the disclosure further provides methods of increasing immune response in tumor microenvironment and methods of activating T cells in tumor microenvironment, spleen, lymph nodes, and/or lymphatic system. These methods involve administering a therapeutically effective amount of the protein complex as described herein to the subject.

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 or an autoimmune disease. An effective amount will vary depending upon, e.g., an age and a body weight of a subject to which the therapeutic agent 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 a protein complex 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 the protein complex may vary, depending on, inter alia, patient history as well as other factors such as the type (and/or dosage) of the therapeutic agent used.

Effective amounts and schedules for administering the protein complex, 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 protein complex, and/or compositions disclosed herein, the route of administration, the particular type of protein complex, and/or compositions disclosed herein used and other drugs being administered to the mammal.

The dosage of an effective amount of the protein complex can be 0.01 mg/kg to 100 mg/kg (mg per kg of patient weight) . 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 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.

As described above, severe side effects were associated with systemic administration of isolated IL-12. The maximal tolerated doses in escalating dose protocols for isolated IL12 range, in relation to the treatment schedule, usually between 250 and 500 ng/kg. The disclosure demonstrates that the protein complex can reduce the toxicity of IL12. Thus, the protein complex can be used as a carrier to deliver a much higher amount of IL12 to a subject. The amount can be significantly above the maximal tolerated doses for isolated IL12. Thus, in one aspect, the disclosure provides methods of reducing IL12 toxicity when IL12 is delivered into a subject and methods of delivering IL12 without causing toxicity to a subject. The equivalent amount IL12 in the protein complex can be significantly above the maximal tolerated doses for isolated IL12. In some embodiments, the dosage for the protein complex 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, or 1 mg/kg. In some embodiments, the equivalent amount of IL12 in the protein complex composition can be calculated (e.g., the dose is divided by the molar mass of the protein complex and then times the molar mass of the IL12 functional domain) . In some embodiments, the dosage for the equivalent amount of IL12 in the protein complex composition is about or more than 500 ng/kg, 600 ng/kg, 700 ng/kg, 800 ng/kg, 900 ng/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, or 5 mg/kg.

In any of the methods described herein, the protein complex, or pharmaceutical composition, 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, the protein complex and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition) . In some embodiments, the protein complex and the at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition containing the protein complex 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 protein complex. In some embodiments, the one or more additional therapeutic agents and the protein complex 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 protein complex in the subject.

In some embodiments, the subject can be administered the protein complex 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) .

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-OX40 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, or an anti-GITR antibody.

In some embodiments, the additional therapeutic agent is an immunotherapy, e.g., a blocking anti-PD-1 antibody (e.g., Pembrolizumab, Nivolumab, or Cemiplimab) or a block anti-PD-L1 antibody (e.g., Atezolizumab, Avelumab, or Durvalumab) .

Pharmaceutical Compositions

Also provided herein are pharmaceutical compositions that contain the protein complex described herein. The pharmaceutical compositions may be formulated in any manner known in the art.

A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is bioactivity acceptable and nontoxic to a subject. Pharmaceutical acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispending agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof. Some suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, coloring agents, emulsifiers or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxanisol, butylated hydroxytoluene, and/or propyl gallate. As disclosed herein, inclusion of one or more antioxidants such as methionine in a pharmaceutical composition provided herein decreases oxidation of the polypeptide complex or the bispecific polypeptide complex. This reduction in oxidation prevents or reduces loss of binding affinity, thereby improving protein stability and maximizing shelf-life. Therefore, in certain embodiments, compositions are provided that comprise the polypeptide complex or the bispecific polypeptide complex disclosed herein and one or more antioxidants such as methionine.

Pharmaceutical compositions are also formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) . 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 protein complex 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) .

Compositions containing one or more of any of the protein complex 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) .

Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration) . For injection, protein complex can be formulated in aqueous solutions, preferably in physiologically-compatible buffers to reduce discomfort at the site of injection.

Exemplary doses include milligram or microgram amounts of any of the protein complex 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; about 1 μg/kg to about 50 μg/kg; about 1 mg/kg to about 10 mg/kg; or about 1 mg/kg to about 5 mg/kg) . While these doses cover a broad range, one of ordinary skill in the art will understand that therapeutic agents, including protein complex, 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 protein complex in 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 protein complex 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. IL12a fused at 3A site of PD-L1 antibody

Plasmids were constructed to express a fusion protein that comprises a mouse interleukin 12 alpha subunit (IL12a, or P35) , fused at the 3A site (position 358 to position 362 according to EU numbering) of each of the two heavy chains of a humanized anti-PD-L1 antibody PDL1-3F2, as shown in FIG. 1A. The PDL1-3F2 IgG1 antibody comprises a heavy chain variable region (VH) with sequence set forth in SEQ ID NO: 13, and a light chain variable region (VL) with sequence set forth in SEQ ID NO: 17. Specifically, the N-terminus of IL12a was linked to position 357 (according to EU numbering) of PDL1-3F2 IgG1 heavy chains via GGGGSGGGGS (SEQ ID NO: 36) , and the C-terminus of IL12a was linked to position 363 (according to EU numbering) of PDL1-3F2 IgG1 heavy chains via GGGGSGGGGS (SEQ ID NO: 36) .

A mouse interleukin 12 beta subunit (IL12b, or P40) and the PDL1-3F2 IgG1 light chains were encoded by separate plasmids. Schematic diagrams of the plasmids are shown in FIG. 1B. The modified PDL1-3F2 IgG1antibody fused with IL12 (including IL12a and IL12b) was named 3F2-mIL12-3A (or 3F2-mIL12-3A-IgG1) . Sequence of the modified heavy chain of 3F2-mIL12-3A is set forth in SEQ ID NO: 39, and sequence of the light chain of 3F2-mIL12-3A is set forth in SEQ ID NO: 40.

Example 2. IFNa4 linked at heavy chain C-terminus of PD-L1 antibody

Plasmids were constructed to express a fusion protein that further comprises a mouse interferon alpha 4 (IFNa4) (SEQ ID NO: 30) , linked to each of the two heavy chain C-terminus of 3F2-mIL12-3A through a linker sequence (SEQ ID NO: 38) . As shown in FIG. 2A, the modified PDL1-3F2 IgG1 antibody fused with IL12 (including IL12a and IL12b) and IFNa4 was named 3F2-mIL12-3A-mIFNa4. Similar to 3F2-mIL12-3A, the IL12b and light chains were encoded by separate plasmids. Schematic diagrams of the plasmids are shown in FIG. 2B. Sequence of the modified heavy chain of 3F2-mIL12-3A-mIFNa4 is set forth in SEQ ID NO: 41, and sequence of the light chain of 3F2-mIL12-3A-mIFNa4 is set forth in SEQ ID NO: 42.

Example 3. Fusion proteins with diverse structures

Plasmids were also constructed to express fusion proteins as described below.

1) 3F2-mIL12-3A2-knob

3F2-mIL12-3A2-knob is a fusion protein that comprises an IL12a fused at the 3A2 site (between position 359 to position 360 according to EU numbering) of the knob heavy chain of PDL1-3F2 IgG1 with knobs-into-holes (KIH) mutations. Specifically, the N-terminus of IL12a was linked to position 359 (according to EU numbering) of the knob heavy chain via GGGGSGGGGS (SEQ ID NO: 36) , and the C-terminus of IL12a was linked to position 360 (according to EU numbering) of the knob heavy chain via GGGGSGGGGS (SEQ ID NO: 36) . Sequence of the modified knob heavy chain of 3F2-mIL12-3A2-knob is set forth in SEQ ID NO: 43, and sequence of the hole heavy chain of 3F2-mIL12-3A2-knob is set forth in SEQ ID NO: 44. Sequence of the light chain of 3F2-mIL12-3A2-knob is set forth in SEQ ID NO: 45. The IL12b and light chains were encoded by separate plasmids.

2) 3F2-mIL12-3A-hole

3F2-mIL12-3A-hole is a fusion protein that comprises an IL12a fused at the 3A site (position 358 to position 362 according to EU numbering) of the hole heavy chain of PDL1-3F2 IgG1 with knobs-into-holes (KIH) mutations. Specifically, the N-terminus of IL12a was linked to position 357 (according to EU numbering) of the hole heavy chain via GGGGSGGGGS (SEQ ID NO: 36) , and the C-terminus of IL12a was linked to position 363 (according to EU numbering) of the hole heavy chain via GGGGSGGGGS (SEQ ID NO: 36) . The IL12b and light chains were encoded by separate plasmids.

3) 3F2-mIL12-HC (3F2-mIL12-HC-IgG1)

3F2-mIL12-HC is a fusion protein that comprises an IL12a linked to each of the two heavy chain C-terminus of PDL1-3F2 IgG1 via a linker sequence (SEQ ID NO: 38) . Sequence of the modified heavy chain of 3F2-mIL12-HC is set forth in SEQ ID NO: 46, and sequence of the light chain of 3F2-mIL12-HC is set forth in SEQ ID NO: 47. The IL12b and light chains were encoded by separate plasmids.

4) 3F2-mIL12-CK (3F2-mIL12-CK-IgG1)

3F2-mIL12-CK is a fusion protein that comprises an IL12a linked to each of the two light chain C-terminus of PDL1-3F2 IgG1 via GGGGSGGGGS (SEQ ID NO: 36) . Sequence of the modified light chain is set forth in SEQ ID NO: 49, and sequence of the heavy chain is set forth in SEQ ID NO: 48. The IL12b was encoded by a separate plasmid.

5) 3F2-mIL12-3A-IgG4

3F2-mIL12-3A-IgG4 is a fusion protein that comprises the same overall structure and sequences as 3F2-mIL12-3A, except that the IgG1 constant regions are replaced by IgG4 constant regions.

6) 3F2-mIL12-3A-knob (3F2-mIL12-3A-IgG1-knob)

3F2-mIL12-3A-knob is a fusion protein that comprises an IL12a fused at the 3A site (position 358 to position 362 according to EU numbering) of the knob heavy chain of PDL1-3F2 IgG1 with knobs-into-holes (KIH) mutations. Specifically, the N-terminus of IL12a was linked to position 357 (according to EU numbering) of the knob heavy chain via GGGGSGGGGS (SEQ ID NO: 36) , and the C-terminus of IL12a was linked to position 363 (according to EU numbering) of the knob heavy chain via GGGGSGGGGS (SEQ ID NO: 36) . The IL12b and light chains were encoded by separate plasmids. Sequence of the light chain is set forth in SEQ ID NO: 89, sequence of the modified knob heavy chain is set forth in SEQ ID NO: 88 and sequence of the hole heavy chain is set forth in SEQ ID NO: 164.

Example 4. Purification of 3F2-mIL12-3A and 3F2-mIL12-3A-mIFNa4

The fusion proteins 3F2-mIL12-3A and 3F2-mIL12-3A-mIFNa4 were purified and analyzed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and size-exclusive chromatography (SEC) . Specifically, plasmids expressing the fusion proteins were constructed and used to transiently transform CHO cells. The transfected CHO cells were then cultured at 37℃ for 6-7 days. Afterwards, the culture supernatant was collected by centrifugation, and the fusion protein was first purified by a Protein A affinity column and then analyzed by SDS-PAGE. Protein purity was further measured by SEC.

Non-reducing SDS-PAGE was performed using a 6%acrylamide gel. The protein samples were prepared as follows. First, the protein samples were diluted to 1 mg/ml. 2.4 μl of the diluted protein sample was mixed with 6 μl Tris-Glycine SDS Sample Buffer (2×) and 3.6 μl distilled water. The mixture was then boiled for 2 minutes and instantly centrifuged before loading. As shown in FIG. 3, both 3F2-mIL12-3A and 3F2-mIL12-3A-mIFNa4 showed a single band with correct molecular weight.

Protein purity was further measured by SEC using the

G3000SW _XL HPLC Column (Tosoh Bioscience LLC) connected to Agilent 1260 series HPCL system. The protein A purified fusion protein samples were analyzed by SEC-HPLC. Before injection, the samples were diluted to 1 mg/ml and then filtered with a 0.22 μm filter. The experimental parameters were set up as: flow rate: 0.7 mL/min; time: 30 minutes; and detection wavelength: 280 nm. Agilent liquid chromatography analysis software was used to analyze the data. Chromatographic curves of the tested fusion proteins were obtained. As shown in FIGS. 4A-4B, the total detected peak area percentage and retention time (RT) were recorded. In general, the purity of the protein A-purified fusion protein 3F2-mIL12-3A was slightly higher than that of 3F2-mIL12-3A-mIFNa4 (86.88%purity, with a small amount of impurities) .

Example 5. Determination of binding affinity to PD-L1

The binding affinity between purified His-tagged human PD-L1 (hPL1-His; ACRO Biosystems) and PDL1-3F2 IgG1, 3F2-mIL12-3A, 3F2-mIL12-3A-mIFNa4 were measured by surface plasmon resonance (SPR) using Biacore ^TM (Biacore Inc., Piscataway N.J. ) T200 biosensor equipped with pre-immobilized protein A sensor chips.

The fusion proteins 3F2-mIL12-3A and 3F2-mIL12-3A-mIFNa4 (1 μg/mL) were injected into Biacore ^TM T200 biosensor at 10 μL/min for 30 seconds to achieve to a desired protein density (about 67 RU) . His-tagged human PD-L1 (hPL1-His) at concentrations of 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, and 1.5625 nM were then injected at 30 μL/min for 100 seconds. Dissociation was monitored for 400 seconds. The chip was regenerated after the last injection of each titration with Glycine (pH 2.0, 30 μL/min for 12 seconds) . As a person of ordinary skill in the art would understand, the same method with appropriate adjustments for parameters (e.g., fusion protein concentration) was performed for each tested fusion protein. The results for the tested fusion proteins are shown in the table below.

Table 4.

Ligand	Analysis	kon (1/Ms)	koff (1/s)	KD (M)	Rmax (RU)
PDL1-3F2 IgG1	hPL1-His	2.32E+06	1.30E-03	5.59E-10	32.37
3F2-mIL12-3A	hPL1-His	2.49E+06	9.52E-04	3.82E-10	16.5
3F2-mIL12-3A-mIFNa4	hPL1-His	2.33E+06	9.22E-04	3.96E-10	14.86

Kinetic association rates (kon) and dissociation rates (koff) were obtained simultaneously by fitting the data globally to a 1: 1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6.99-110) using Biacore ^TM T200 Evaluation Software 3.0. Affinities were calculated from the quotient of the kinetic rate constants (KD=koff/kon) . The results showed that all three tested fusion proteins PDL1-3F2 IgG1, 3F2-mIL12-3A, and 3F2-mIL12-3A-mIFNa4 can bind to hPL1-His with comparable binding affinities. Non-specific effects were not observed.

Example 6. Determination of binding affinity to IL12

The binding affinity between purified His-tagged recombinant mouse IL12RB2 protein (mIL12R-B2-His; Sino Biological Inc., Cat#: 50099-M08H) and 3F2-mIL12-3A, 3F2-mIL12-3A-mIFNa4 were measured by SPR using Biacore ^TM (Biacore Inc., Piscataway N.J. ) T200 biosensor equipped with pre-immobilized protein A sensor chips. The experiment was performed by a similar method as described in Example 5. The results for the tested fusion proteins are shown in the table below.

Table 5.

Ligand	Analysis	kon (1/Ms)	koff (1/s)	KD (M)	Rmax (RU)
3F2-mIL12-3A	mIL12R-B2-His	5.73E+04	3.35e-03	5.85E-08	56.2
3F2-mIL12-3A-mIFNa4	mIL12R-B2-His	5.29E+04	2.99e-03	5.65E-08	42.3

The results showed that both tested fusion proteins 3F2-mIL12-3A and 3F2-mIL12-3A-mIFNa4 can bind to mIL12R-B2-His with comparable binding affinities. Non-specific effects were not observed.

Example 7. In vivo pharmacological validation

A hPD-1/hPD-L1 mouse model (obtained from Biocytogen Pharmaceuticals (Beijing) Co., Ltd., Cat#: 120522) was engineered to express a chimeric PD-1 protein and a chimeric PD-L1 protein. The chimeric PD-1 protein (SEQ ID NO: 22) includes a replacement of a part of the extracellular region of the mouse PD-1 protein with the corresponding human PD-1 extracellular region. The amino acid residues 31-141 of mouse PD-1 (SEQ ID NO: 21) were replaced by amino acid residues 31-141 of human PD-1 (SEQ ID NO: 20) . The chimeric PD-L1 protein (SEQ ID NO: 25) includes a replacement of a part of the extracellular region of the mouse PD-L1 protein with the corresponding human PD-L1 extracellular region. The amino acid residues 21-128 of mouse PD-L1 (SEQ ID NO: 24) were replaced by amino acid residues 21-128 of human PD-L1 (SEQ ID NO: 23) .

The humanized mouse model (hPD-1/hPD-L1) provides a tool for testing new therapeutic treatments in a clinical setting by significantly decreasing the difference between clinical outcome in human and in ordinary mice expressing mouse PD-1 or PD-L1. A detailed description regarding humanized PD-1 mouse, PD-L1 mouse and hPD-1/hPD-L1 mouse model can be found in PCT/CN2017/090320, PCT/CN2017/099574, CN Application No. 201710505554.0 and CN Application No. 201710757022.6, each of which is incorporated herein by reference in its entirety.

PD-1/PD-L1 double humanized mice (5-8 weeks) were subcutaneously injected with MC-38 cancer tumor cells (colon adenocarcinoma cell) expressing human PD-L1 (MC38-hPD-L1) (5 × 10 ⁵/100 μl PBS) , and when the tumor volume grew to about 100-150 mm ³, the mice were divided to a control group and six treatment groups based on tumor size (n = 7/group) . The treatment groups were randomly selected for humanized anti-human PD-L1 antibody PDL1-3F2 IgG1 treatment (1 mg/kg or 3 mg/kg) , fusion protein 3F2-mIL12-3A treatment (1.82 mg/kg or 5.46 mg/kg) , or fusion protein 3F2-mIL12-3A-mIFNa4 treatment (2.11 mg/kg or 6.34 mg/kg) . The molar dosages (e.g., mole/kg) of 3F2-mIL12-3A and 3F2-mIL12-3A-mIFNa4 were equal to that of PDL1-3F2 IgG1. The control group mice were injected with an equal volume of physiological saline (PS) . The frequency of administration was twice a week (6 times of administrations in total) . The tumor volume was measured twice a week and the body weight of the mice was weighed as well. Euthanasia was performed when the tumor volume of the mouse reached 3000 mm ³.

Table 6.

Overall, the mice in each group were healthy. The body weight of all the treatment and control group mice increased, and the body weight were not obviously different from each other (FIGS. 5 and 6) . As shown in FIG. 7, the tumor in the control group continued growing during the experimental period. When compared with the control group mice, the tumor volumes in the treatment groups were smaller than the control group. Thus, the anti-human PD-L1 antibody PDL1-3F2 IgG1 and the two fusion proteins 3F2-mIL12-3A, 3F2-mIL12-3A-mIFNa4 were well tolerated, and inhibited the tumor growth in mice.

The table below summarizes the results for this experiment, including the tumor volumes on the day of grouping (Day 0) , 14 days after the grouping (Day 14) , and at the end of the experiment (Day 25) ; number of tumor-free mice; the survival rate of the mice; the Tumor Growth Inhibition value (TGI _TV) ; and the statistical differences (P value) of tumor volume between the treatment and control groups.

Table 7.

At the end of the experiment (Day 25) , the body weight of each group increased and there was no significant difference between the treatment group (except G5) and control group mice. Although the body weight of the G5 group mice was lower than that of the G1 group mice, the G5 group mice continued to gain weight throughout the experimental period, and the body weight increased by about 10%at the end of the experiment. No obvious difference in body weight change was observed. Considering that the mice in the G5 group had a lower tumor volume at the end of the experiment, the difference in body weight between the G5 group and the G1 group was mainly caused by the tumor weight. The results indicate that the anti-human PD-L1 antibody PDL1-3F2 IgG1 and the two fusion proteins 3F2-mIL12-3A, 3F2-mIL12-3A-mIFNa4 were well tolerated by mice.

The tumor volumes in all treatment groups (G2-G7) were smaller than those in the control group (G1) . The results also showed that anti-human PD-L1 antibody PDL1-3F2 IgG1 and the two fusion proteins 3F2-mIL12-3A, 3F2-mIL12-3A-mIFNa4 had different tumor inhibitory effects, which was dosage-dependent. Under the same condition (e.g., administration dosage and frequency) , the inhibitory effects of the fusion proteins (G4-G7) were better than those of the anti-PD-L1 antibody (G2 and G3) . This indicates that fusion of IL12 to an anti-PD-L1 antibody can improve the in vivo anti-tumor efficacy of PD-L1 antibody. In addition, at the same dose level, the effect of 3F2-mIL12-3A was slightly better than that of 3F2-mIL12-3A-mIFNa4, indicating that fusion of interferon alpha (e.g., IFNa4) may not further promote the tumor inhibitory effect of 3F2-mIL12-3A.

The above results showed that the two fusion proteins (3F2-mIL12-3A and 3F2-mIL12-3A-mIFNa4) exhibited a significantly better tumor growth inhibitory effect than PDL1-3F2 IgG1, in PD-1/PD-L1 double humanized mice. In addition, these fusion proteins had no obvious toxic effects in mice.

In a different experiment, the in vivo efficacy of 3F2-mIL12-3A was determined by similar experiments as described above. PD-1/PD-L1 double humanized mice (5-8 weeks) injected with mouse colon cancer cell MC38-hPD-L1 were used to construct the tumor-bearing mouse model. The grouping and dosing schedule are shown in the table below.

Table 8.

Consistent results were obtained as compared to the previous experiment. The weight of the mice was monitored during the entire treatment period. The weight of mice in different groups all increased, with no statistically significant differences (P > 0.05) between groups. On the grouping day (Day 0; or “D0” ) , the average weight of each group was in a range of 19.2 g-19.3 g. On Day 21 (21 days after grouping) , the average weight of each group was in a range of 22.8 g-24.2 g, making the weight changes being in a range of 113.8%-125.6%. The weight change results confirmed that the antibodies and fusion proteins used were non-toxic. According to the tumor size (FIG. 8) on Day 21, the fusion protein (3F2-mIL12-3A) exhibited significantly better tumor growth inhibitory effect as compared to PDL1-3F2 IgG1, in the PD-1/PD-L1 double humanized mice.

The mouse tumor volume and survival rate after Day 21 were continued to be monitored. Euthanasia was performed when the tumor volume of the mouse reached 3000 mm ³. On Day 35, all mice in G1-G3 groups died; while the G4 and G5 groups had better survival rates, with tumor disappeared in 1 and 3 mice, respectively. At the end of the experiment on Day 70, all mice in G1-G3 group died; in G4 group, 2 mice survived and tumor disappeared in 1 mouse; in G5 group, 4 mice survived and tumor disappeared in all 4 mice. The survival curves were drawn according to the Kaplan-Meier rule (shown in FIG. 9) , which indicated that the fusion protein (3F2-mIL12-3A) can significantly improve the survival period of tumor-bearing mice as compared to PDL1-3F2 IgG1, and the effect of the high-dosage group (G5) was better than that of the low-dosage group (G4) .

In another similar experiment, the MC38-hPDL1 cells were replaced with B16F10-hPDL1 (a murine melanoma cell expressing human PD-L1) for injection, to evaluate the in vivo efficacy of 3F2-mIL12-3A in a melanoma model. The grouping and dosing schedule are shown in the table below.

Table 9.

The weight of the mice was monitored during the entire treatment period. The weight of mice in different groups all increased, with no statistically significant differences (P > 0.05) between groups. On the day of grouping (Day 0; or “D0” ) , the average weight of each group was in a range of 19.7 g-20.0 g. On Day 20 (20 days after grouping) , the average weight of each group was in a range of 23.1 g-26.7 g, making the weight changes being in a range of 117.3%-136.0%. Thus, the results confirmed again that the used antibodies and fusion proteins were non-toxic.

The table below summarizes the results for this experiment, including the tumor volumes on the day of grouping (Day 0) , 13 days after the grouping (Day 13) , and 20 days after the grouping (Day 20) ; the survival rate of the mice; the Tumor Growth Inhibition value (TGI _TV) ; and the statistical differences (P value) of tumor volume between the treatment and control groups.

Table 10.

According to the tumor size (FIG. 10) on Day 20, the fusion protein (3F2-mIL12-3A) exhibited significantly better tumor growth inhibitory effect than PDL1-3F2 IgG1, in the PD-

1/PD-L1 double humanized mice.

The mouse tumor volume and survival after Day 20 were continued to be monitored. Euthanasia was performed when the tumor volume of the mouse reached 3000 mm ³. The survival curves were drawn according to the Kaplan-Meier rule (shown in FIG. 11) . Consistent results were obtained as compared to the previous experiment, which indicated that the fusion protein (3F2-mIL12-3A) can significantly improve the survival period of tumor-bearing mice as compared to PDL1-3F2 IgG1.

Example 8. In vivo pharmacological validation

Replacement of variable regions from different anti-PD-L1 antibodies

Two of the marketed anti-PD-L1 monoclonal antibodies, Avelumab (developed by Genentech, VH SEQ ID NO: 60; VL SEQ ID NO: 61) and Atezolizumab (developed by Merck/Pfizer, VH SEQ ID NO: 58; VL SEQ ID NO: 59) , were selected, and their variable regions were used to replace the anti-PD-L1 variable regions of the 3F2-mIL12-3A fusion protein according to their sequences. The fusion proteins were named Avelumab-mIL12-3A IgG1 (with heavy chain sequence set forth in SEQ ID NO: 62 and light chain sequence set forth in SEQ ID NO: 63) and Atezolizumab-mIL12-3A IgG1 (with heavy chain sequence set forth in SEQ ID NO: 64 and light chain sequence set forth in SEQ ID NO: 65) , respectively.

Similar to the previous in vivo drug efficacy experiments, the fusion proteins Avelumab-mIL12-3A IgG1 and Atezolizumab-mIL12-3A IgG1 were tested for their effect on tumor growth in vivo in a mouse model of colon carcinoma. MC38-hPD-L1 cells were injected subcutaneously in hPD-1/hPD-L1 mice. When the tumors in the mice reached about 100 mm ³, the mice were randomly placed into different groups (7 mice per group) based on the tumor volume.

In each group, hPD-1/hPD-L1 mice were injected with phosphate-buffered saline (PBS, G1) , 1 mg/kg anti-PD-L1 monoclonal antibody PDL1-3F2 IgG1 (G2) , 1 mg/kg anti-PD-L1 monoclonal antibody Avelumab IgG1 (G3) , 1 mg/kg anti-PD-L1 monoclonal antibody Atezolizumab IgG1 (G4) , 1.82 mg/kg (based on similar molar amount) 3F2-mIL12-3A (G5) , 1.82 mg/kg Avelumab-mIL12-3A IgG1 (G6) , or 1.82 mg/kg Atezolizumab-mIL12-3A IgG1 (G7) , by intraperitoneal (i.p. ) administration. The frequency of administration was twice a week (4 administrations in total) . Details are shown in the table below.

Table 11.

The weight of the mice was monitored during the entire treatment period. The weight of mice in different groups all increased, with no statistically significant differences (P > 0.05) between groups. On the day of grouping (Day 0; or “D0” ) , the average weight of each group was in a range of 19.4 g-19.8 g. On Day 25 (25 days after grouping) , the average weight of each group was in a range of 21.8 g-25.0 g, making the weight changes being in a range of 108.5%-121.1%. The results indicated that the anti-PD-L1 antibodies and fusion proteins were well tolerated and not toxic to the mice.

The tumor size in groups treated with the antibodies is shown in FIG. 12. The table below summarizes the results for this experiment, including the tumor volumes on the day of grouping (Day 0) , 14 days after the grouping (Day 14) , and 25 days after the grouping (Day 25) ; number of tumor-free mice; the survival rate of the mice; the Tumor Growth Inhibition value (TGI _TV) ; and the statistical differences (P value) of body weight and tumor volume between the treatment and control groups.

Table 12.

The results showed that, all fusion proteins 3F2-mIL12-3A (G5) , Avelumab-mIL12-3A IgG1 (G6) and Atezolizumab-mIL12-3A IgG1 (G7) inhibited tumor growth with a higher TGI _TV% (e.g., on Day 25) than that of the monoclonal antibodies (G2, G3, and G4) . In particular, all mice in the G5, G6, and G7 groups survived on Day 25 and the tumor in one mouse in G6 group disappeared completely. The results indicate that fusion proteins having different anti-PD-L1 antigen-binding fragments (e.g., VH/VL) that are fused with IL12 can be generated to obtain better anti-tumor efficacy.

The mouse tumor volume and survival after Day 25 were continued to be monitored. Euthanasia was performed when the tumor volume of the mouse reached 3000 mm ³. At the end of the experiment on Day 46, all mice in groups G1-G5 died; 6 mice survived in G6 group with tumor disappeared in 2 mice; 5 mice survived in G7 mice with tumor disappeared in 2 mice. The survival curves were drawn according to the Kaplan-Meier rule (shown in FIG. 13) . Consistent results were obtained as compared to the previous experiments, which indicated that all the fusion proteins (G5, G6, and G7) can significantly improve the survival period of tumor-bearing mice as compared to the monoclonal antibodies (G2, G3, and G4) .

Replacement of variable regions from anti-PD-L1 antibodies with different PD-L1-binding affinities

Anti-human PD-L1 antibodies with different binding affinities to human PD-L1 (hPD-L1) were selected: 30-2C12-IgG1 (VH SEQ ID NO: 66; VL SEQ ID NO: 67, KD against hPD-L1: 5.85 × 10 ^-8 M) , 23-2A6-IgG1 (VH SEQ ID NO: 68; VL SEQ ID NO: 69, KD against hPD-L1: 5.19 × 10 ^-9 M) , and 23-10A11-IgG1 (VH SEQ ID NO: 70; VL SEQ ID NO: 71, KD against hPD-L1: 6.36 × 10 ^-8 M) . The VH and VL of these antibodies were used to replace the corresponding anti-PD-L1 variable regions of the 3F2-mIL12-3A fusion protein according to their sequences. The fusion proteins were named PL1-2C12-mIL12-3A-IgG1 (with heavy chain sequence set forth in SEQ ID NO: 72 and light chain sequence set forth in SEQ ID NO: 73) , PL1-2A6-mIL12-3A-IgG1 (with heavy chain sequence set forth in SEQ ID NO: 74 and light chain sequence set forth in SEQ ID NO: 75) , and PL1-10A11-mIL12-3A-IgG1 (with heavy chain sequence set forth in SEQ ID NO: 76 and light chain sequence set forth in SEQ ID NO: 77) , respectively.

Similar to the previous in vivo drug efficacy experiments, the fusion proteins 3F2-mIL12-3A, PL1-2C12-mIL12-3A-IgG1, PL1-2A6-mIL12-3A-IgG1, and PL1-10A11-mIL12-3A-IgG1 were tested for their effect on tumor growth in vivo in a mouse model of colon carcinoma. MC38-hPD-L1 cells were injected subcutaneously in hPD-1/hPD-L1 mice. When the tumors in the mice reached about 200 mm ³, the mice were randomly placed into different groups (7 mice per group) based on the tumor volume.

In each group, hPD-1/hPD-L1 mice were injected with phosphate-buffered saline (PBS, G1) , 1 mg/kg anti-PD-L1 monoclonal antibody PDL1-3F2 IgG1 (G2) , 1 mg/kg anti-PD-L1 monoclonal antibody 30-2C12-IgG1 (G3) , 1 mg/kg anti-PD-L1 monoclonal antibody 23-2A6-IgG1 (G4) , 1 mg/kg anti-PD-L1 monoclonal antibody 23-10A11-IgG1 (G5) , 1.82 mg/kg (based on similar molar amount) 3F2-mIL12-3A (G6) , 1.82 mg/kg PL1-2C12-mIL12-3A-IgG1 (G7) , 1.82 mg/kg PL1-2A6-mIL12-3A-IgG1 (G8) , or 1.82 mg/kg PL1-10A11-mIL12-3A-IgG1 (G9) , by intraperitoneal (i.p. ) administration. The frequency of administration was twice a week (4 administrations in total) . Details are shown in the table below.

Table 13.

The weight of the mice was monitored during the entire treatment period. The weight of mice in different groups all increased, with no statistically significant differences (P > 0.05) between groups. On the day of grouping (Day 0; or “D0” ) , the average weight of each group was in a range of 19.4 g-19.7 g. On Day 21 (21 days after grouping) , the average weight of each group was in a range of 21.3 g-24.7 g, making the weight changes being in a range of 109.1%-125.5%. The results indicated that the anti-PD-L1 antibodies and fusion proteins were well tolerated and not toxic to the mice.

The tumor size in groups treated with the antibodies and fusion proteins is shown in FIG. 14. The table below summarizes the results for this experiment, including the tumor volumes on the day of grouping (Day 0) , 14 days after the grouping (Day 14) , and 21 days after the grouping (Day 21) ; the survival rate of the mice; the Tumor Growth Inhibition value (TGI _TV) ; and the statistical differences (P value) of body weight and tumor volume between the treatment and control groups.

Table 14.

The results showed that, all fusion proteins 3F2-mIL12-3A (G6) , PL1-2C12-mIL12-3A-IgG1 (G7) , PL1-2A6-mIL12-3A-IgG1 (G8) , and PL1-10A11-mIL12-3A-IgG1 (G9) inhibited tumor growth with a higher TGI _TV% (e.g., on Day 21) than that of the monoclonal antibodies (G2, G3, G4, and G5) . In particular, all mice in the G6-G8 group survived and the tumor in one mouse in G5 disappeared completely. The results indicate that anti-PD-L1 monoclonal antibodies with different PD-L1-binding affinities, after fused with IL12, can effectively improve the anti-tumor efficacy as compared with the monoclonal antibodies.

The mouse tumor volume and survival after Day 21 were continued to be monitored. Euthanasia was performed when the tumor volume of the mouse reached 3000 mm ³. On Day 35, all mice died in groups G1-G3; mice in groups G7-G9 survived better, with tumor disappeared in 1 mouse in G7 group, 2 mice in G8 group, and 2 mice in G9 group. At the end of the experiment on Day 81, all mice died in groups G1-G6; 4 mice survived in group G7 with tumor disappeared in 3 mice; 3 mice survived in group G8 with tumor disappeared in 2 mice; 6 mice survived in group G9 with tumor disappeared in 5 mice. The survival curves were drawn according to the Kaplan-Meier rule (shown in FIG. 15) . Consistent results were obtained as compared to the previous experiments, which indicated that all the fusion proteins (G6, G7, G8, and G9) can significantly improve the survival period of tumor-bearing mice as compared to the monoclonal antibodies (G2, G3, G4, and G5) .

Replacement of variable regions from anti-PD-L1 antibodies with different antigen-binding epitopes

Anti-human PD-L1 antibodies with different antigen-binding epitopes than PDL1-3F2 IgG1 were selected: 30-2C12-IgG1 (VH SEQ ID NO: 66; VL SEQ ID NO: 67) , 23-2A5-IgG1 (VH SEQ ID NO: 78; VL SEQ ID NO: 79) , and 17-1C9-IgG1 (VH SEQ ID NO: 80; VL SEQ ID NO: 81) . The VH and VL of these antibodies were used to replace the corresponding anti-PD-L1 variable regions of the 3F2-mIL12-3A fusion protein according to their sequences. As shown in FIG. 16 and FIG. 17, the epitopes of these antibodies are not identical or do not completely overlap with PDL1-3F2 IgG1. The fusion proteins were named PL1-2C12-mIL12-3A-IgG1, PL1-2A5-mIL12-3A-IgG1 (with heavy chain sequence set forth in SEQ ID NO: 82 and light chain sequence set forth in SEQ ID NO: 83) , and PL1-1C9-mIL12-3A-IgG1 (with heavy chain sequence set forth in SEQ ID NO: 84 and light chain sequence set forth in SEQ ID NO: 85) , respectively.

Similar to the previous in vivo drug efficacy experiments, the fusion proteins 3F2-mIL12-3A, PL1-2C12-mIL12-3A-IgG1, PL1-2A5-mIL12-3A-IgG1, and PL1-1C9-mIL12-3A-IgG1 were tested for their effect on tumor growth in vivo in a mouse model of colon carcinoma. MC38-hPD-L1 cells were injected subcutaneously in hPD-1/hPD-L1 mice. When the tumors in the mice reached about 100 mm ³, the mice were randomly placed into different groups (7 mice per group) based on the tumor volume.

In each group, hPD-1/hPD-L1 mice were injected with phosphate-buffered saline (PBS, G1) , 1 mg/kg anti-PD-L1 monoclonal antibody PDL1-3F2 IgG1 (G2) , 1 mg/kg anti-PD-L1 monoclonal antibody 30-2C12-IgG1 (G3) , 1 mg/kg anti-PD-L1 monoclonal antibody 23-2A5-IgG1 (G4) , 1 mg/kg anti-PD-L1 monoclonal antibody 17-1C9-IgG1 (G5) , 1.82 mg/kg (based on similar molar amount) 3F2-mIL12-3A (G6) , 1.82 mg/kg PL1-2C12-mIL12-3A-IgG1 (G7) , 1.82 mg/kg PL1-2A5-mIL12-3A-IgG1 (G8) , or 1.82 mg/kg PL1-1C9-mIL12-3A-IgG1 (G9) , by intraperitoneal (i.p. ) administration. The frequency of administration was twice a week (4 administrations in total) . Details are shown in the table below.

Table 15.

The weight of the mice was monitored during the entire treatment period. The weight of mice in different groups all increased, with no statistically significant differences (P > 0.05) between groups. On the day of grouping (Day 0; or “D0” ) , the average weight of each group was in a range of 18.5 g-19.2 g. On Day 28 (28 days after grouping) , the average weight of each group was in a range of 21.9 g-24.3 g, making the weight changes being in a range of 116.3%-127.9%. The results indicated that the anti-PD-L1 antibodies and fusion proteins were well tolerated and not toxic to the mice.

The tumor size in groups treated with the antibodies and fusion proteins is shown in FIG. 18. The table below summarizes the results for this experiment, including the tumor volumes on the day of grouping (Day 0) , 17 days after the grouping (Day 17) , and 28 days after the grouping (Day 28) ; the survival rate of the mice; the Tumor Growth Inhibition value (TGI _TV) ; and the statistical differences (P value) of body weight and tumor volume between the treatment and control groups.

Table 16.

The results showed that, all fusion proteins 3F2-mIL12-3A (G6) , PL1-2C12-mIL12-3A-IgG1 (G7) , PL1-2A5-mIL12-3A-IgG1 (G8) , and PL1-1C9-mIL12-3A-IgG1 (G9) inhibited tumor growth with a higher TGI _TV% (e.g., on Day 28) than that of the monoclonal antibodies (G2, G3, G4, and G5) . The results indicate that anti-PD-L1 monoclonal antibodies with different antigen-binding epitopes, after fused with IL12, can effectively improve the anti-tumor efficacy as compared with the monoclonal antibodies.

The mouse tumor volume and survival after Day 28 were continued to be monitored. On Day 49, all mice died in groups G1, G4, and G5; in contrast, G7, G8, and G9 groups had better survival rates with tumor disappeared in 4, 3, and 3 mice, respectively. At the end of the experiment on Day 80, all mice died in groups G1-G5; 2 mice survived in group G6 with tumor disappeared in 1 mouse; 4 mice survived in group G7 with tumor disappeared in all 4 mice; 4 mice survived in group G8 with tumor disappeared in 3 mice; 3 mice survived in group G9 with tumor disappeared in 2 mice. The survival curves were drawn according to the Kaplan-Meier rule (shown in FIG. 19) . Consistent results were obtained as compared to the previous experiments, which indicated that all the fusion proteins (G6, G7, G8, and G9) can significantly improve the survival period of tumor-bearing mice as compared to the monoclonal antibodies (G2, G3, G4, and G5) .

Dose tolerance and rechallenge test

Similar to the previous in vivo drug efficacy experiments, the fusion protein 3F2-mIL12-3A was tested for its inhibitory effect on tumor growth in vivo in a mouse model of colon carcinoma. MC38-hPD-L1 cells were injected subcutaneously in hPD-1/hPD-L1 mice. When the tumors in the mice reached a volume of about 150 mm ³, the mice were randomly placed into different groups (5 mice per group) based on the tumor volume.

In each group, hPD-1/hPD-L1 mice were injected with phosphate-buffered saline (PBS, G1) , 0.1-30 mg/kg 3F2-mIL12-3A, by intraperitoneal (i.p. ) administration. The frequency of administration was twice a week (3 administrations in total) . Details are shown in the table below.

Table 17.

The weight of the mice was monitored during the entire treatment period. The weight of mice in different groups all increased (FIG. 20) , but the high-dosage group (G6, G7, and G8) showed significant weight loss under the dosing conditions described above. On the day of grouping (Day 0; or “D0” ) , the average weight of each group was in a range of 20.2 g-20.6 g. On Day 24 (24 days after grouping) , the average weight of each group was in a range of 20.8 g-25.6 g, making the weight changes being in a range of 116.3%-127.9%. The results indicated that the fusion protein 3F2-mIL12-3A at doses below 10 mg/kg was well tolerated and not toxic to the mice, while high dosage group (≥ 10 mg/kg) showed toxicity after administration. The mouse serum samples on Day 3 were collected to determine the IFN-γ level by ELISA (e.g., using Mouse Interferon gamma ELISA Kit (IFNG) , ABCAM, Cat#: ab100689) . As shown in FIG. 21, the results showed that the level of IFN-γ in the control group (G1) and the treatment groups with a dose level no higher than 3 mg/kg (G2-G5) was lower, while the level of IFN-γ in the high dosage groups (G6-G8) increased significantly, indicating a higher toxicity.

The tumor size in groups treated with the fusion proteins is shown in FIG. 22. The results showed that, fusion protein 3F2-mIL12-3A was almost ineffective at a dose level of 0.1 mg/kg (G2) , was effective at a dose level above 0.3 mg/kg, and showed a significant dose-response relationship with an increase of dosing. However, when the dose level was increased to 10 mg/kg or higher, the tumor suppressive effect of the high dosage groups (G6, G7, and G8) was similar.

The mouse tumor volume and survival after Day 24 were continued to be monitored. At the end of the experiment on Day 45, all mice in G1 and G2 groups died; there were some mice survived in the G4-G8 groups, and tumor disappeared in at least one mouse in these groups. The survival curves were drawn according to the Kaplan-Meier rule (shown in FIG. 23) . The results indicate that the fusion protein 3F2-mIL12-3A can significantly improve the survival period of tumor-bearing mice when administered at a dose level no less than 0.3 mg/kg.

The table below summarizes the results for this experiment, including the tumor volumes on the day of grouping (Day 0) , 17 days after the grouping (Day 17) , and 24 days after the grouping (Day 24, or “D24” ) ; number of tumor-free mice on Day 45 (D45) ; the survival rate of the mice on Day 45; the Tumor Growth Inhibition value (TGI _TV) ; and the statistical differences (P value) of body weight and tumor volume between the treatment and control groups.

Table 18.

The results showed that fusion protein 3F2-mIL12-3A can inhibit tumor growth with a TGI _TV% (e.g., on Day 24) higher than 90%when administered with a dose level of 3 mg/kg or higher (G5, G6, G7, and G8) .

In a different experiment, 6 hPD-1/hPD-L1 mice (G1) and 6 of the tumor-regression mice obtained above (G2) were selected. 5 × 10 ⁵ MC38-hPD-L1 cells were injected subcutaneously to each mouse and tumor growth in each mouse was observed (FIG. 24) . The observation lasted for 25 days. The results showed that the tumors in the G1 group mice could grow normally, but no tumor growth was observed in the G2 group mice. The results indicate that the immune response elicited by 3F2-mIL12-3A treatment has a long-term protective effect (with immune memory) .

Replacement of variable regions from anti-PD-L1 antibodies with minimal or no blocking activity

Anti-human PD-L1 antibodies that can interfere with the activity to the PD-1/PD-L1 interaction (BC-B2E5) or with no blocking activity to the PD-1/PD-L1 interaction (BC-B1C7, and BC-A8C2) were selected. The VH and VL of these antibodies were used to replace the corresponding anti-PD-L1 variable regions of the 3F2-mIL12-3A fusion protein according to their sequences. The fusion proteins were named PL1-B2E5-mIL12-3A-IgG1, PL1-B1C7-mIL12-3A-IgG1, and PL1-A8C2-mIL12-3A-IgG1, respectively.

Similar to the previous in vivo drug efficacy experiments, the fusion proteins PL1-B2E5-mIL12-3A-IgG1, PL1-B1C7-mIL12-3A-IgG1, and PL1-A8C2-mIL12-3A-IgG1 were tested for their in vivo toxicity in a mouse model of colon carcinoma. MC38-hPD-L1 cells were injected subcutaneously in hPD-1/hPD-L1 mice. When the tumors in the mice reached about 100 mm ³, the mice were randomly placed into different groups based on the tumor volume.

The body weight results showed that PL1-B2E5-mIL12-3A-IgG1 was well tolerated and not toxic to the mice. However, treatment of PL1-B1C7-mIL12-3A-IgG1 and PL1-A8C2-mIL12-3A-IgG1 showed some toxicity. The results indicate that IL12 conjugated to an PD-L1 targeting moiety with an interference of the PD-1/PD-L1 interaction can reduce the toxicity of the IL12. It is contemplated that blocking (or interfering) the PD-1/PD-L1 interaction can activate the PD-1-expressing T cells, which may increase the expression of IL12R. These cells can be further activated by IL12. However, because PL1-B1C7-mIL12-3A-IgG1 and PL1-A8C2-mIL12-3A-IgG1 cannot block the PD-1/PD-L1 interaction, the PD-1-expressing T cells were not activated and the off-target IL12 exhibited similar toxicity as compared to MOCK-mIL12-3A-IgG1.

Example 9. IL12 fused at different positions of anti-PD-L1 antibodies

Antibody preparation

In addition to the schematic structure shown in FIG. 1 where IL12 is fused to the 3A site of an anti-PD-L1 antibody, IL12 can also be fused at other positions of an anti-PD-L1 antibody, e.g., the C-terminus of the heavy chain (schematic diagram shown in FIG. 25A) , the C-terminus of the light chain (schematic diagram shown in FIG. 25B) , the N-terminus of both the heavy chain and the light chain (schematic diagram shown in FIG. 25C) , and the N-terminus of the light chain (schematic diagram shown in FIG. 25D) . For example, when mIL12 was fused to PDL1-3F2 IgG1 with a schematic structure shown in FIG. 25A (the HC structure) , the obtained fusion protein was named 3F2-mIL12-HC-IgG1 (as discussed in Example 3) . When mIL12 was fused to PDL1-3F2 IgG1 with a schematic structure shown in FIG. 25B (the CK structure) , the obtained fusion protein was named 3F2-mIL12-CK-IgG1 (as discussed in Example 3) . When mIL12 was fused to PDL1-3F2 IgG1 with a schematic structure shown in FIG. 25C (the NHK structure) , the obtained fusion protein was named mIL12-3F2-IgG1 (with heavy chain sequence set forth in SEQ ID NO: 90 and light chain sequence set forth in SEQ ID NO: 91) . When mIL12 was fused to PDL1-3F2 IgG1 with a schematic structure shown in FIG. 25D (the NK structure) , the obtained fusion protein was named mIL12-NK-3F2-IgG1 (with heavy chain sequence set forth in SEQ ID NO: 92 and light chain sequence set forth in SEQ ID NO: 93) .

The fusion proteins were purified by protein A chromatography and analyzed by a native gel. As shown in FIGS. 26A-26D, 3F2-mIL12-CK-IgG1 (FIG. 26A, Lane 1) , 3F2-mIL12-HC-IgG1 (FIG. 26A, Lane 5) , mIL12-3F2-IgG1 (FIG. 26B, Lane 10) and mIL12-NK-3F2-IgG1 (FIG. 26C, Lane 1) each presented a clear band, indicating that these modified antibodies had relatively high purity.

In vivo pharmacological validation

Similar to the previous in vivo drug efficacy experiments, the fusion proteins 3F2-mIL12-3A, mIL12-3F2-IgG1, mIL12-NK-3F2-IgG1, 3F2-mIL12-HC-IgG1, 3F2-mIL12-CK-IgG1 and MOCK-mIL12-3A-IgG1 were tested for their effect on tumor growth in vivo in a mouse model of colon carcinoma. MOCK-mIL12-3A-IgG1 was obtained by replacing the anti-PD-L1 antibody variable region sequences of 3F2-mIL12-3A with the VH and VL sequences of an unrelated antibody (i.e., an antibody that does not target PD-L1 or PD-1) .

MC38-hPD-L1 cells were injected subcutaneously in hPD-1/hPD-L1 mice. When the tumors in the mice reached a volume of about 100 mm ³, the mice were randomly placed into different groups (7 mice per group) based on the tumor volume.

In each group, hPD-1/hPD-L1 mice were injected with phosphate-buffered saline (PBS, G1) , 1 mg/kg anti-PD-L1 monoclonal antibody PDL1-3F2 IgG1 (G2) , 1.82 mg/kg (based on similar molar amount) 3F2-mIL12-3A (G3) , 1.82 mg/kg mIL12-3F2-IgG1 (G4) , 1.82 mg/kg mIL12-NK-3F2-IgG1 (G5) , 1.82 mg/kg 3F2-mIL12-HC-IgG1 (G6) , 1.82 mg/kg 3F2-mIL12-CK-IgG1 (G7) , or 1.82 mg/kg MOCK-mIL12-3A-IgG1 (G8) , by intraperitoneal (i.p. ) administration. The frequency of administration was twice a week (4 administrations in total) . Details are shown in the table below.

Table 19.

The weight of the mice was monitored during the entire treatment period. The weight of mice in different groups all increased (FIG. 27) . The results showed that MOCK-mIL12-3A-IgG1 (G8) exhibited significant weight loss under the above-mentioned dosing regimen, indicating that MOCK-mIL12-3A-IgG1 was toxic in mice. On the day of grouping (Day 0; or “D0” ) , the average weight of each group was in a range of 20.8 g-21.1 g. On Day 31 (31 days after grouping) , the average weight of each group was in a range of 23.5 g-27.8 g, making the weight changes being in a range of 114.2%-132.5%. The results indicate that all the anti-PD-L1 antibody and fusion proteins (other than MOCK-mIL12-3A-IgG1 in group G8) were well tolerated and not toxic to the mice.

The tumor size in groups treated with the antibodies and fusion proteins is shown in FIG. 28. The table below summarizes the results for this experiment, including the tumor volumes on the day of grouping (Day 0) , 17 days after the grouping (Day 17) , and 31 days after the grouping (Day 31) ; number of tumor-free mice; the survival rate of the mice; the Tumor Growth Inhibition value (TGI _TV) ; and the statistical differences (P value) of tumor volume between the treatment and control groups.

Table 20.

The results showed that, all fusion proteins 3F2-mIL12-3A (G3) , mIL12-3F2-IgG1 (G4) , mIL12-NK-3F2-IgG1 (G5) , 3F2-mIL12-HC-IgG1 (G6) , 3F2-mIL12-CK-IgG1 (G7) , and MOCK-mIL12-3A-IgG1 (G8) can inhibited tumor growth with a higher TGI _TV% (e.g., on Day 31) than that of the monoclonal antibody (G2) . In particular, all mice in the G3-G7 group survived and the tumor in at least one mouse in this groups disappeared completely. The results indicate that fusion of IL12 at various positions of an anti-PD-L1 antibody can exert better anti-tumor effect. In addition, fusion protein MOCK-mIL12-3A-IgG1 (G8) is functionally equivalent to an IL12 protein, which also showed good anti-tumor efficacy in mice, but with obvious toxicity.

The mouse tumor volume and survival after Day 31 were continued to be monitored. On Day 45, all mice died in groups G1 and G2; in the G3-G8 groups, only one mouse died in the G5 and G6 groups, while the rest of the mice survived and the tumors in many mice completely disappeared. At the end of the experiment on Day 77, all mice died in groups G1 and G2; 1 mouse survived in group G3 with tumor disappeared; 2 mice survived in group G4 with tumor disappeared in all 2 mice; 4 mice survived in group G5 with tumor disappeared in all 4 mice; 3 mice survived in group G6 with tumor disappeared in all 3 mice; 4 mice survived in group G7 with tumor disappeared in all 4 mice; 2 mice survived in group G8 with tumor disappeared in all 2 mice. The survival curves were drawn according to the Kaplan-Meier rule (shown in FIG. 29) .

Example 10. Replacement of anti-PD-L1 antibody sequences with anti-PD-1 antibody sequences

In this experiment, the VH and VL sequences of the anti-PD-L1 arm of the fusion proteins described herein (e.g., as shown in FIG. 1) were replaced with the VH and VL sequences of anti-PD-1 antibody 25-1A7 (PD1-25-1A7, VH SEQ ID NO: 94; VL SEQ ID NO: 95) . The fusion proteins were named PD1-1A7-mIL12-3A-IgG4 (with heavy chain sequence set forth in SEQ ID NO: 96 and light chain sequence set forth in SEQ ID NO: 97) .

Similar to the previous in vivo drug efficacy experiments, the fusion proteins PD1-1A7-mIL12-3A-IgG4 and MOCK-mIL12-3A-IgG4-S228P were tested for their effect on tumor growth in vivo in a mouse model of colon carcinoma. MOCK-mIL12-3A-IgG4-S228P was obtained by replacing the anti-PD-L1 antibody variable region sequences of 3F2-mIL12-3A with the VH and VL sequences of an unrelated antibody (i.e., an antibody that does not target PD-L1 or PD-1) , and also replacing the IgG1 subclass to IgG4 having a S228P mutation (IgG4-S228P) .

MC38-hPD-L1 cells were injected subcutaneously in hPD-1/hPD-L1 mice. When the tumors in mice reached a volume of about 100 mm ³, the mice were randomly placed into different groups (7 mice per group) based on the tumor volume.

In each group, hPD-1/hPD-L1 mice were injected with phosphate-buffered saline (PBS, G1) , 1 mg/kg anti-PD1 monoclonal antibody PD1-25-1A7-IgG4-S228P (G2) , 1.82 mg/kg (based on similar molar amount) MOCK-mIL12-3A-IgG4-S228P (G3) , a combination of 1 mg/kg PD1-25-1A7-IgG4-S228P and 1.82 mg/kg MOCK-mIL12-3A-IgG4-S228P (G4) , or 1.82 mg/kg PD1-1A7-mIL12-3A-IgG4 (G5) , by intraperitoneal (i.p. ) administration. The frequency of administration was twice a week (6 administrations in total) . Details are shown in the table below.

Table 21.

The weight of the mice was monitored during the entire treatment period. On the day grouping (Day 0; or “D0” ) , the average weight of each group was in a range of 19.2 g-19.4 g. At the end of the experiment (20 days after grouping; Day 20) , the average weight of each group was in a range of 20.2 g-22.5 g, making the weight changes being in the range of 104.1%-117.0%. The weight of mice in different groups all increased, but weight loss of mice was observed in G3, G4, and G5 groups (FIG. 30) . The results showed that the anti-PD-1 monoclonal antibody (G2) was well tolerated and not toxic to the mice. However, treatment of MOCK-mIL12-3A-IgG4-S228P (G3 and G4) and PD1-1A7-mIL12-3A-IgG4 (G5) showed some toxicity.

The tumor size in groups treated with the antibodies, fusion proteins, or combinations thereof is shown in FIG. 31. The table below summarizes the results for this experiment, including the tumor volumes on the day of grouping (Day 0) , 16 days after the grouping (Day 16) , and at the end of the experiment (Day 20) ; the survival rate of the mice; the Tumor Growth Inhibition value (TGI _TV) ; and the statistical differences (P value) of body weight and tumor volume between the treatment and control groups.

Table 22.

The results showed that, fusion proteins MOCK-mIL12-3A-IgG4-S228P (G3) and PD1-1A7-mIL12-3A-IgG4 (G5) can inhibit tumor growth with a higher TGI _TV% (e.g., on Day 20) than the monoclonal antibody PD1-25-1A7-IgG4-S228P (G2) . The results indicate that fusion of IL12 can increase the anti-tumor efficacy of the anti-PD-1 antibody, but obvious toxicity as MOCK-mIL12-3A-IgG4-S228P (G3) was also observed for PD1-1A7-mIL12-3A-IgG4 (G5) .

Example 11. In vivo toxicity of IL12 and efficacy of various fusion proteins

Validation of IL12 toxicity in vivo was performed. To increase the half-life of IL12, mIL12 was fused to the C-terminus of Fc (one mIL12 fused to the C-terminus of each of the two Fc heavy chains) to obtain a fusion protein named Fc-mIL12-HC. In addition, a glycosylation modification site was introduced in the Fc region of the fusion protein. For example, a N297A mutation (according to EU numbering) was introduced in the Fc region of 3F2-mIL12-3A, to reduce or eliminate the interaction of Fc with FcγR and Fc effector functions (e.g., CDC and ADCC) . The fusion protein was named 3F2-mIL12-3A-IgG1-N297A (with heavy chain sequence set forth in SEQ ID NO: 86 and light chain sequence set forth in SEQ ID NO: 87) .

Similar to the previous in vivo drug efficacy experiments, MC38-hPD-L1 cells were injected subcutaneously in hPD-1/hPD-L1 mice. When the tumors in the mice reached a volume of about 100 mm ³, the mice were randomly placed into different groups (6 mice per group) based on the tumor volume.

In each group, hPD-1/hPD-L1 mice were injected with phosphate-buffered saline (PBS, G1) , 1 mg/kg anti-PD-L1 monoclonal antibody PDL1-3F2 (PDL1-3F2 IgG1, G2) , 1.15 mg/kg (based on similar molar amount) Fc-mIL12-HC (G3) , a combination of 1 mg/kg PDL1-3F2 IgG1 and 1.15 mg/kg Fc-mIL12-HC (G4) , 1.82 mg/kg (based on similar molar amount) 3F2-mIL12-3A (G5) , 1.82 mg/kg 3F2-mIL12-3A-IgG1-N297A (G6) , or 1.82 mg/kg 3F2-mIL12-3A-IgG1-knob (G7) , by intraperitoneal (i.p. ) administration. The frequency of administration was twice a week (6 administrations in total) . Details are shown in the table below.

Table 23.

Table 24.

The weight of the mice and tumor size were monitored during the entire treatment period. As shown in FIGS. 32-34, consistent results were obtained as compared to previous experiments. Specifically, good tumor growth inhibitory effects were observed when the mice were treated with the following antibodies, fusion proteins, or combinations thereof: Fc-mIL12- HC (G3) , Fc-mIL12-HC combined with PDL1-3F2 IgG1 (G4) , 3F2-mIL12-3A (G5) , 3F2-mIL12-3A-IgG1-N297A (G6) , and 3F2-mIL12-3A-IgG1-knob (G7) . However, only the Fc-mIL12-HC treatment group (G3) and the Fc-mIL12-HC combined with PDL1-3F2 IgG1 group (G4) showed an obvious decrease in body weight, indicating that Fc-mIL12-HC has some toxicity (FIG. 32) .

In addition, 3F2-mIL12-3A-IgG1-N297A (G6) and 3F2-mIL12-3A-IgG1-knob (G7) showed slightly better tumor-suppressing effect than 3F2-mIL12-3A (G5) . On Day 3 (3 days after grouping) , mouse serum samples were collected to detect INF-γ levels by ELISA. As shown in FIG. 35, G3 and G4 groups had higher levels of INF-γ, while G2 and G5-G7 groups had the same level as G1 group. The results confirmed that the IL12 fusion protein 3F2-mIL12-3A and its various fusion protein forms can have good safety and therapeutic effects.

The mouse tumor volume and survival after Day 21 were continued to be monitored. On Day 38, all mice died in groups G1 and G2. At the end of the experiment on Day 91, all mice died in group G5; 3 mice survived in group G6 with tumor disappeared in all 3 mice; and 1 mouse survived with tumor disappeared in group G7. The survival curves were drawn according to the Kaplan-Meier rule (shown in FIG. 36) . The results indicate that in the long term, 3F2-mIL12-3A-IgG1-N297A (G6) and 3F2-mIL12-3A-IgG1-knob (G7) are significantly more potent than 3F2-mIL12-3A (G5) , indicating that weaker Fc effector functions and/or asymmetric (e.g., single or single-sided) IL12 fusion can increase the anti-tumor efficacy of the IL12 fusion proteins.

Example 12. Mechanism verification

In vivo efficacy determination in immuno-compromised mice

MC38-hPD-L1 cells were injected subcutaneously in immuno-compromised B-NDG mice (Biocytogen, Cat#: B-CM-001. When tumors in the mice reached a volume of about 100 mm ³, the mice were randomly placed into different groups (7 mice per group) based on the tumor volume.

In each group, B-NDG mice were injected with phosphate-buffered saline (PBS, G1) , 1 mg/kg anti-PD-L1 monoclonal antibody PDL1-3F2 IgG1 (G2) , 1 mg/kg Atezolizumab (Atezolizumab IgG1, G3) , 1.82 mg/kg (based on similar molar amount) 3F2-mIL12-3A (3F2-mIL12-3A IgG1, G4) , or 1.82 mg/kg Atezolizumab-mIL12-3A IgG1 (G5) by intraperitoneal (i.p. ) administration. The frequency of administration was twice a week (3 administrations in total) . Details are shown in the table below.

Table 25.

The weight of the mice was monitored during the entire treatment period (FIG. 37) . The weight of mice in different groups all increased, with no statistically significant differences (P >0.05) between groups. On the day of grouping (Day 0; or “D0” ) , the average weight of each group was in a range of 21.1 g-21.2 g. At the end of the experiment (24 days after grouping; or Day 24) , the average weight of each group was in a range of 20.1 g-21.5 g, making the weight changes being in a range of 95.7%-102.0%. The results showed that the anti-PD-L1 antibodies and fusion proteins were well tolerated and not toxic to the mice.

The tumor size in groups treated with the antibodies and fusion proteins is shown in FIG. 38. The results indicate that all the anti-PD-L1 antibodies and fusion proteins have no tumor inhibitory effect in B-NDG mice.

In vivo efficacy determination in wild-type mice

The same in vivo efficacy experiment was performed with identical dosing scheme and MC38-hPD-L1 cells were injected, but the B-NDG mice were replaced with C57BL/6 wild-type mice. The weight of the mice was monitored during the entire treatment period (FIG. 39) . The weight of mice in different groups all increased. However, the body weight of 3F2-mIL12-3A IgG1 (G4) decreased significantly. On the day of grouping (Day 0; or “D0” ) , the average weight of each group was in a range of 19.6 g-19.9 g. At the end of the experiment (28 days after grouping; or Day 28) , the average weight of each group was in a range of 20.6 g-24.7 g, making the weight changes being in a range of 105.2%-118.7%. The results indicate that, except for 3F2- mIL12-3A IgG1 (G4) , all the other anti-PD-L1 antibodies and fusion proteins were well tolerated and not toxic to the mice.

The tumor size in groups treated with the antibodies was shown in FIG. 40. The results showed that in immuno-competent C57BL/6 mice, the anti-PD-L1 antibody Atezolizumab IgG1 (G3) and its fusion protein Atezolizumab-mIL12-3A IgG1 (G5) had different degrees of tumor inhibition, and the fusion protein was more effective than the corresponding monoclonal antibody. Further, the anti-PD-L1 antibody PDL1-3F2 IgG1 (G2) exhibited no significant anti-tumor efficacy and no toxicity, while its fusion protein 3F2-mIL12-3A IgG1 (G4) was toxic and exhibited good tumor inhibition effect.

Determination of pharmacological effects in double humanized mice injected with wild-type MC38

The same in vivo efficacy experiment was performed, except that wild-type MC38 cells were injected subcutaneously in hPD-1/hPD-L1 mice. The weight of the mice was monitored during the entire treatment period (FIG. 41) . The weight of mice in different groups all increased, with no statistically significant differences (P > 0.05) between groups. On the day of grouping (Day 0; or “D0” ) , the average weight of each group was in a range of 19.2 g-19.4 g. At the end of the experiment (25 days after grouping; or Day 25) , the average weight of each group was in a range of 21.2 g-23.4 g, making the weight changes being in the range of 108.9%-121.8%. The results showed that the anti-PD-L1 antibodies and fusion proteins were well tolerated and not toxic to the mice.

The tumor size in groups treated with the antibodies was shown in FIG. 42. The results showed that in immuno-competent hPD-1/hPD-L1 mice, the anti-PD-L1 antibodies and fusion protein had different degrees of tumor inhibitory effect, and the effect of fusion protein was better than that of the corresponding monoclonal antibody.

Conclusion

The in vivo efficacy experiments as described herein demonstrated that in the hPD-1/hPD-L1 mice injected with MC38-hPD-L1 (that is, in immuno-competent mice having immune cells, and having tumor cells expressing both human and mouse PD-L1) , the tested anti-PD-L1 monoclonal antibodies and fusion proteins can exhibit pharmacological effects. However, when IL12 is not fused to an anti-PD-L1 monoclonal antibody (e.g., fusion protein Fc-mIL12-HC where IL12 is fused to Fc; fusion protein PD1-1A7-mIL12-3A-IgG4 where IL12 is fused to an anti-PD-1 monoclonal antibody; or fusion proteins MOCK-mIL12-3A-IgG1 and MOCK-mIL12-3A-IgG4-S228P, where IL12 is fused to an irrelevant antibody) , all fusion proteins are effective but also exhibit significant toxicity.

The three in vivo efficacy experiments further indicate that in the B-NDG mice injected with hPDL1-MC38 (that is, in immuno-compromised mice having no immune cells, and having tumor cells only expressing human PD-L1) , neither the monoclonal antibodies nor the fusion proteins were effective.

In addition, Atezolizumab IgG1 is a humanized therapeutic anti-PD-L1 antibody that can cross-react with both human and mouse PD-L1, while PDL1-3F2 IgG1 can only bind to human PD-L1 but not to mouse PD-L1. Thus, in wild-type C57BL/6 mice injected with MC38-hPD-L1 (that is, in immuno-competent mice having immune cells expressing mouse PD-L1, and having tumor cells expressing both human and mouse PD-L1) , the monoclonal antibody Atezolizumab IgG1 can bind to mouse PD-L1, and its fusion protein Atezolizumab-mIL12-3A IgG1 showed anti-tumor efficacy and no toxicity. However, the monoclonal antibody PDL1-3F2 IgG1, which only binds to human PD-L1, did not show significant anti-tumor efficacy or toxicity under the above dosing regimen, whereas its fusion protein 3F2-mIL12-3A IgG1 showed anti-tumor efficacy and toxicity.

In hPD-1/hPD-L1 mice injected with wild-type MC38 (that is, in immuno-competent animals having immune cells expressing human PD-L1, and having tumor cells expressing mouse PD-L1 but not human PD-L1) , similar results were obtained as compared to the hPD-1/hPD-L1 mice injected with MC38-hPD-L1, in which all anti-PD-L1 monoclonal antibodies and fusion proteins exhibited anti-tumor effects with no toxicity. Thus, the results indicate that the safety and mechanism of IL12 fusion proteins only rely on the expression of PD-L1 on immune cells (e.g., antigen-presenting cells) , but not the expression of PD-L1 on tumor cells.

Example 13. IL12 reporter cell line activation assay

Anti-PD-L1 antibody BC-2H9-IgG1 (VH SEQ ID NO: 98; VL SEQ ID NO: 99) was selected to generate a fusion protein with a schematic structure shown in FIG. 1A: PL1-2H9-mIL12-3A-IgG1 (with heavy chain sequence set forth in SEQ ID NO: 100, and light chain sequence set forth in SEQ ID NO: 101) . In this experiment, the IL12 activity in fusion proteins PL1-2H9-mIL12-3A-IgG1, Fc-mIL12-HC, and recombinant mouse IL-12 was compared. Fc-mIL12-HC and recombinant mouse IL12 (IL12A &IL12B Heterodimer Protein, Sino Biological, Cat#: CT022-9160) were serially diluted (2-fold) with the highest concentration of 150 μg/ml. PL1-2H9-mIL12-3A-IgG1 was serially diluted (5-fold) with the highest concentration of 50 μg/ml. Reporter cell line HEK-BLUE ^TM IL12 (InvivoGen, Cat#: hkb-il12) was used and cells were seeded in a 96-well plate (cell density 5×10 ⁴ cells/well) . 20 μl proteins were added to each well and incubated at 37 ℃ overnight. On the next day, QUANTI-Blue ^TM Solution (InvivoGen, Cat#: Rep-qblb3) was used to detect the OD ₆₃₀ value after processing the samples according to the manufacturer’s protocol. EC50 was calculated after data analysis, and results are shown as follows.

Table 26.

Protein Name	EC50 (nmol/L)	R2
Fc-mIL12-HC	0.231	0.9997
Recombinant Mouse IL-12	0.288	0.9861
PL1-2H9-mIL12-3A-IgG1	0.071	0.9987

The above results show that compared with the recombinant mouse IL-12, the fusion protein Fc-mIL12-HC had a slightly higher activity (i.e., a lower EC50) . However, after fusion with an anti-PD-L1 antibody at the 3A site, the IL12 activity was significantly higher than that of the free protein.

Example 14. Depletion of Fc effector function

A glycosylation modification site can also be introduced into the Fc region of the fusion proteins. For example, a N297A mutation (according to EU numbering) was introduced in the Fc region of PL1-2H9-mIL12-3A-IgG1. The fusion protein was named PL1-2H9-mIL12-3A-IgG1-N297A (with heavy chain sequence set forth in SEQ ID NO: 102, and light chain sequence set forth in SEQ ID NO: 103) .

Similar to the previous in vivo drug efficacy experiments, fusion proteins PL1-2H9-mIL12-3A-IgG1, PL1-2H9-mIL12-3A-IgG1-N297A, PD1-1A7-mIL12-3A-IgG4, Fc-mIL12- HC, and MOCK-mIL12-3A-IgG1 were tested for their inhibitory effects on tumor growth in vivo in a mouse model of colon carcinoma.

MC38-hPD-L1 cells were injected subcutaneously in hPD-1/hPD-L1 mice. When the tumors in the mice reached a volume of about 100 mm ³, the mice were randomly placed into different groups (5 mice per group) based on the tumor volume.

In each group, hPD-1/hPD-L1 mice were injected with phosphate-buffered saline (PBS, G1) , 1.82 mg/kg MOCK-mIL12-3A-IgG1 (G2) , 1.82 mg/kg (based on similar molar amount) PL1-2H9-mIL12-3A-IgG1 (G3) , 1.82 mg/kg PL1-2H9-mIL12-3A-IgG1-N297A (G4) , 1.82 mg/kg PD1-1A7-mIL12-3A-IgG4 (G5) , 1.15 mg/kg Fc-mIL12-HC (G6) , or 1 mg/kg anti-PD-L1 monoclonal antibody BC-2H9-IgG1 (G7) , by intraperitoneal (i.p. ) administration. The frequency of administration was twice a week (4 administrations in total) . Details are shown in the table below.

Table 27.

The weight of the mice was monitored during the entire treatment period. The weight of mice in different groups all increased, but G2, G5, and G6 groups showed a trend of obvious weight loss and recovery after administration (FIGS. 43) . The results indicate that PD1-1A7-mIL12-3A-IgG4 (G5) exhibited some toxicity, similar to MOCK-mIL12-3A-IgG1 (G2) and Fc-mIL12-HC (G6) . The anti-PD-L1 monoclonal antibody BC-2H9-IgG1, and other IL12 fusion proteins were well tolerated and not toxic to the mice. In addition, the blood biochemical test results (FIGS. 44A-44B) on Day 7 (7 days after grouping) also supported the above conclusion.

The tumor size in groups treated with the antibodies and fusion proteins is shown in FIG. 45. The table below summarizes the results for this experiment, including the tumor volumes on the day of grouping (Day 0) , 14 days after the grouping (Day 14) , and at the end of the experiment (Day 21) ; the survival rate of the mice; the Tumor Growth Inhibition value (TGI _TV) ; and the statistical differences (P value) of tumor volume between the treatment and control groups.

Table 28.

The results showed that, all IL12 fusion proteins including PL1-2H9-mIL12-3A-IgG1 (G3) and PL1-2H9-mIL12-3A-IgG1-N297A (G4) inhibited tumor growth with a higher TGI _TV%(e.g., on Day 21) than that of the monoclonal antibody BC-2H9-IgG1 (G7) . More specifically, PL1-2H9-mIL12-3A-IgG1 (G3) exhibited a better anti-tumor efficacy than PL1-2H9-mIL12-3A-IgG1-N297A (G4) .

Another similar experiment was performed where the MC38-hPDL1 cells were replaced with B16F10-hPDL1. The body weight measurement results are shown in FIG. 46, and the tumor volume over time is shown in FIG. 47. The blood biochemical test results on Day 7 (7 days after grouping) are shown in FIGS. 48A-48B. Consistent with previous results, fusion proteins having IL12 not fused to an anti-PD-L1 antibody (e.g., PD1-1A7-mIL12-3A-IgG4 (G5) , MOCK-mIL12-3A-IgG1 (G2) , and Fc-mIL12-HC (G6) ) showed obvious toxicity. By contrast, fusion proteins having IL12 fused to an anti-PD-L1 antibody (e.g., PL1-2H9-mIL12-3A-IgG1 (G3) , and PL1-2H9-mIL12-3A-IgG1-N297A (G4) ) showed a better anti-tumor efficacy with no toxicity. Further, the results showed that PL1-2H9-mIL12-3A-IgG1-N297A (G4) exhibited a higher tumor growth inhibitory effect than PL1-2H9-mIL12-3A-IgG1 (G3) . Given that B16F10 tumors are more malignant than MC38, B16F10-generated tumors are more difficult to treat. Thus, the results suggest that fusion proteins having an IgG subtype with weaker Fc effector functions may have a better anti-tumor efficacy.

Example 15. Binding affinity of monoclonal antibodies

PD-1/PD-L1 blocking assays (30-2C12, 23-10A11, 23-2A5, and 17-1C9)

Blocking assays were performed to determine whether the anti-PD-L1 antibodies can block the binding between PD-L1 and its ligand PD-1. Specifically, 25 μl CHO-S cells (2 × 10 ⁴ cells) transiently transfected to express human PD-L1 were added to each well in a plate. The purified antibodies were titrated to final concentrations of 50, 5, 0.5, 0.05, and 0.005 μg/ml. The titrated antibodies were added to each well at 25 μl per well at 4 ℃ and incubated for 30 minutes. After the incubation, 50 μl of Biotin-hPD-1 (Acro Biosystems, Cat#: PD1-H82E4) was added to each well (with a final concentration of 0.4 μg/ml in each well) . The cells with Biotin-hPD-1 and the antibodies were incubated at 4 ℃ for 15 minutes. After two washes with phosphate-buffered saline (PBS) , 50 μl of FITC-labeled anti-mouse IgG Fc antibody (FITC Anti-mIgG Fc, Jackson ImmunoResearch, Cat#: 115-095-003) or FITC-labeled anti-human IgG Fc antibody (FITC Anti-hIgG Fc, Jackson ImmunoResearch, Cat#: 109-096-008) at a 1: 100 dilution ratio, and PE-labeled Streptavidin (PE Streptavidin, BioLegend, Cat #: 405204) at a 1: 100 dilution ratio were added to each well, and incubated for 15 minutes at 4 ℃, followed by a PBS wash. The signals for Streptavidin (PE) and FITC were determined by flow cytometry (Thermo Attune ^TM NX) .

The table below shows the percentage of tested cells that had Streptavidin signals in the flow cytometry analysis. An antibody has blocking affinity (indicating strong binding affinity) if the percentage of tested cells that had Streptavidin signals (PE) increases while the antibody concentration decreases. Based on the data, 17-1C9-mHvKv-IgG1, 23-2A5, 23-10A11 and 30-2C12 had blocking effects.

Table 29.

Chimeric antibody affinity

The binding affinity between purified His-tagged human PD-L1 (hPL1-His; ACRO Biosystems) and 17-1C9-mHVKV-IgG1, 23-2A5-mHvKv-IgG1, 23-10A11-mHvKv-IgG1, and 30-2C12-mHvKv-IgG1 were measured by surface plasmon resonance (SPR) using Biacore ^TM (Biacore Inc., Piscataway N.J. ) T200 biosensor equipped with pre-immobilized protein A sensor chips as described in Example 5. The results for the tested fusion proteins are shown in the table below.

Table 30.

Ligand	Analysis	kon (1/Ms)	koff (1/s)	KD (M)
17-1C9-mHVKV-IgG1	hPL1-His	2.20E+05	7.08E-05	3.22E-10
23-2A5-mHvKv-IgG1	hPL1-His	1.15E+05	1.85E-04	1.61E-09
23-10A11-mHvKv-IgG1	hPL1-His	4.42E+05	6.20E-02	1.40E-07
30-2C12-mHvKv-IgG1	hPL1-His	1.09E+05	3.89E-03	3.57E-08

2H9 in vitro blocking activity and IC50 determination

The experiment was performed to test whether the antibody BC-2H9-IgG1 can block the PD-1/PD-L1 pathway.

Basal cells CHO-aAPC-hPD-L1 (Promega, Cat#: J1255) were seeded in a 96-well plate (cell density: 4×10 ⁴ cells/well) and incubated at 37 ℃ overnight. The anti-PD-L1 monoclonal antibody BC-2H9-IgG1 and Atezolizumab-IgG1-N297A were serially diluted (2-fold) with the highest concentration at 20 μg/ml. The assay buffer was RPMI 1640 medium supplemented with 10%fetal bovine serum (FBS) . On the next day, effector cells Jurkat-Luc-hPD-1 (Promega, Cat#: J1255) were centrifuged at 120 g for 10 minutes, and then seeded in a 96-well plate (cell density: 5 × 10 ⁴ cells/well) . Next, supernatant from the plate seeded with CHO-aAPC-hPD-L1 was discarded, and then 50 μl Jurkat-Luc-hPD-1 cells together with 25 μl antibody were added to each well. The above 96-well plate was incubated in a 37 ℃ incubator for 6 hours. After the incubation, the plate was taken out, and 75 μl of Bio-lite ^TM Luciferase Assay Reagent (Vazyme Biotech Co., Ltd., Cat#: DD1201-02-AB) was added to incubate at room temperature for 5-10 minutes. The plate was then placed in a luminescence detector to detect the fluorescence signal. The effector cells can report a signal if the antibody can block the interaction between PD-1 and PD-L1.

As shown in the table below, the anti-PD-L1 monoclonal antibody BC-2H9-IgG1 and Atezolizumab-IgG1-N297A exhibited a blocking effect to the PD-1/PD-L1 pathway. And the IC50 value of Atezolizumab-IgG1-N297A was relatively higher than BC-2H9-IgG1, indicating that BC-2H9-IgG1 had a more potent PD-1/PD-L1 blocking effect than Atezolizumab-IgG1-N297A.

Table 31.

Protein	IC50 (ng/mL)	R ²
BC-2H9-IgG1	65.33	0.9742
Atezolizumab-IgG1-N297A	117.9	0.9743

Additional affinity data of the antibodies or fusion proteins used in the present disclosure are also summarized in FIGS. 49-53.

OTHER EMBODIMENTS

Embodiment 1. A method of activating T cells (e.g., in a tumor microenvironment, spleen, lymph nodes, and/or lymphatic system) in a subject, the method comprising administering a therapeutically effective amount of a composition comprising a protein complex to the subject in need thereof, wherein the protein complex comprises or consists of (a) a first portion targeting Interleukin-12 receptor (IL12R) ; and (b) a second portion targeting PD-L1; wherein the protein complex activates IL12 signaling pathway in T cells that are in close proximity of tumor cells, thereby activating T cells (e.g., in the tumor microenvironment, spleen, lymph nodes, and/or lymphatic system) .

Embodiment 2. The method of embodiment 1, wherein the second portion targeting PD-L1 blocks (e.g., interferes) or does not block the interaction between PD-1 and PD-L1.

Embodiment 3. The method of

embodiment

1 or 2, wherein the second portion comprises or consists of an antibody or antigen-binding fragment.

Embodiment 4. The method of embodiment 3, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain, wherein a polypeptide of the first portion is linked to the N-terminus or C-terminus of the heavy chain.

Embodiment 5. The method of embodiment 3, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain, wherein a polypeptide of the first portion is fused to the heavy chain at a region from position 344 to position 382 of the heavy chain CH3 domain according to EU numbering.

Embodiment 6. The method of any one of embodiments 3-5, wherein the antibody or antigen-binding fragment thereof further comprises a light chain, wherein a polypeptide of the first portion is linked to the N-terminus or C-terminus of the light chain.

Embodiment 7. The method of any one of embodiments 1-6, wherein a polypeptide of the first portion is linked to a polypeptide of the second portion.

Embodiment 8. The method of any one of embodiments 1-7, wherein the protein complex further comprises a third portion (e.g., albumin) , wherein a polypeptide of the first portion and/or a polypeptide of the second portion are linked to the third portion.

Embodiment 9. The method of any one of embodiments 1-8, wherein the first portion comprises or consists of an anti-IL12R antibody or antigen-binding fragment thereof.

Embodiment 10. The method of any one of embodiments 1-8, wherein the first portion comprises or consists of IL12a and IL12b.

Embodiment 11. The method of any one of embodiments 1-10, wherein the protein complex further comprises a fourth portion, wherein the fourth portion is linked to a polypeptide of the first portion and/or a polypeptide of the second portion, optionally via a linker sequence.

Embodiment 12. The method of embodiment 11, wherein the fourth portion comprises or consists of a cytokine (e.g., IL2, IL7, IL15, IL18, and/or IL21) or an interferon (e.g., IFNa1, IFNa2, IFNa3, IFNa4, and/or IFNγ) .

Embodiment 13. A protein complex comprising or consisting of: (a) a first portion targeting Interleukin-12 receptor (IL12R) ; and (b) a second portion targeting PD-L1.

Embodiment 14. The protein complex of embodiment 13, wherein the second portion comprises or consists of an antibody or antigen-binding fragment.

Embodiment 15. The protein complex of embodiment 14, wherein the antibody or antigen-binding fragment thereof comprises or consists of a VHH.

Embodiment 16. The protein complex of embodiment 14, wherein the antibody or antigen-binding fragment thereof comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH and VH associate with each other, forming an antigen-binding site targeting PD-L1.

Embodiment 17. The protein complex of any one of embodiments 14-16, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain, wherein the heavy chain comprises an optional immunoglobulin hinge region, a CH2 domain and a CH3 domain.

Embodiment 18. The protein complex of embodiment 17, wherein a polypeptide of the first portion is linked to the N-terminus of the heavy chain, optionally via a linker sequence.

Embodiment 19. The protein complex of

embodiments

17 or 18, wherein a polypeptide of the first portion is linked to the C-terminus of the heavy chain, optionally via a linker sequence.

Embodiment 20. The protein complex of any one of embodiments 17-19, wherein a polypeptide of the first portion is fused to the heavy chain at a region from position 344 to position 382 of the heavy chain CH3 domain according to EU numbering, optionally via a linker sequence.

Embodiment 21. The protein complex of any one of embodiments 17-20, wherein the antibody or antigen-binding fragment thereof further comprises a light chain.

Embodiment 22. The protein complex of embodiment 21, wherein a polypeptide of the first portion is linked to the N-terminus of the light chain, optionally via a linker sequence.

Embodiment 23. The protein complex of

embodiment

21 or 22, wherein a polypeptide of the first portion is linked to the C-terminus of the light chain, optionally via a linker sequence.

Embodiment 24. The protein complex of any one of embodiments 13-16, wherein a polypeptide of the first portion is linked to a polypeptide of the second portion, optionally via a linker sequence.

Embodiment 25. The protein complex of any one of embodiments 13-16, further comprising a third portion (e.g., albumin) , wherein a polypeptide of the first portion and/or a polypeptide of the second portion are linked to the third portion, optionally via a linker sequence.

Embodiment 26. The protein complex of any one of embodiments 13-25, wherein the first portion comprises or consists of an anti-IL12R antibody or antigen-binding fragment thereof.

Embodiment 27. The protein complex of any one of embodiments 13-25, wherein the first portion comprises or consists of IL12a and IL12b.

Embodiment 28. The protein complex of any one of embodiments 13-17, further comprising a fourth portion, wherein the fourth portion is linked to a polypeptide of the first portion and/or a polypeptide of the second portion, optionally via a linker sequence.

Embodiment 29. The protein complex of embodiment 28, wherein the fourth portion comprises or consists of a cytokine (e.g., IL2, IL7, IL15, IL18, and/or IL21) or an interferon (e.g., IFNa1, IFNa2, IFNa3, IFNa4, and/or IFNγ) .

Embodiment 30. A fusion protein comprising IL12, wherein the fusion protein is engineered to target a tumor-associated antigen (TAA) .

Embodiment 31. The fusion protein of embodiment 30, wherein the tumor associated antigen is PD-L1.

Embodiment 32. The fusion protein of

embodiment

30 or 31, wherein the fusion protein comprises an Fc region.

Embodiment 33. The fusion protein of any one of embodiments 30-32, wherein the IL12 comprises or consists of IL12a and IL12b.

Embodiment 34. The fusion protein of embodiment 33, wherein IL12a is fused to a region from position 344 to position 382 of a CH3 domain of the Fc region according to EU numbering, and IL12b associates with IL12a.

Embodiment 35. The fusion protein of embodiment 33, wherein IL12b is linked to a region from position 344 to position 382 of a CH3 domain of the Fc region according to EU numbering, and IL12a associates with IL12b.

Embodiment 36. A protein complex, comprising or consisting of an anti-PD-L1 antibody or antigen-binding fragment thereof, and an IL12.

Embodiment 37. The protein complex of embodiment 36, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof is an immunoglobulin (e.g., IgM, IgA, IgE, IgD, or IgG) .

Embodiment 38. The protein complex of embodiment 37, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof is an IgG (e.g., IgG1 or IgG4) .

Embodiment 39. The protein complex of any one of embodiments 36-38, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises or consists of a heavy chain variable region and a light variable region.

Embodiment 40. The protein complex of any one of embodiments 36-38, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises or consists of a VHH.

Embodiment 41. The protein complex of any one of embodiments 36-40, wherein the IL12 is linked to the anti-PD-L1 antibody or antigen-binding fragment thereof.

Embodiment 42. The protein complex of any one of embodiments 36-40, wherein the IL12 is linked to a heavy chain of the anti-PD-L1 antibody or antigen-binding fragment thereof.

Embodiment 43. The protein complex of any one of embodiments 36-40, wherein the IL12 is linked to a light chain of the anti-PD-L1 antibody or antigen-binding fragment thereof.

Embodiment 44. The protein complex of any one of embodiments 36-43, wherein the IL12 comprises or consists of IL12a and IL12b.

Embodiment 45. The protein complex of embodiment 44, wherein IL12a is linked to a heavy chain of the anti-PD-L1 antibody or antigen-binding fragment thereof, and IL12b associates with IL12a.

Embodiment 46. The protein complex of embodiment 44, wherein IL12b is linked to a heavy chain of the anti-PD-L1 antibody or antigen-binding fragment thereof, and IL12a associates with IL12b.

Embodiment 47. The protein complex of embodiment 44, wherein IL12a is linked to a light chain of the anti-PD-L1 antibody or antigen-binding fragment thereof, and IL12b associates with IL12a.

Embodiment 48. The protein complex of embodiment 44, wherein IL12b is linked to a light chain of the anti-PD-L1 antibody or antigen-binding fragment thereof, and IL12a associates with IL12b.

Embodiment 49. The protein complex of any one of embodiments 36-48, wherein the protein complex further comprises an interferon.

Embodiment 50. The protein complex of embodiment 49, wherein the interferon is IFNa4.

Embodiment 51. The protein complex of

embodiment

49 or 50, wherein the interferon is linked to a heavy chain of the anti-PD-L1 antibody or antigen-binding fragment thereof.

Embodiment 52. The protein complex of

embodiment

49 or 50, wherein the interferon is linked to a light chain of the anti-PD-L1 antibody or antigen-binding fragment thereof.

Embodiment 53. The protein complex of any one of embodiments 36-38, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises or consists of: a first heavy chain polypeptide comprising a first heavy chain CH3 domain, a second heavy chain polypeptide comprising a second heavy chain CH3 domain, a first light chain polypeptide, and a second light chain polypeptide; wherein the first heavy chain polypeptide and the first light chain polypeptide associate with each other, forming a first antigen-binding region targeting PD-L1; wherein the second heavy chain polypeptide and the second light chain polypeptide associate with each other, forming a second antigen-binding region targeting PD-L1; wherein the IL12 comprises or consists of: a first interleukin 12 subunit alpha (IL12a) ; and a first interleukin 12 subunit beta (IL12b) that associates with the first IL12a.

Embodiment 54. The protein complex of embodiment 53, wherein the first IL12a is fused to the first heavy chain polypeptide at a region from position 344 to position 382 of the first heavy chain CH3 domain according to EU numbering.

Embodiment 55. The protein complex of embodiment 53, wherein the first IL12b is fused to the first heavy chain polypeptide at a region from position 344 to position 382 of the first heavy chain CH3 domain according to EU numbering.

Embodiment 56. The protein complex of embodiment 54, wherein the first IL12a is linked to a first amino acid residue and a second amino acid residue of the first heavy chain CH3 domain, wherein the first and the second amino acid residues are selected from the group consisting of amino acid residues at positions 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, and 383 of the first heavy chain CH3 domain according to EU numbering.

Embodiment 57. The protein complex of embodiment 54, wherein the first IL12a is fused to the first heavy chain polypeptide at a region from position 351 to position 362 of the first heavy chain CH3 domain according to EU numbering.

Embodiment 58. The protein complex of embodiment 57, wherein the first IL12a is linked to a first amino acid residue and a second amino acid residue of the first heavy chain CH3 domain, wherein the first and the second amino acid residues are selected from the group consisting of amino acid residues at positions 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, and 363 of the first heavy chain CH3 domain according to EU numbering.

Embodiment 59. The protein complex of embodiment 54, wherein the first IL12a is fused to the first heavy chain polypeptide at a region from position 358 to position 362 of the first heavy chain CH3 domain according to EU numbering.

Embodiment 60. The protein complex of embodiment 59, wherein the first IL12a is linked to a first amino acid residue and a second amino acid residue of the first heavy chain CH3 domain, wherein the first and the second amino acid residues are selected from the group consisting of amino acid residues at positions 357, 358, 359, 360, 361, 362, and 363 of the first heavy chain CH3 domain according to EU numbering.

Embodiment 61. The protein complex of any one of

embodiments

56, 58, and 60, wherein one or more amino acid residues in a wildtype CH3 domain between the first and the second amino acid residues in the first heavy chain CH3 domain are deleted.

Embodiment 62. The protein complex of any one of embodiments 53-61, wherein the first IL12a is a human IL12a and the first IL12b is a human IL12b.

Embodiment 63. The protein complex of any one of embodiments 53-62, wherein the first IL12a is fused to the first heavy chain CH3 domain through a linker sequence.

Embodiment 64. The protein complex of embodiment 63, wherein the linker sequence comprises or consists of a sequence that is at least 80%identical to GGGGSGGGGS (SEQ ID NO: 36) .

Embodiment 65. The protein complex of any one of embodiments 53-64, further comprising a second IL12, wherein the second IL12 comprises or consists of: a second IL12a; and a second IL12b that associates with the second IL12a.

Embodiment 66. The protein complex of embodiment 65, wherein the second IL12a is fused to the second heavy chain polypeptide at a region from position 344 to position 382 of the second heavy chain CH3 domain according to EU numbering.

Embodiment 67. The protein complex of embodiment 65, wherein the second IL12b is fused to the second heavy chain polypeptide at a region from position 344 to position 382 of the second heavy chain CH3 domain according to EU numbering.

Embodiment 68. The protein complex embodiment 66, wherein the second IL12a is linked to a first amino acid residue and a second amino acid residue of the second heavy chain CH3 domain, wherein the first and the second amino acid residues are selected from the group consisting of amino acid residues at positions 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, and 383 of the second heavy chain CH3 domain according to EU numbering.

Embodiment 69. The protein complex of embodiment 66, wherein the second IL12a is fused to the second heavy chain polypeptide at a region from position 351 to position 362 of the second heavy chain CH3 domain according to EU numbering.

Embodiment 70. The protein complex of embodiment 69, wherein the second IL12a is linked to a first amino acid residue and a second amino acid residue of the second heavy chain CH3 domain, wherein the first and the second amino acid residues are selected from the group consisting of amino acid residues at positions 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, and 363 of the second heavy chain CH3 domain according to EU numbering.

Embodiment 71. The protein complex of embodiment 66, wherein the second IL12a is fused to the second heavy chain polypeptide at a region from position 358 to position 362 of the second heavy chain CH3 domain according to EU numbering.

Embodiment 72. The protein complex of embodiment 71, wherein the second IL12a is linked to a first amino acid residue and a second amino acid residue of the second heavy chain CH3 domain, wherein the first and the second amino acid residues are selected from the group consisting of amino acid residues at positions 357, 358, 359, 360, 361, 362, and 363 of the second heavy chain CH3 domain according to EU numbering.

Embodiment 73. The protein complex of any one of

embodiments

68, 70, and 72, wherein one or more amino acid residues in a wildtype CH3 domain between the first and the second amino acid residues in the second heavy chain CH3 domain are deleted.

Embodiment 74. The protein complex of any one of embodiments 65-73, wherein the second IL12a is a human IL12a and the second IL12b is a human IL12b.

Embodiment 75. The protein complex of any one of embodiments 65-74, wherein the second IL12a is fused to the second heavy chain CH3 domain through a linker sequence.

Embodiment 76. The protein complex of embodiment 75, wherein the linker sequence comprises or consists of a sequence that is at least 80%identical to GGGGSGGGGS (SEQ ID NO: 36) .

Embodiment 77. The protein complex of any one of embodiments 53-76, further comprising a first Interferon alpha-4 (IFNa4) that is linked to the C-terminus of the first heavy chain polypeptide.

Embodiment 78. The protein complex of embodiment 77, wherein the first IFNa4 is linked to the C-terminus of the first heavy chain polypeptide through a linker sequence.

Embodiment 79. The protein complex of embodiment 78, wherein the linker sequence comprises or consists of a sequence that is at least 80%identical to SEQ ID NO: 38.

Embodiment 80. The protein complex of any one of embodiments 77-79, wherein the first IFNa4 is a human IFNa4.

Embodiment 81. The protein complex of any one of embodiments 77-80, further comprising a second IFNa4 that is linked to the C-terminus of the second heavy chain polypeptide.

Embodiment 82. The protein complex of embodiment 81, wherein the second IFNa4 is fused to the C-terminus of the second heavy chain polypeptide through a linker sequence.

Embodiment 83. The protein complex of embodiment 82, wherein the linker sequence comprises or consists of a sequence that is at least 80%identical to SEQ ID NO: 38.

Embodiment 84. The protein complex of any one of embodiments 81-83, wherein the second IFNa4 is a human IFNa4.

Embodiment 85. A protein complex of any one of embodiments 36-38, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises or consists of: a first heavy chain polypeptide, a second heavy chain polypeptide, a first light chain polypeptide, and a second light chain polypeptide; wherein the first heavy chain polypeptide and the first light chain polypeptide associate with each other, forming a first antigen-binding region targeting PD-L1; wherein the second heavy chain polypeptide and the second light chain polypeptide associate with each other, forming a second antigen-binding region targeting PD-L1; wherein the IL12 comprises or consists of: a first interleukin 12 subunit alpha (IL12a) ; and a first interleukin 12 subunit beta (IL12b) that associates with the first IL12a.

Embodiment 86. The protein complex of embodiment 85, wherein the first IL12a is linked to the C-terminus of the first heavy chain polypeptide.

Embodiment 87. The protein complex of embodiment 85, wherein the first IL12b is linked to the C-terminus of the first heavy chain polypeptide.

Embodiment 88. The protein complex of embodiment 86, wherein the first IL12a is linked to the C-terminus of the first heavy chain polypeptide through a linker sequence.

Embodiment 89. The protein complex of embodiment 88, wherein the linker sequence comprises or consists of a sequence that is at least 80%identical to SEQ ID NO: 38.

Embodiment 90. The protein complex of any one of embodiments 85-89, further comprising a second IL12, wherein the second IL12 comprises or consists of: a second IL12a; and a second IL12b that associates with the second IL12a.

Embodiment 91. The protein complex of embodiment 90, wherein the second IL12a is linked to the C-terminus of the second heavy chain polypeptide.

Embodiment 92. The protein complex of embodiment 90, wherein the second IL12b is linked to the C-terminus of the second heavy chain polypeptide.

Embodiment 93. The protein complex of embodiment 91, wherein the second IL12a is fused to the C-terminus of the second heavy chain polypeptide through a linker sequence.

Embodiment 94. The protein complex of embodiment 93, wherein the linker sequence comprises or consists of a sequence that is at least 80%identical to SEQ ID NO: 38.

Embodiment 95. The protein complex of embodiment 85, wherein the first IL12a is linked to the C-terminus of the first light chain polypeptide

Embodiment 96. The protein complex of embodiment 85, wherein the first IL12b is linked to the C-terminus of the first light chain polypeptide

Embodiment 97. The protein complex of embodiment 95, wherein the first IL12a is linked to the C-terminus of the first light chain polypeptide through a linker sequence.

Embodiment 98. The protein complex of embodiment 97, wherein the linker sequence comprises or consists of a sequence that is at least 80%identical to GGGGSGGGGS (SEQ ID NO: 36) .

Embodiment 99. The protein complex of any one of embodiments 85, and 95-98, further comprising a second IL12, wherein the second IL12 comprises or consists of: a second IL12a; and a second IL12b that associates with the second IL12a.

Embodiment 100. The protein complex of embodiment 99, wherein the second IL12a is linked to the C-terminus of the second light chain polypeptide.

Embodiment 101. The protein complex of embodiment 99, wherein the second IL12b is linked to the C-terminus of the second light chain polypeptide.

Embodiment 102. The protein complex of embodiment 100, wherein the second IL12a is fused to the C-terminus of the second light chain polypeptide through a linker sequence.

Embodiment 103. The protein complex of embodiment 102, wherein the linker sequence comprises or consists of a sequence that is at least 80%identical to GGGGSGGGGS (SEQ ID NO: 36) .

Embodiment 104. The protein complex of any one of embodiments 53-103, wherein the first heavy chain polypeptide and the second heavy chain polypeptide form a heterodimer through knobs-in-holes (KIH) technique.

Embodiment 105. The protein complex of any one of embodiments 36-104, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises or consists of: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises or consists of an amino acid sequence that is at least 80%identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises or consists of an amino acid sequence that is at least 80%identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises or consists of 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 or consists of an amino acid sequence that is at least 80%identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises or consists of an amino acid sequence that is at least 80%identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises or consists of an amino acid sequence that is at least 80%identical to a selected VL CDR3 amino acid sequence,

wherein the selected

VH CDRs

(1) the selected

VH CDRs

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

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, 6, respectively;

(2) the selected

VH CDRs

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

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10, 11, 12, respectively;

(3) the selected

VH CDRs

VL CDRs

(4) the selected

VH CDRs

VL CDRs

(5) the selected

VH CDRs

VL CDRs

(6) the selected

VH CDRs

VL CDRs

(7) the selected

VH CDRs

VL CDRs

(8) the selected

VH CDRs

VL CDRs

(9) the selected

VH CDRs

VL CDRs

(10) the selected

VH CDRs

VL CDRs

(11) the selected

VH CDRs

VL CDRs

(12) the selected

VH CDRs

VL CDRs

Embodiment 106. The protein complex of embodiment 105, wherein the VH comprises

CDRs

1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3 respectively, and the VL comprises

CDRs

1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively.

Embodiment 107. The protein complex of embodiment 105, wherein the VH comprises

CDRs

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

CDRs

1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively.

Embodiment 108. The protein complex of any one of embodiments 36-107, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises or consists of a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 90%identical to SEQ ID NO: 13, 14, 15, 16, 66, 68, 70, 78, 80, or 98, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 90%identical to SEQ ID NO: 17, 18, 19, 67, 69, 71, 79, 81, or 99.

Embodiment 109. The protein complex of any one of embodiments 36-108, wherein the protein complex binds to PD-L1 (e.g., human PD-L1) with a KD value less than 1 × 10 ^-7 M, less than 1 × 10 ^-8 M, less than 1 × 10 ^-9 M, less than 5 × 10 ^-10 M, less than 4.5 × 10 ^-10 M, or less than 4 × 10 ^-10 M.

Embodiment 110. The protein complex of any one of embodiments 36-109, wherein the protein complex binds to IL12 receptor (e.g., human IL12 receptor) with a KD value less than 7 × 10 ^-8 M, less than 6.5 × 10 ^-8 M, or less than 6 × 10 ^-8 M.

Embodiment 111. The protein complex of embodiment 110, wherein the protein complex binds to IL12 receptor (e.g., human IL12 receptor) with a KD value more than 4 × 10 ^-10 M, more than 4.5 × 10 ^-10 M, more than 5 × 10 ^-10 M, more than 1 × 10 ^-9 M, or more than 1 × 10 ^-8 M.

Embodiment 112. A fusion heavy chain polypeptide, comprising or consisting of from N-terminus to C-terminus: a first region comprising CH2, and a first portion of CH3; a second region comprising an interleukin 12 polypeptide; and a third region comprising a second portion of CH3; wherein the first portion of CH3 comprises or consists of amino acid residues 341-343 of a CH3 domain according to EU numbering, and the second portion of CH3 comprises or consists of amino acid residues 383-447 of a CH3 domain according to EU numbering.

Embodiment 113. The fusion heavy chain polypeptide of embodiment 112, wherein the interleukin 12 polypeptide is IL12a.

Embodiment 114. The fusion heavy chain polypeptide of embodiment 112, wherein the interleukin 12 polypeptide is IL12b.

Embodiment 115. The fusion heavy chain polypeptide of embodiment 112, wherein the interleukin 12 polypeptide is an IL12a and IL12b fusion protein.

Embodiment 116. The fusion heavy chain polypeptide of any one of embodiments 112-115, wherein the first portion of CH3 comprises or consists of amino acid residues 341-350 of a CH3 domain according to EU numbering, and the second portion of CH3 comprises or consists of amino acid residues 363-447 of a CH3 domain according to EU numbering.

Embodiment 117. The fusion heavy chain polypeptide of any one of embodiments 112-116, wherein the first portion of CH3 comprises or consists of amino acid residues 341-357 of a CH3 domain according to EU numbering, and the second portion of CH3 comprises or consists of amino acid residues 363-447 of a CH3 domain according to EU numbering.

Embodiment 118. The fusion heavy chain polypeptide of any one of embodiments 112-117, wherein the second region further comprises a first linker sequence and a second linker sequence, wherein the second region is linked to the first region through the first linker sequence, wherein the second region is linked to the third region through the second linker sequence.

Embodiment 119. The fusion heavy chain polypeptide of embodiment 118, wherein the first and/or the second linker sequence comprise a sequence that is at least 80%identical to GGGGSGGGGS (SEQ ID NO: 36) .

Embodiment 120. The fusion heavy chain polypeptide of any one of embodiments 112-119, wherein the first region further comprises VH and/or CH1.

Embodiment 121. The fusion heavy chain polypeptide of any one of

embodiments

112, 113, and 116-120, wherein the fusion heavy chain polypeptide comprises or consists of a sequence that is at least 80%identical to SEQ ID NO: 39.

Embodiment 122. The fusion heavy chain polypeptide of any one of embodiments 112-121, wherein the fusion heavy chain polypeptide further comprises a fourth region comprising Interferon alpha-4 (IFNa4) .

Embodiment 123. The fusion heavy chain polypeptide of embodiment 122, wherein the fourth region is linked to the third region through a third linker sequence.

Embodiment 124. The fusion heavy chain polypeptide of embodiment 123, wherein the third linker sequence comprises or consists of a sequence that is at least 80%identical to SEQ ID NO: 38.

Embodiment 125. The fusion heavy chain polypeptide of any one of embodiments 122-124, wherein the fusion heavy chain polypeptide comprises or consists of a sequence that is at least 80%identical to SEQ ID NO: 41.

Embodiment 126. A fusion heavy chain polypeptide, comprising or consisting of from N-terminus to C-terminus: a first region comprising VH, CH1, CH2, and CH3; and a second region comprising an interleukin 12 polypeptide; wherein the first region is linked to the second region through a linker sequence.

Embodiment 127. The fusion heavy chain polypeptide of embodiment 126, wherein the interleukin 12 polypeptide is IL12a or IL12b.

Embodiment 128. The fusion heavy chain polypeptide of embodiment 126, wherein the interleukin 12 polypeptide is an IL12a-IL12b fusion protein.

Embodiment 129. The fusion heavy chain polypeptide of any one of embodiments 126-128, wherein the linker sequence comprises or consists of a sequence that is at least 80%identical to SEQ ID NO: 38.

Embodiment 130. The fusion heavy chain polypeptide of any one of

embodiments

126, 127, or 129, wherein the fusion heavy chain polypeptide comprises or consists of a sequence that is at least 80%identical to SEQ ID NO: 46.

Embodiment 131. A modified antibody comprising or consisting of two light chain polypeptides; a heavy chain polypeptide; and the fusion heavy chain polypeptides of any one of embodiments 112-130.

Embodiment 132. A modified antibody comprising or consisting of two light chain polypeptides; and two of the fusion heavy chain polypeptides of any one of embodiments 112-130.

Embodiment 133. The fusion heavy chain polypeptide of any one of embodiments 112-130, wherein the fusion heavy chain polypeptide comprises one or more knobs-into-holes (KIH) modifications.

Embodiment 134. The fusion heavy chain polypeptide of embodiment 133, wherein the fusion heavy chain polypeptide comprises one or more knob modifications, and the fusion heavy chain polypeptide comprises or consists of a sequence that is at least 80%identical to SEQ ID NO: 43.

Embodiment 135. A modified antibody comprising or consisting of two light chain polypeptides; the fusion heavy chain polypeptide of embodiment 134; and a heavy chain polypeptide comprising or consisting of a sequence that is at least 80%identical to SEQ ID NO: 44.

Embodiment 136. The modified antibody of any one of

embodiments

131, 132, and 135, wherein the two light chain polypeptides comprise a sequence that is at least 80%identical to SEQ ID NO: 40.

Embodiment 137. A fusion light chain polypeptide, comprising or consisting of from N-terminus to C-terminus: a first region comprising VL and CL; and a second region comprising an interleukin 12 polypeptide; wherein the first region is linked to the second region through a linker sequence.

Embodiment 138. The fusion heavy chain polypeptide of embodiment 137, wherein the interleukin 12 polypeptide is IL12a or IL12b.

Embodiment 139. The fusion heavy chain polypeptide of embodiment 137, wherein the interleukin 12 polypeptide is an IL12a-IL12b fusion protein.

Embodiment 140. The fusion light chain polypeptide of any one of embodiments 137-139, wherein the linker sequence comprises or consists of a sequence that is at least 80%identical to GGGGSGGGGS (SEQ ID NO: 36) .

Embodiment 141. The fusion light chain polypeptide of any one of embodiments 137-140, wherein the fusion light chain polypeptide comprises or consists of a sequence that is at least 80%identical to SEQ ID NO: 49.

Embodiment 142. A modified antibody comprising or consisting of two heavy chain polypeptides; a light chain polypeptide; and the fusion light chain polypeptides of any one of embodiments 137-141.

Embodiment 143. A modified antibody comprising or consisting of two heavy chain polypeptides; and two of the fusion light chain polypeptides of any one of embodiments 137-141.

Embodiment 144. The modified antibody of

embodiment

142 or 143, wherein the two heavy chain polypeptides comprise a sequence that is at least 80%identical to SEQ ID NO: 48.

Embodiment 145. A method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the protein complex of any one of embodiments 13-29 and 36-111, the fusion protein of any one of embodiments 30-35, or the modified antibody of any one of

embodiments

131, 132, 135, 136, and 142-144, to the subject.

Embodiment 146. The method of embodiment 145, wherein the subject has a PD-L1-expressing cancer.

Embodiment 147. The method of

embodiment

145 or 146, wherein the cancer is resistant to anti-PD-1 antibody or anti-PD-L1 antibody treatment.

Embodiment 148. A method of increasing immune response (e.g., in tumor microenvironment, spleen, lymph nodes, and/or lymphatic system) in a subject, the method comprising administering a therapeutically effective amount of a composition comprising the protein complex of any one of embodiments 13-29 and 36-111, the fusion protein of any one of embodiments 30-35, or the modified antibody of any one of

embodiments

131, 132, 135, 136, and 142-144, to the subject.

Embodiment 149. A method of activating T cells (e.g., in tumor microenvironment, spleen, lymph nodes, and/or lymphatic system) of a subject, the method comprising administering a therapeutically effective amount of a composition comprising the protein complex of any one of embodiments 13-29 and 36-111, the fusion protein of any one of embodiments 30-35, or the modified antibody of any one of

embodiments

131, 132, 135, 136, and 142-144, to the subject.

Embodiment 150. A method of reducing IL12 toxicity when delivered into a subject, the method comprising administering a therapeutically effective amount of a composition comprising the protein complex of any one of embodiments 13-29 and 36-111, the fusion protein of any one of embodiments 30-35, or the modified antibody of any one of

embodiments

131, 132, 135, 136, and 142-144, to the subject.

Embodiment 151. A method of delivering IL12 without causing toxicity to a subject, the method comprising administering a therapeutically effective amount of a composition comprising the protein complex of any one of embodiments 13-29 and 36-111, the fusion protein of any one of embodiments 30-35, or the modified antibody of any one of

embodiments

131, 132, 135, 136, and 142-144, to the subject.

Embodiment 152. The method of embodiment 151, wherein the composition comprises no interferon.

Embodiment 153. A method of blocking PD-1/PD-L1 interaction in a subject, the method comprising administering a therapeutically effective amount of a composition comprising the protein complex of any one of embodiments 13-29 and 36-111, the fusion protein of any one of embodiments 30-35, or the modified antibody of any one of

embodiments

131, 132, 135, 136, and 142-144, to the subject; wherein the protein complex or modified antibody specifically binds to PD-L1 (e.g., human PD-L1) with a KD value less than 1 × 10 ^-7 M, less than 1 × 10 ^-8 M, less than 1 × 10 ^-9 M, less than 5 × 10 ^-10 M, less than 4.5 × 10 ^-10 M, or less than 4 × 10 ^-10 M.

Embodiment 154. The method of any one of embodiments 145-153, wherein the protein complex, the fusion protein, or the modified antibody is administered at a dose level of at least 0.1-20000 μg/kg (μg per kilogram of subject body weight) .

Embodiment 155. The method of any one of embodiments 145-154, wherein the subject is a human patient.

Embodiment 156. A method of treating cancer, the method comprising administering an anti-PD-1 antibody and a composition comprising the protein complex of any one of embodiments 13-29 and 36-111, the fusion protein of any one of embodiments 30-35, or the modified antibody of any one of

embodiments

131, 132, 135, 136, and 142-144, to a patient in need of such treatment.

Embodiment 157. An antibody-drug conjugate comprising the protein complex of any one of embodiments 13-29 and 36-111, the fusion protein of any one of embodiments 30-35, or the modified antibody of any one of

embodiments

131, 132, 135, 136, and 142-144; covalently bound to a therapeutic agent.

Embodiment 158. The antibody-drug conjugate of embodiment 157, wherein the therapeutic agent is a cytotoxic or cytostatic agent.

Embodiment 159. A pharmaceutical composition comprising the protein complex of any one of embodiments 13-29 and 36-111, the fusion protein of any one of embodiments 30-35, or the modified antibody of any one of

embodiments

131, 132, 135, 136, and 142-144; and a pharmaceutically acceptor carrier.

Embodiment 160. A pharmaceutical composition comprising the antibody-drug conjugate of

embodiment

157 or 158, and a pharmaceutically acceptable carrier.

Embodiment 161. A nucleic acid encoding the protein complex of any one of embodiments 13-29 and 36-111, the fusion protein of any one of embodiments 30-35, or the modified antibody of any one of

embodiments

131, 132, 135, 136, and 142-144.

Embodiment 162. A vector comprising the nucleic acid of embodiment 161.

Embodiment 163. A host cell comprising the nucleic acid of embodiment 161.

Embodiment 164. A method for producing a protein complex or a modified antibody, the method comprising culturing the host cell of embodiment 163 under conditions suitable to produce the protein complex or the modified antibody.

Embodiment 165. A method of delivering IL12 to a tumor in a subject, the method comprising administering a therapeutically effective amount of a composition comprising a protein complex to the subject in need thereof, wherein IL12 is conjugated to an anti-PD-L1 antibody or antigen-binding fragment thereof in the protein complex, wherein IL12 binds to T cells and activates T cells (e.g., in the tumor microenvironment, spleen, lymph nodes, and/or lymphatic system) .

Embodiment 166. The method of embodiment 165, wherein the subject is a human patient, and the protein complex is administered at a dose level of at least 0.1-20000 μg/kg (μg per kg of subject body weight) .

Embodiment 167. The method of

embodiment

165 or 166, wherein the protein complex has a binding affinity to hPD-L1 with a KD value less than 1 × 10 ^-7 M, less than 1 × 10 ^-8 M, less than 1 × 10 ^-9 M, less than 5 × 10 ^-10 M, less than 4.5 × 10 ^-10 M, or less than 4 × 10 ^-10 M.

Embodiment 168. The method of any one of embodiments 165-167, wherein the effects of IL12 is limited to the tumor microenvironment, spleen, lymph nodes, and/or lymphatic system.

Embodiment 169. The method of any one of embodiments 165-168, wherein the protein complex tethers T cells with tumor cells expressing PD-L1.

Embodiment 170. The method of any one of embodiments 165-169, wherein the anti-PD-L1 antibody or antigen-binding fragment is an immunoglobulin (e.g., IgM, IgA, IgE, IgD, or IgG) .

Embodiment 171. The method of embodiment 170, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof is an IgG (e.g., IgG1 or IgG4) .

Embodiment 172. The method of any one of embodiments 165-171, wherein the IL12 is linked to a heavy chain of the anti-PD-L1 antibody or antigen-binding fragment thereof.

Embodiment 173. The method of any one of embodiments 165-171, wherein the IL12 is linked to a light chain of the anti-PD-L1 antibody or antigen-binding fragment thereof.

Embodiment 174. The method of any one of embodiments 165-173, wherein the IL12 comprises or consists of: an interleukin 12 subunit alpha (IL12a) ; and an interleukin 12 subunit beta (IL12b) that associates with the IL12a.

Embodiment 175. The method of embodiment 174, wherein the IL12a is fused to the heavy chain at a region from position 344 to position 382 of the heavy chain CH3 domain according to EU numbering.

Embodiment 176. The method of embodiment 174, wherein the IL12b is fused to the heavy chain at a region from position 344 to position 382 of the heavy chain CH3 domain according to EU numbering.

Embodiment 177. The method of any one of embodiments 165-176, wherein the protein complex further comprises an interferon (e.g., IFNa4) .

Embodiment 178. A method of activating T cells (e.g., in a tumor microenvironment, spleen, lymph nodes, and/or lymphatic system) , the method comprising delivering a therapeutically effective amount of a protein complex to a tumor microenvironment, spleen, lymph nodes, and/or lymphatic system, wherein IL12 is conjugated to an anti-PD-L1 antibody or antigen-binding fragment thereof in the protein complex, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof limits the distribution of the IL12 to the tumor microenvironment, spleen, lymph nodes, and/or lymphatic system, and the activation of T cells (e.g., in the tumor microenvironment, spleen, lymph nodes, and/or lymphatic system) depends on the presence of IL12 and the anti-PD-L1 antibody or antigen-binding fragment thereof.

Embodiment 179. The method of embodiment 178, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof is an immunoglobulin (e.g., IgM, IgA, IgE, IgD, or IgG) .

Embodiment 180. The method of embodiment 179, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof is an IgG (e.g., IgG1 or IgG4) .

Embodiment 181. The method of any one of embodiments 178-180, wherein the IL12 is linked to a heavy chain of the anti-PD-L1 antibody or antigen-binding fragment thereof.

Embodiment 182. The method of any one of embodiments 178-180, wherein the IL12 is linked to a light chain of the anti-PD-L1 antibody or antigen-binding fragment thereof.

Embodiment 183. The method of any one of embodiments 178-182, wherein the IL12 comprises or consists of: an interleukin 12 subunit alpha (IL12a) ; and an interleukin 12 subunit beta (IL12b) that associates with the first IL12a.

Embodiment 184. The method of embodiment 183, wherein the IL12a is fused to the heavy chain at a region from position 344 to position 382 of the first heavy chain CH3 domain according to EU numbering.

Embodiment 185. The method of embodiment 183, wherein the IL12b is fused to the heavy chain at a region from position 344 to position 382 of the first heavy chain CH3 domain according to EU numbering.

Embodiment 186. A method of improving the efficacy of an anti-PD-L1 antibody or antigen binding fragment thereof in a subject, comprising administering a therapeutically effective amount of an anti-PD-L1 antibody or antigen binding fragment thereof to the subject in need thereof, wherein IL12 is conjugated to the anti-PD-L1 antibody or antigen-binding fragment thereof.

Embodiment 187. The method of embodiment 186, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof blocks (e.g., interferes) or does not block the interaction between PD-L1 on tumor cells and PD-1 on T cells, and IL12 activates T cells that are adjacent to the tumor cells.

Embodiment 188. The method of embodiment 186 or 187, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof is an immunoglobulin (e.g., IgM, IgA, IgE, IgD, or IgG) .

Embodiment 189. The method of embodiment 188, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof is an IgG (e.g., IgG1 or IgG4) .

Embodiment 190. The method of any one of embodiments 186-189, wherein the IL12 is linked to a heavy chain of the anti-PD-L1 antibody or antigen-binding fragment thereof.

Embodiment 191. The method of any one of embodiments 186-189, wherein the IL12 is linked to a light chain of the anti-PD-L1 antibody or antigen-binding fragment thereof.

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

A protein complex comprising a targeting moiety fused with an immunomodulatory moiety, wherein (a) the targeting moiety specifically binds to a PD-1 ligand; and (b) the immunomodulatory moiety specifically binds to interleukin-12 receptor (IL12R) .
The protein complex of claim 1, wherein the immunomodulatory moiety comprises an antibody or antigen-binding fragment, a single chain variable fragment (scFv) , a Fc-containing polypeptide, or a fusion protein that specifically binds IL12R.
The protein complex of claim 1 or 2, wherein the immunomodulatory moiety is an IL12R agonist.
The protein complex of any one of claims 1-3, wherein the immunomodulatory moiety comprises IL12.
The protein complex of claim 4, wherein the IL12 comprises a IL12a (e.g., a human IL12a) , a IL12b (e.g., a human IL12b) , and/or a variant thereof.
The protein complex of any one of claims 1-5, wherein the targeting moiety comprises an antibody or antigen-binding fragment, a single chain variable fragment (scFv) , a Fc-containing polypeptide, or a fusion protein that specifically binds to the PD-1 ligand.
The protein complex of any one of claims 1-6, wherein the targeting moiety comprises a full-length antibody.
The protein complex of any one of claims 1-7, wherein the targeting moiety comprises an extracellular domain of PD-1.
The protein complex of any one of claims 1-8, wherein the PD-1 ligand is PD-L1 or PD-L2.
The protein complex of any one of claims 1-9, wherein the targeting moiety blocks or interferes the interaction between PD-1 and the PD-1 ligand.
The protein complex of any one of claims 1-10, wherein targeting moiety has a KD of less than 1 x 10 ^-7 M, less than 1 x 10 ^-8 M, or less than 1 x 10 ^-9 M with the PD-1 ligand.
The protein complex of any one of claims 1-11, wherein the targeting moiety comprises a polypeptide, wherein the immunomodulatory moiety is fused to the N-terminus or the C-terminus of the polypeptide.
The protein complex of any one of claims 1-11, wherein the immunomodulatory moiety comprises a polypeptide, wherein the targeting moiety is fused to the N-terminus or the C-terminus of the polypeptide.
The protein complex of any one of claims 1-11, wherein the targeting moiety comprises a polypeptide comprising a CH3 domain, wherein the immunomodulatory moiety is fused to the CH3 domain.
The protein complex of any one of claims 1-11, wherein the targeting moiety and the immunomodulatory moiety are fused to a scaffold protein (e.g., an albumin) .
The protein complex of any one of claims 1-11, wherein the protein complex comprises a bispecific antibody, wherein the bispecific antibody binds to the PD-1 ligand and the IL12R.
The protein complex of any one of claims 1-16, wherein the protein complex comprises two or more immunomodulatory moieties that specifically bind to IL12R.
The protein complex of any one of claims 1-17, wherein the protein complex comprises two or more targeting moieties that specifically bind to a PD-1 ligand.
The protein complex of any one of claims 1-18, wherein the protein complex comprises an Fc comprising two CH3 domains.
The protein complex of claim 19, wherein the immunomodulatory moiety is linked to a CH3 domain of the two CH3 domains in the Fc.
The protein complex of claim 19, wherein the immunomodulatory moiety is linked to the C-terminus of a CH3 domain of the two CH3 domains.
The protein complex of claim 19, wherein the immunomodulatory moiety is fused to a CH3 domain of the two CH3 domains in the Fc at a region from position 344 to position 382 of the CH3 domain according to EU numbering.
The protein complex of claim 19, wherein the immunomodulatory moiety comprises IL12a and IL12b, wherein IL12a is linked to the Fc.
The protein complex of claim 19, wherein the immunomodulatory moiety comprises IL12a and IL12b, wherein IL12b is linked to the Fc.
The protein complex of claim 19, wherein the targeting moiety is linked to a CH3 domain of the two CH3 domains in the Fc.
The protein complex of claim 19, wherein the targeting moiety is linked to the C-terminus of a CH3 domain of the two CH3 domains in the Fc.
The protein complex of claim 19, wherein the targeting moiety is fused to a CH3 domain of the two CH3 domains in the Fc at a region from position 344 to position 382 of the CH3 domain according to EU numbering.
The protein complex of claim 19, wherein the targeting moiety comprises a scFv (e.g., a scFv targeting the PD-1 ligand) .
The protein complex of any one of claims 1-28, wherein the protein complex comprises two light chains.
The protein complex of claim 29, wherein the immunomodulatory moiety is linked to one of the two light chains.
The protein complex of claim 29, wherein the targeting moiety is linked to one of the two light chains.
The protein complex of any one of claims 1-31, wherein the protein complex further comprises an interferon (e.g., IFNa1, IFNa2, IFNa3, IFNa4, and/or IFNγ) .
The protein complex of any one of claims 1-32, wherein the protein complex further comprises a cytokine (e.g., IL2, IL7, IL15, IL18, and/or IL21) .
The protein complex of claim 32 or 33, wherein the interferon or the cytokine is linked to the protein complex by a linker sequence.
The protein complex of any one of claims 1-34, wherein the protein complex comprises a linker sequence (GGGGS) n, wherein n can be 1, 2, 3, 4, 5, 6, 7 or 8.
The protein complex of any one of claims of 1-35, wherein the targeting moiety comprises an anti-PD-L1 antibody or antigen-binding fragment thereof.
The protein complex of claim 36, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof is an IgG (e.g., IgG1, IgG2 or IgG4) .
The protein complex of any one of claims of 1-37, wherein the targeting moiety comprises 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 the selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are one of the following:

(1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 2, and 3, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, and 6, respectively;

(2) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 7, 8, and 9, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10, 11, and 12, respectively;

(3) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 104, 105, and 106, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 107, 108, and 109 respectively;

(4) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 134, 135, and 136, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 137, 138, and 139, respectively;

(5) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 110, 111, and 112, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 113, 114, and 115, respectively;

(6) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 140, 141, and 142, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 143, 144, and 145, respectively;

(7) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 116, 117, and 118, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 119, 120, and 121, respectively;

(8) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 146, 147, and 148, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 149, 150, and 151, respectively;

(9) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 122, 123, and 124, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 125, 126, and 127, respectively;

(10) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 152, 153, and 154, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 155, 156, and 157, respectively;

(11) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 128, 129, and 130, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 131, 132, and 133, respectively; and

(12) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 158, 159, and 160, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 161, 162, and 163, respectively.
The protein complex of any one of claims of 1-38, wherein the targeting moiety comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%identical to SEQ ID NO: 13, 14, 15, or 16, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90%identical to SEQ ID NO: 17, 18, or 19.
The protein complex of any one of claims of 1-39, wherein the immunomodulatory moiety comprises a sequence that is at least 80%identical to SEQ ID NO: 28 and a sequence that is at least 80%identical to SEQ ID NO: 29.
The protein complex of any one of claims 1-38, wherein the targeting moiety comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%identical to SEQ ID NO: 66, 68, 70, 78, 80, or 98, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90%identical to SEQ ID NO: 67, 69, 71, 79, 81, or 99.
An antibody or antigen-binding fragment thereof that binds to PD-L1, comprising:

a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence 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 the selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are one of the following:

(1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 104, 105, and 106, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 107, 108, and 109 respectively;

(2) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 134, 135, and 136, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 137, 138, and 139, respectively;

(3) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 110, 111, and 112, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 113, 114, and 115, respectively;

(4) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 140, 141, and 142, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 143, 144, and 145, respectively;

(5) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 116, 117, and 118, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 119, 120, and 121, respectively;

(6) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 146, 147, and 148, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 149, 150, and 151, respectively;

(7) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 122, 123, and 124, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 125, 126, and 127, respectively;

(8) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 152, 153, and 154, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 155, 156, and 157, respectively;

(9) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 128, 129, and 130, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 131, 132, and 133, respectively; and

(10) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 158, 159, and 160, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 161, 162, and 163, respectively.
The antibody or antigen-binding fragment thereof of claim 42, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 104, 105, and 106 respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 107, 108, and 109, respectively.
The antibody or antigen-binding fragment thereof of claim 42, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 110, 111, and 112, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 113, 114, and 115, respectively.
The antibody or antigen-binding fragment thereof of claim 42, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 116, 117, and 118, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 119, 120, and 121, respectively.
The antibody or antigen-binding fragment thereof of claim 42, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 122, 123, and 124, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 125, 126, and 127, respectively.
The antibody or antigen-binding fragment thereof of claim 42, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 128, 129, and 130, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 131, 132, and 133, respectively.
The antibody or antigen-binding fragment thereof of any one of claims 42-47, wherein the antibody or antigen-binding fragment specifically binds to human PD-L1.
The antibody or antigen-binding fragment thereof of any one of claims 42-48, wherein the antibody or antigen-binding fragment is a human or humanized antibody or antigen-binding fragment thereof.
The antibody or antigen-binding fragment thereof of any one of claims 42-49, wherein the antibody or antigen-binding fragment is a single-chain variable fragment (scFV) , or a multi-specific antibody (e.g., a bispecific antibody) .
A nucleic acid comprising a polynucleotide encoding a polypeptide comprising:

(1) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 104, 105 and 106, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 81, binds to PD-L1;

(2) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 107, 108, and 109, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 80, binds to PD-L1;

(3) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 110, 111, and 112, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 79, binds to PD-L1;

(4) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 113, 114, and 115, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 78, binds to PD-L1;

(5) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 116, 117, and 118, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 71, binds to PD-L1;

(6) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 119, 120, and 121, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 70, binds to PD-L1;

(7) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 122, 123, and 124, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 67, binds to PD-L1;

(8) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 125, 126, and 127, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 66, binds to PD-L1;

(9) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 128, 129, and 130, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 99, binds to PD-L1; or

(10) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 131, 132, and 133, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 98, binds to PD-L1.
The nucleic acid of claim 51, wherein the VH when paired with a VL specifically binds to human PD-L1, or the VL when paired with a VH specifically binds to human PD-L1.
The nucleic acid of claim 51 or 52, wherein the immunoglobulin heavy chain or the fragment thereof is a human or humanized immunoglobulin heavy chain or a fragment thereof, and the immunoglobulin light chain or the fragment thereof is a human or humanized immunoglobulin light chain or a fragment thereof.
The nucleic acid of any one of claims 51-53, wherein the nucleic acid encodes a single-chain variable fragment (scFv) .
The nucleic acid of any one of claims 51-54, wherein the nucleic acid is cDNA.
A vector comprising one or more of the nucleic acids of any one of claims 51-55.
A vector comprising two of the nucleic acids of any one of claims 51-55, wherein the vector encodes the VL region and the VH region that together bind to PD-L1.
A pair of vectors, wherein each vector comprises one of the nucleic acids of any one of claims 51-55, wherein together the pair of vectors encodes the VL region and the VH region that together bind to PD-L1.
A cell comprising the vector of claim 56 or 57, or the pair of vectors of claim 58.
The cell of claim 59, wherein the cell is a CHO cell.
A cell comprising one or more of the nucleic acids of any one of claims 51-55.
A cell comprising two of the nucleic acids of any one of claims 51-55.
The cell of claim 62, wherein the two nucleic acids together encode the VL region and the VH region that together bind to PD-L1.
A method of producing an antibody or an antigen-binding fragment thereof, the method comprising

(a) culturing the cell of any one of claims 59-63 under conditions sufficient for the cell to produce the antibody or the antigen-binding fragment; and

(b) collecting the antibody or the antigen-binding fragment produced by the cell.
An antibody or antigen-binding fragment thereof that binds to PD-L1 comprising a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%identical to a selected VH sequence, and a 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 is SEQ ID NO: 66, 70, 78, 80, or 98, and the selected VL sequence is SEQ ID NO: 67, 71, 79, 81, or 99.
The antibody or antigen-binding fragment thereof of claim 65, wherein the VH comprises the sequence of SEQ ID NO: 66 and the VL comprises the sequence of SEQ ID NO: 67.
The antibody or antigen-binding fragment thereof of claim 65, wherein the VH comprises the sequence of SEQ ID NO: 70 and the VL comprises the sequence of SEQ ID NO: 71.
The antibody or antigen-binding fragment thereof of claim 65, wherein the VH comprises the sequence of SEQ ID NO: 78 and the VL comprises the sequence of SEQ ID NO: 79.
The antibody or antigen-binding fragment thereof of claim 65, wherein the VH comprises the sequence of SEQ ID NO: 80 and the VL comprises the sequence of SEQ ID NO: 81.
The antibody or antigen-binding fragment thereof of claim 65, wherein the VH comprises the sequence of SEQ ID NO: 98 and the VL comprises the sequence of SEQ ID NO: 99.
A method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the protein complex of any one of claims 1-41 or the antibody or antigen-binding fragment thereof of any one of claims 42-50 and 65-70, to the subject.
The method of claim 71, wherein the subject has a cancer that expresses PD-L1 or does not express PD-L1.
The method of claim 71, wherein the cancer is resistant to anti-PD-1 antibody or anti-PD-L1 antibody treatment.
The method of any one of claims 71-73, further comprising administering an effective amount of an anti-PD-1 antibody or an anti-PD-L1 antibody to the subject.
A method of increasing immune response (e.g., in tumor microenvironment, spleen, lymph nodes, and/or lymphatic system) in a subject, the method comprising administering a therapeutically effective amount of a composition comprising the protein complex of any one of claims 1-41 or the antibody or antigen-binding fragment thereof of any one of claims 42-50 and 65-70 to the subject.
A method of activating T cells of a subject, the method comprising administering a therapeutically effective amount of a composition comprising the protein complex of any one of claims 1-41 or the antibody or antigen-binding fragment thereof of any one of claims 42-50 and 65-70 to the subject.
A method of reducing IL12 toxicity when delivering IL12 into a subject, the method comprising administering a therapeutically effective amount of a composition comprising the protein complex of any one of claims 1-41, to the subject.
An isolated molecule comprising the protein complex of any one of claims 1-41 or the antibody or antigen-binding fragment thereof of any one of claims 42-50 and 65-70; covalently bound to a therapeutic agent.
The isolated molecule of claim 78, wherein the therapeutic agent is a cytotoxic or cytostatic agent.
A pharmaceutical composition comprising the protein complex of any one of claims 1-41 or the antibody or antigen-binding fragment thereof of any one of claims 42-50 and 65-70; and a pharmaceutically acceptor carrier.
A nucleic acid encoding the protein complex of any one of claims 1-41 or the antibody or antigen-binding fragment thereof of any one of claims 42-50 and 65-70.
A vector comprising the nucleic acid of claim 81.
A host cell comprising the nucleic acid of claim 81.
A method for producing a protein complex, the method comprising culturing the host cell of claim 83 under conditions suitable to produce the protein complex.
A method of reducing IL12 toxicity when delivering an agent comprising IL12 into a subject, wherein the IL12 is conjugated (e.g., fused) to an PD-L1 targeting moiety.
The method of claim 85, wherein the PD-L1 targeting moiety is an anti-PD-L1 antibody or antigen-binding fragment thereof.
The method of claim 86, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof can block or interfere the interaction between PD-1 and PD-L1.