Fusion proteins and uses thereof

Abstract

The present application provides fusion proteins having extracellular domains of TGFβ superfamily receptors (e.g., fusion proteins having extracellular domains of TGFβ superfamily receptors and binding to PD-L1), nucleic acids encoding these fusion proteins or portions thereof, vectors comprising these nucleic acids, host cells containing these vectors, methods of making these fusion proteins, pharmaceutical compositions comprising any of these fusion proteins, and methods of using these fusion proteins or compositions.

Description

[0001] The sequence list is submitted as an ASCII text file.

[0002] The following content, submitted as an ASCII text file, is incorporated herein by reference in its entirety as a sequence list in computer-readable form (CRF) (filename: 759892000640SEQLIST.TXT, record date: February 14, 2020, size: 125KB). Technical Field

[0003] This application relates to fusion proteins comprising one or more TGFβ superfamily receptor extracellular domains and binding to PD-L1, methods of their manufacture, and uses, including the treatment of diseases or conditions. Background Technology

[0004] In cancer treatment, it has long been recognized that chemotherapy is associated with high toxicity and can lead to the emergence of resistant cancer cell variants. Even with targeted therapies targeting overexpressed or activated oncoproteins important for tumor survival and growth, cancer cells can always mutate and adapt to reduce their dependence on that targeted pathway, such as by utilizing redundant pathways. Cancer immunotherapy is a new paradigm in cancer treatment that focuses not on targeting cancer cells, but on activating the immune system. Its principle is to re-establish an immune response in the host, especially an adaptive T-cell response, to provide immune surveillance to kill cancer cells, particularly minimal residual disease that has escaped other forms of treatment, thereby achieving long-term protective immunity.

[0005] Programmed death 1 (PD-1) is a member of the CD28 superfamily. PD-1 is expressed in activated T cells, B cells, and bone marrow cells, which possess two ligands, programmed death ligand 1—PD-L1 and PD-L2. PD-L1 interacts with its receptor PD-1 on T cells and plays an important role in the negative regulation of the immune response. PD-L1 protein expression can be detected in many tumor tissues. The tumor site microenvironment can induce PD-L1 expression on tumor cells. PD-L1 expression promotes tumorigenesis and growth and induces anti-tumor activity. In clinical trials, anti-PD-1 or anti-PD-L1 therapies have shown encouraging results in melanoma, renal cell carcinoma, and lung cancer. However, these therapies are also limited by their toxicity profile and limited therapeutic efficacy. For example, inhibiting the PD-L1 / PD-1 interaction leads to the dis-inhibition of the existing chronic immune response in exhausted T cells, which are essentially antiviral or anticancer (Wherry, EJ, Nat Immunol. 2011; 12:492-9). However, anti-PD-1 therapy can sometimes lead to potentially fatal lung-related autoimmune adverse events.

[0006] All publications, patents, patent applications, and disclosures of published patent applications mentioned herein are hereby incorporated in their entirety by reference. Summary of the Invention

[0007] This application provides a fusion protein comprising the extracellular domain of a TGFβ superfamily receptor. In some embodiments, the fusion protein comprises a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V). H Furthermore, each of the two light chains contains a light chain variable region (V). L b) TGFβ superfamily receptor extracellular domain.

[0008] In one aspect, this application provides a fusion protein comprising: a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V... H Furthermore, each of the two light chains contains a light chain variable region (V). L a) TGFβ superfamily receptor extracellular domain; wherein the TGFβ superfamily receptor extracellular domain is fused to at least one of the heavy or light chains of the anti-PD-L1 antibody. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to at least one or both of the two light chains. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to the C-terminus of at least one or both of the two light chains. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to the N-terminus of at least one or both of the two light chains. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to at least one or both of the two heavy chains. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to the C-terminus of at least one or both of the two heavy chains. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to the N-terminus of at least one or both of the two heavy chains.

[0009] In some embodiments of any of the above-described fusion proteins, the TGFβ superfamily receptor extracellular domain comprises the TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the TGFβ receptor type II extracellular domain or a variant thereof comprises the human TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type II extracellular domain or a variant thereof comprises a variant containing an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:2, wherein the variant contains one or more amino acid substitutions at positions N47, N71, and / or N131 according to the numbers in SEQ ID NO:2. In some embodiments, the variant comprises the amino acid sequence of SEQ ID NO:3.

[0010] In some embodiments of any of the above-described fusion proteins, the TGFβ superfamily receptor extracellular domain comprises the TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the TGFβ receptor type I extracellular domain or a variant thereof comprises the human TGFβ receptor type I extracellular domain or a variant thereof.

[0011] In some embodiments of any of the above-described fusion proteins, V H It includes: i) HC-CDR1 containing the amino acid sequence of SEQ ID NO:6; ii) HC-CDR2 containing the amino acid sequence of SEQ ID NO:7; and iii) HC-CDR3 containing the amino acid sequence of SEQ ID NO:8.

[0012] In some embodiments of any of the above-described fusion proteins, V L It includes: i) LC-CDR1 containing the amino acid sequence of SEQ ID NO:9; ii) LC-CDR2 containing the amino acid sequence of SEQ ID NO:10; and iii) LC-CDR3 containing the amino acid sequence of SEQ ID NO:11.

[0013] In some embodiments of any of the above-described fusion proteins, the extracellular domain of the TGFβ superfamily receptor is fused to at least one of the heavy or light chains of the anti-PD-L1 antibody via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is about 2 to about 25 amino acids in length. In some embodiments, the peptide linker is about 2 to about 10 amino acids in length. In some embodiments, the linker is a GS linker. In some embodiments, the linker comprises a modified sequence derived from the IgG hinge region. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 56-68.

[0014] In some embodiments of any of the above-described fusion proteins, V H The amino acid sequence comprising SEQ ID NO:12 or a variant thereof having at least about 80% (including, for example, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and wherein V L The amino acid sequence comprising SEQ ID NO:13 or its variants having at least about 80% (including, for example, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0015] In some embodiments of any of the above-described fusion proteins, the heavy chain comprises the amino acid sequence of any one of SEQ ID NO:14-17 or a variant thereof having at least about 80% (including, for example, any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and wherein the light chain comprises the amino acid sequence of SEQ ID NO:18 or a variant thereof having at least about 80% (including, for example, any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:16 or 17, and wherein the light chain comprises the amino acid sequence of SEQ ID NO:18.

[0016] In some embodiments of any of the above-described fusion proteins, the extracellular domain or polypeptide of the TGFβ superfamily receptor fused with at least one or both of the two light chains comprises an amino acid sequence of any one of SEQ ID NO:27-38 or a variant thereof having at least about 80% (including, for example, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0017] In some embodiments of any of the above-described fusion proteins, the extracellular domain of the TGFβ superfamily receptor fused with at least one or both of the two heavy chains comprises an amino acid sequence of any one of SEQ ID NO:22-26 or a variant thereof having at least about 80% (including, for example, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0018] In some embodiments of any of the above-described fusion proteins, the extracellular domain of the TGFβ superfamily receptor is fused to both heavy chains to contain the amino acid sequence of SEQ ID NO:22, and the two light chains contain the amino acid sequence of SEQ ID NO:18.

[0019] In some embodiments of any of the above-described fusion proteins, the extracellular domain of the TGFβ superfamily receptor is fused to both heavy chains to contain the amino acid sequence of SEQ ID NO:23, and the two light chains contain the amino acid sequence of SEQ ID NO:18.

[0020] In some embodiments of any of the above-described fusion proteins, the extracellular domain of the TGFβ superfamily receptor is fused to both light chains to contain the amino acid sequence of SEQ ID NO:27, and both heavy chains contain the amino acid sequence of SEQ ID NO:16 or 17.

[0021] In some embodiments of any of the above-described fusion proteins, the extracellular domain of the TGFβ superfamily receptor is fused to both light chains to contain the amino acid sequence of SEQ ID NO:28, and both heavy chains contain the amino acid sequence of SEQ ID NO:16 or 17.

[0022] In some embodiments of any of the above-described fusion proteins, the extracellular domain of the TGFβ superfamily receptor is fused with an amino acid sequence comprising SEQ ID NO:22, and the extracellular domain of the TGFβ superfamily receptor is also fused with an amino acid sequence comprising SEQ ID NO:28, comprising two light chains.

[0023] In some embodiments of any of the above-described fusion proteins, the extracellular domain of the TGFβ superfamily receptor is fused with both heavy chains to form an amino acid sequence comprising SEQ ID NO:23, and the extracellular domain of the TGFβ superfamily receptor is also fused with both light chains to form an amino acid sequence comprising SEQ ID NO:27.

[0024] In some embodiments of any of the above-described fusion proteins, the extracellular domain of the TGFβ superfamily receptor is fused with both heavy chains to form an amino acid sequence comprising SEQ ID NO:22, and the extracellular domain of the TGFβ superfamily receptor is also fused with both light chains to form an amino acid sequence comprising SEQ ID NO:27.

[0025] In some embodiments of any of the above-described fusion proteins, the extracellular domain of the TGFβ superfamily receptor is fused with an amino acid sequence comprising SEQ ID NO:23, and the extracellular domain of the TGFβ superfamily receptor is also fused with an amino acid sequence comprising SEQ ID NO:28, comprising two light chains.

[0026] In some embodiments of any of the above-described fusion proteins, the polypeptide is fused with both heavy chains to an amino acid sequence comprising SEQ ID NO:24, and both light chains comprise an amino acid sequence comprising SEQ ID NO:18.

[0027] In some embodiments of any of the above-described fusion proteins, the extracellular domain of the TGFβ superfamily receptor is fused to both heavy chains to contain the amino acid sequence of SEQ ID NO:25, and both light chains contain the amino acid sequence of SEQ ID NO:18.

[0028] In some embodiments of any of the above-described fusion proteins, the extracellular domain of the TGFβ superfamily receptor is fused to both heavy chains to contain the amino acid sequence of SEQ ID NO:26, and both light chains contain the amino acid sequence of SEQ ID NO:18.

[0029] In some embodiments of any of the above-described fusion proteins, the extracellular domain of the TGFβ superfamily receptor is fused to both light chains to form an amino acid sequence containing SEQ ID NO:29, and both heavy chains contain an amino acid sequence containing SEQ ID NO:16 or 17.

[0030] In some embodiments of any of the above-described fusion proteins, the extracellular domain of the TGFβ superfamily receptor is fused to both light chains to contain the amino acid sequence of SEQ ID NO:30, and both heavy chains contain the amino acid sequence of SEQ ID NO:16 or 17.

[0031] In some embodiments of any of the above-described fusion proteins, the extracellular domain of the TGFβ superfamily receptor is fused to both light chains to contain the amino acid sequence of SEQ ID NO:31, and both heavy chains contain the amino acid sequence of SEQ ID NO:16 or 17.

[0032] In some embodiments of any of the above-described fusion proteins, the extracellular domain of the TGFβ superfamily receptor is fused to both light chains to contain the amino acid sequence of SEQ ID NO:32, and both heavy chains contain the amino acid sequence of SEQ ID NO:16 or 17.

[0033] In some embodiments of any of the above-described fusion proteins, the extracellular domain of the TGFβ superfamily receptor is fused to both light chains to contain the amino acid sequence of SEQ ID NO:33, and both heavy chains contain the amino acid sequence of SEQ ID NO:16 or 17.

[0034] In some embodiments of any of the above-described fusion proteins, the extracellular domain of the TGFβ superfamily receptor is fused to both light chains to contain the amino acid sequence of SEQ ID NO:34, and both heavy chains contain the amino acid sequence of SEQ ID NO:16 or 17.

[0035] In some embodiments of any of the above-described fusion proteins, the extracellular domain of the TGFβ superfamily receptor is fused to both light chains to contain the amino acid sequence of SEQ ID NO:35, and both heavy chains contain the amino acid sequence of SEQ ID NO:16 or 17.

[0036] In some embodiments of any of the above-described fusion proteins, the extracellular domain of the TGFβ superfamily receptor is fused to both light chains to contain the amino acid sequence of SEQ ID NO:36, and both heavy chains contain the amino acid sequence of SEQ ID NO:16 or 17.

[0037] In some embodiments of any of the above-described fusion proteins, the extracellular domain of the TGFβ superfamily receptor is fused to both light chains to contain the amino acid sequence of SEQ ID NO:37, and both heavy chains contain the amino acid sequence of SEQ ID NO:16 or 17.

[0038] In another aspect, this application provides a polypeptide comprising a variant of the human TGFβ receptor type II extracellular domain, the variant having at least about 90% (including, for example, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:2, wherein the variant contains one or more amino acid substitutions at positions N47, N71, and / or N131 according to the numbers in SEQ ID NO:2. In some embodiments, the variant comprises the amino acid sequence of SEQ ID NO:3.

[0039] In another aspect, this application provides a fusion protein comprising a) any one of the aforementioned polypeptides; and b) a second portion. In some embodiments, the second portion comprises a half-life extension domain. In some embodiments, the second portion comprises an Fc fragment. In some embodiments, the second portion comprises an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor comprises an anti-PD-L1 antibody portion.

[0040] On the other hand, this application provides a pharmaceutical composition comprising either a fusion protein or a polypeptide.

[0041] On the other hand, this application provides a nucleic acid that encodes a fusion protein or a portion thereof, or any of the aforementioned polypeptides.

[0042] On the other hand, this application provides a nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:19-21 and 38-53.

[0043] On the other hand, this application provides a vector comprising any one of the aforementioned nucleic acids.

[0044] On the other hand, this application provides a host cell containing any one of the aforementioned nucleic acids or vectors.

[0045] On the other hand, this application provides a method for generating any one of the above-mentioned fusion proteins or polypeptides, the method comprising: a) culturing the host cells under conditions that effectively express the fusion protein or polypeptide; and b) obtaining the expressed fusion protein or polypeptide from the host cells.

[0046] On the other hand, this application provides a method for treating an individual's disease or condition, the method comprising administering to the individual an effective amount of any of the above-described fusion protein or pharmaceutical composition. In some embodiments, the disease or condition is cancer. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), cervical cancer, oropharyngeal cancer, anal cancer, vaginal or penile cancer, biliary tract cancer, bile duct cancer, gallbladder cancer, head and neck cancer, pancreatic cancer, prostate cancer, urothelial carcinoma, bladder cancer, urinary system tumors, urogenital tract cancer, gastric cancer, breast cancer, or colorectal cancer. In some embodiments, the cancer is advanced or metastatic cancer. In some embodiments, the method further comprises administering a second agent or therapy. In some embodiments, the fusion protein or pharmaceutical composition is administered parenterally to the individual. In some embodiments, the individual is a person.

[0047] On the other hand, this application provides a kit for treating a disease or condition, comprising any one of the pharmaceutical compositions and instructions for use. Attached Figure Description

[0048] Figure 1 The binding affinity of various fusion proteins to TGFβ1 was demonstrated.

[0049] Figure 2 The binding affinity of various fusion proteins to TGFβ2 was demonstrated.

[0050] Figure 3 The binding affinity of various fusion proteins to TGFβ3 was demonstrated.

[0051] Figure 4 The binding affinity of various fusion proteins to PD-L1 was demonstrated.

[0052] Figure 5 The results of the PD-1 / PD-L1 blocking assay are shown in Example 2.

[0053] Figure 6 The binding affinity of various fusion proteins to TGFβ1 was demonstrated.

[0054] Figure 7 The binding affinity of various fusion proteins to TGFβ2 was demonstrated.

[0055] Figure 8 The binding affinity of various fusion proteins to TGFβ3 was demonstrated.

[0056] Figure 9 The binding affinity of various fusion proteins to PD-L1 was demonstrated.

[0057] Figure 10 The results of the PD-1 / PD-L1 blocking assay are shown in Example 2.

[0058] Figure 11 The results of the PD-1 / PD-L1 blocking assay are shown in Example 2. Detailed Implementation

[0059] This application provides a fusion protein comprising one or more TGFβ superfamily receptor extracellular domains. In some embodiments, the fusion protein comprises a) a full-length anti-PD-L1 antibody and b) a TGFβ superfamily receptor extracellular domain (such as a type I or type II extracellular domain or a variant thereof). In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to the C-terminus of one or both of the light chains of the full-length anti-PD-L1 antibody. In some embodiments, the full-length anti-PD-L1 antibody comprises a) two heavy chains, each comprising a heavy chain variable region (V... H The heavy chain variable region comprises: i) HC-CDR1 containing the amino acid sequence of SEQ ID NO:6; ii) HC-CDR2 containing the amino acid sequence of SEQ ID NO:7; and iii) HC-CDR3 containing the amino acid sequence of SEQ ID NO:8, and b) two light chains, each containing a light chain variable region (V L The light chain variable region comprises: i) LC-CDR1 containing the amino acid sequence of SEQ ID NO:9; ii) LC-CDR2 containing the amino acid sequence of SEQ ID NO:10; and iii) LC-CDR3 containing the amino acid sequence of SEQ ID NO:11. This application also provides a human TGFβ receptor type II extracellular domain variant comprising an amino sequence having at least about 90% sequence identity with SEQ ID NO:2, wherein the variant contains one or more amino acid substitutions at positions N47, N71, and / or N131 according to the numbering of SEQ ID NO:2. This application also provides methods and therapeutic methods for preparing the fusion proteins or peptides described herein.

[0060] This application is based, at least in part, on the advantageous effects shown by the fusion proteins described herein (such as those comprising a full-length anti-PD-L1 antibody and a TGFβ type II receptor fused to the light chain of the anti-PD-L1 antibody) compared to reference fusion proteins (such as M7824). Specifically, the fusion proteins described herein exhibit a lower binding affinity for TGFβ2, which plays a physiological role in normal cardiac function and early hematopoiesis, thereby reducing toxicity. See, for example, Bhandary et al., J AmHeart Assoc. 2018 Oct 16; 7(20):e010013.

[0061] I. Definition

[0062] The techniques and procedures described or referenced herein include those well understood and / or commonly employed by those skilled in the art using conventional methods, such as those widely used methods described in: Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition, 2001); Current Protocols in Molecular Biology (edited by Ausubel et al., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (edited by An, 2009); Monoclonal Antibodies Methods and Protocols (Edited by Albitar 2010); and Antibody Engineering Vols 1 and 2 (edited by Kontermann and Dübel, 2nd edition 2010).

[0063] Unless otherwise defined herein, the technical and scientific terms used in this invention have the meanings commonly understood by one of ordinary skill in the art. For the purposes of interpreting this specification, the following description of terms will apply, and where appropriate, terms used in the singular also include the plural and vice versa. In the event of any conflict between any description of the listed terms and any reference incorporated herein by reference, the description of the listed terms shall prevail.

[0064] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a binding protein, such as an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a binding molecule X for its binding partner Y can typically be expressed using the dissociation constant (K). D ( ) indicates. Affinity can be measured by methods commonly known in the art, including those described herein. Low-affinity antibodies typically bind to antigens slowly and tend to dissociate easily, while high-affinity antibodies typically bind to antigens more quickly and tend to maintain binding for a longer period. A variety of methods for measuring binding affinity are known in the art, any of which can be used for the purposes of this disclosure. Specific illustrative embodiments include the following. In one embodiment, “K D "or "K D The value can be measured by methods known in the art (e.g., by combination assays). K DIt can be measured in RIA, for example, using the Fab version of the target antibody and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81). K D or K D Values ​​can also be obtained by using (e.g., using) Red96 system) or (e.g., using) TM-2000 or The biolayer interferometry (BLI) or surface plasmon resonance (SPR) measurement of the TM-3000 can be used for measurement. The "association rate (on-rate or rate of association or association rate or kon)" can also be measured using, for example, as described above. Red96 TM-2000, or The same biolayer interferometry (BLI) or surface plasmon resonance (SPR) technique used in the TM-3000 system is used to determine this.

[0065] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length. Polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acid groups. These terms also cover amino acid polymers that have been naturally modified or modified by interventions such as disulfide bond formation, glycosylation, lipoylation, acetylation, phosphorylation, or any other manipulation or modification. This definition also includes polypeptides containing, for example, one or more amino acid analogs (including, but not limited to, non-natural amino acids) and other modifications known in the art. It should be understood that, because the polypeptides disclosed herein may be based on antibodies or other members of the immunoglobulin superfamily, in some embodiments, a “polypeptide” may exist as a single chain or two or more related chains.

[0066] The term "antibody" is used in its broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and their antigen-binding fragments, provided they exhibit the desired antigen-binding activity. The term "antibody moiety" refers to a full-length antibody or its antigen-binding fragment.

[0067] Full-length antibodies consist of two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. These variable domains can be referred to as "V" domains, respectively. H " and "V LThe variable regions in both chains typically contain three highly variable loops called complementarity-determining regions (CDRs) (including light chain (LC) CDRs of LC-CDR1, LC-CDR2, and LC-CDR3, and heavy chain (HC) CDRs of HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibody-antigen binding fragments disclosed herein can be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat (1991). The three CDRs of the heavy or light chain are located between flanking segments called the framework region (FR), which is more conserved than the CDRs and forms a scaffold to support the hypervariable loop. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are classified based on the amino acid sequence of the antibody heavy chain constant region. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are subclassed, such as lgG1 (γ1 heavy chain), lgG2 (γ2 heavy chain), lgG3 (γ3 heavy chain), lgG4 (γ4 heavy chain), lgA1 (α1 heavy chain), or lgA2 (α2 heavy chain).

[0068] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, including, for example, biantibodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized biantibodies (ds biantibodies), single-chain Fv (scFv), scFv dimers (bivalent biantibodies), multispecific antibodies formed from a portion of an antibody containing one or more CDRs, single-domain antibodies (e.g., camelified single-domain antibodies), nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds an antigen but does not contain the complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen that binds to a parent antibody or a parent antibody fragment (e.g., a parental scFv). In some embodiments, an antigen-binding fragment may contain one or more CDRs from a particular human antibody grafted onto a frame region from one or more different human antibodies.

[0069] A "single-chain Fv" (also abbreviated as "sFv" or "scFv") is a V-shaped structure containing a single polypeptide chain. H and V L Antibody fragments containing antibody domains. In some embodiments, the scFv polypeptide further includes V H and V LA polypeptide linker between the domains allows scFv to form the desired structure for antigen binding. For a review of scFv, see Plückthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994).

[0070] As used herein, the term “CDR” or “complementarity-determining region” is intended to refer to a discontinuous antigen-binding site found within the variable region of heavy and light chain polypeptides. These specific regions have been described by the following: Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., USDept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc MP et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001), where the definitions include overlap or subsets when comparing amino acid residues with each other. However, the application of any definition to refer to an antibody or transplanted antibody or its variants is intended to fall within the scope of the terminology as defined and used herein. For comparison, Table 1 below lists the amino acid residues that cover the CDRs defined in each of the above-cited references. CDR prediction algorithms and interfaces are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38:D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43:D432-D438 (2015). The references cited in this paragraph are incorporated herein by reference in their entirety for use in this application and may be included in one or more claims herein.

[0071] Table 1: CDR Definition

[0072]

[0073] 1 Residue numbering follows the nomenclature of Kabat et al., as above.

[0074] 2Residue numbering follows the nomenclature of Chothia et al., as above.

[0075] 3 Residue numbering follows the nomenclature of MacCallum et al., as above.

[0076] 4 Residue numbering follows the nomenclature of Lefranc et al., as above.

[0077] 5 Residue numbering follows the Honegger and Plückthun nomenclature, as above.

[0078] The expressions “as in Kabat variable domain residue numbering” or “as in Kabat amino acid position numbering” and their variations refer to the numbering system used for the heavy chain variable domain or light chain variable domain in the antibody compilation by Kabat et al. above. Using this numbering system, the actual straight-chain amino acid sequence may contain fewer or additional amino acids corresponding to the shortened or inserted FR or hypervariable region (HVR) of the variable domain. For example, the heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (according to residue 52a in Kabat) and an inserted residue after FR residue 82 of the heavy chain (e.g., residues 82a, 82b, and 82c, etc., according to Kabat). The Kabat number of a given antibody residue can be determined by comparing the antibody sequence with homology regions of a “standard” Kabat numbered sequence.

[0079] Unless otherwise stated herein, the residue numbers in the immunoglobulin heavy chain are those of the EU index described above by Kabat et al., but with slight modifications. In short, we added more than five residues to the hypervariable loop preceding CDR1 in the heavy chain. "EU index as described by Kabat" refers to the residue numbers of the human IgG1 EU antibody.

[0080] “Frame” or “FR” residues are those variable domain residues other than CDR residues as defined in this paper.

[0081] The “humanized” form of non-human (e.g., rodent) antibodies is a chimeric antibody containing a minimal sequence derived from the non-human antibody. In most cases, humanized antibodies are human immunoglobulins (recipient antibodies), where residues from the recipient hypervariable region (HVR) are replaced by residues from a hypervariable region (donor antibody) of a non-human species (e.g., mouse, rat, rabbit, or non-human primate) with the desired antibody specificity, affinity, and capacity. In some cases, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in either the recipient or donor antibody. These modifications are made to further improve antibody performance. Typically, humanized antibodies will contain at least one, and typically substantially all, of the two variable domains, where all or substantially all of the hypervariable loops correspond to those of the non-human immunoglobulin, and all or substantially all of the FRs are those of the human immunoglobulin sequence. Humanized antibodies will also optionally contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of human immunoglobulins. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0082] A “human antibody” is an antibody that has an amino acid sequence corresponding to that of an antibody produced by humans and / or has been produced using any of the techniques disclosed herein for the manufacture of human antibodies. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Methods also used to prepare human monoclonal antibodies are those described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared by administering antigens to transgenic animals (e.g., immunized xenomic mice) that have been modified to produce such antibodies in response to antigen challenge but whose endogenous loci have been deactivated (see, for example, XENOMOUSE).TM The technology is referenced in U.S. patents 6,075,181 and 6,150,584. For further information on human antibodies produced via human B-cell hybridoma technology, see, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006).

[0083] The “percentage of amino acid sequence identity (%)” or “homology” for the peptide and antibody sequences identified herein is defined as the percentage of amino acid residues in the candidate sequence that are identical to amino acid residues in the compared peptide after sequence alignment (with any conserved substitutions as part of the sequence identity). For the purpose of determining the percentage of amino acid sequence identity, alignment can be performed in a variety of ways in the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm that requires maximizing alignment across the full length of the compared sequences. However, for the purposes of this article, the amino acid sequence identity % value was generated using the sequence comparison computer program MUSCLE (Edgar, RC, Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5(1):113, 2004).

[0084] "Homologous" refers to the sequence similarity or identity between two polypeptides or two nucleic acid molecules. Two compared sequences are homologous when a position is occupied by the same base or amino acid monomer subunit; for example, if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared multiplied by 100. For example, if 6 out of 10 positions in two sequences are matching or homologous, then the two sequences are 60% homologous. For instance, the DNA sequences ATTGCC and TATGGC have 50% homology. Typically, two sequences are compared when the alignment yields the greatest possible homology.

[0085] The term "constant domain" refers to a portion of an immunoglobulin molecule that, compared to another portion, the variable domain, has a more conserved amino acid sequence containing antigen-binding sites. Constant domains contain the C16 heavy chain. H 1. C H 2 and C H 3. Structural domains (collectively referred to as C) H) and light chain CHL (or C L ) structural domain.

[0086] The "light chain" of antibodies (immunoglobulins) in any mammalian species can be designated as one of two distinct types based on the amino acid sequence of their constant domains, referred to as kappa ("κ") and lambda ("λ").

[0087] The “CH1 domain” (also known as the “C1” of the “H1” domain) typically ranges from about amino acid 118 to about amino acid 215 (EU numbering system).

[0088] The "hinge region" is generally defined as the region in IgG corresponding to Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). Hinge regions of other IgG isotypes can be compared with the IgG1 sequence by placing the first and last cysteine ​​residues that form the heavy chain SS bond in the same position.

[0089] The CH2 domain of the human IgG Fc region (also known as the “C2” domain) typically extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique because it does not pair tightly with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the intact native IgG molecule. It is speculated that the carbohydrates can provide a substitute for domain-domain pairing and contribute to the stability of the CH2 domain. (Burton, Molec Immunol. 22:161-206 (1985)).

[0090] The “CH3 domain” (also known as the “C2” domain) contains the extension of the CH2 domain from the C-terminal residue to the Fc region (i.e. from about amino acid residue 341 to the C-terminus of the antibody sequence (typically at amino acid residues 446 or 447 of IgG)).

[0091] The term "Fc region" or "fragment crystallizable region" used herein is used to define the C-terminal region of the immunoglobulin heavy chain, including the native sequence Fc region and variant Fc regions. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary, the Fc region of the human IgG heavy chain is generally defined as an amino acid residue extending from the Cys226 position or from Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during antibody production or purification or by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Thus, compositions of complete antibodies can comprise antibody populations with all K447 residues removed, antibody populations without K447 residues removed, and antibody populations having a mixture of antibodies with and without K447 residues. Suitable native sequence Fc regions for the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0092] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. Preferred FcRs are naturally occurring human FcRs. Furthermore, preferred FcRs are those that bind IgG antibodies (γ receptors) and include FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences, differing primarily in their intracellular domains. The activating receptor FcγRIIA contains an activation motif (ITAM) based on the immunoreceptor tyrosine residue in its intracellular domain. The inhibitory receptor FcγRIIB contains an inhibitory motif (ITIM) based on the immunoreceptor tyrosine residue in its intracellular domain. (See M.) Annu.Rev.Immunol.15:203-234 (1997)). FcR is reviewed in Ravetch and Kinet, Annu.Rev.Immunol.9:457-92 (1991); Capel et al., Immunomethods4:25-34 (1994); and de Haas et al., J.Lab.Clin.Med.126:330-41 (1995). The term “FcR” used in this paper covers other FcRs, including those to be identified in the future.

[0093] As used herein, the term "epitope" refers to a specific atom or amino acid group on an antigen to which an antibody or antibody moiety binds. If two antibodies or antibody moietyes competitively bind to an antigen, they can bind to the same epitope within the antigen.

[0094] As used herein, when a first antibody or fragment thereof, in the presence of an equimolar concentration, inhibits the binding of a second antibody or fragment thereof to the target antigen by at least about 50% (e.g., at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%), the first antibody or fragment thereof “competes” with the second antibody or fragment thereof to bind to the target antigen, or vice versa. A high-throughput method for “binding” these antibodies based on antibody cross-competition is described in PCT Publication No. WO 03 / 48731.

[0095] As used herein, the terms “specific binding,” “specific recognition,” and “specific to” refer to measurable and reproducible interactions, such as the binding between a target and an antibody or antibody moiety, which determines the presence of the target in the presence of a population of heterogeneous molecules, including biomolecules. For example, an antibody or antibody moiety that specifically recognizes a target (which may be an epitope) is an antibody or antibody moiety that binds to that target with superior affinity, avidity, readiness, and / or duration of binding to other targets. In some embodiments, the degree of binding of an antibody to an unrelated target is less than, for example, about 10% of the antibody-target binding as measured by radioimmunoassay (RIA). In some embodiments, the dissociation constant (Ki) of an antibody that specifically binds to a target is... D ≤10 -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M, or ≤10 -12 M. In some embodiments, antibody-specific binding occurs on epitopes of proteins that are conserved for proteins from different species. In some embodiments, specific binding may include, but is not required to be, exclusive binding. The binding specificity of the antibody or antigen-binding domain can be determined experimentally by methods known in the art. Such methods include, but are not limited to, Western blotting, ELISA, RIA, ECL, IRMA, EIA, and BIACORE. TM - Inspection and peptide scanning.

[0096] "Isolated" antibodies (or constructs) are antibodies that have been identified, isolated, and / or recovered from components (e.g., natural or recombinant) in their production environment. Preferably, the isolated peptide does not associate with any other components in its production environment.

[0097] Where used interchangeably herein, “polynucleotide” and “nucleic acid” refer to polymers of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogues, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and their analogues. As used herein, “oligonucleotide” refers to a short, usually single-stranded, synthetic polynucleotide whose length is typically, but not necessarily, less than about 200 nucleotides. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The above description of polynucleotides applies equivalently and entirely to oligonucleotides.

[0098] The “isolated” nucleic acid molecule encoding the constructs, antibodies, or antigen-binding fragments thereof described herein is a nucleic acid molecule identified and isolated from at least one contaminant nucleic acid molecule typically associated with it in its production environment. Preferably, the isolated nucleic acid does not associate with any components related to the production environment. The isolated nucleic acid molecule encoding the polypeptides and antibodies described herein is in a form different from its naturally occurring form or background. Therefore, the isolated nucleic acid molecule is different from the nucleic acid encoding the polypeptides and antibodies described herein that are naturally present in cells. The isolated nucleic acid includes the nucleic acid molecule typically contained in cells, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

[0099] When a nucleic acid is functionally related to another nucleic acid sequence, that nucleic acid is "operably linked." For example, if the DNA of a pre-sequence or secretory leader sequence is expressed as a pre-protein involved in polypeptide secretion, then the DNA of the pre-sequence or secretory leader sequence is operably linked to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to that sequence; or if a ribosome binding site is localized to facilitate translation, then the ribosome binding site is operably linked to the coding sequence. Generally, "operably linked" means that the linked DNA sequences are contiguous, and in the case of a secretory leader sequence, they are contiguous and within the reading frame. However, enhancers do not need to be contiguous. Linkage is achieved by linking at a convenient restriction site. If such a site is not available, synthetic oligonucleotide adaptors or linkers are used according to conventional practice.

[0100] As used herein, the term "vector" refers to a nucleic acid molecule capable of replicating another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures, as well as vectors incorporated into the genome of a host cell (in which the vector has been introduced). Some vectors are capable of directing the expression of the nucleic acid to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0101] As used herein, the terms “transfected,” “transformed,” or “transduced” refer to the process of transferring or introducing exogenous nucleic acids into host cells. “Transfected,” “transformed,” or “transduced” cells are cells that have been transfected, transformed, or transduced using exogenous nucleic acids. These cells include primary target cells and their progeny.

[0102] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including progeny of such cells. Host cells include “transformations” and “transformed cells,” encompassing primary transformed cells and their derived progeny, regardless of passage number. The nucleic acid content of progeny cells may not be identical to that of the parent cells, and they may contain mutations. Progeny cells with mutations exhibiting the same function or biological activity as those screened or selected in the original transformed cells are included herein.

[0103] As used herein, “treatment” is a method for achieving a beneficial or desired outcome (including clinical outcomes). For the purposes of this application, beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: relieving one or more symptoms caused by a disease, reducing the severity of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, providing remission of the disease (partial or complete), reducing the dosage of one or more other medications required to treat the disease, delaying the progression of the disease, increasing or improving quality of life, increasing weight, and / or prolonging survival. “Treatment” also encompasses reducing pathological outcomes of cancer (e.g., tumor volume). The methods of this application consider any one or more of these treatment aspects.

[0104] In the context of cancer, the term "treatment" includes any or all of the following: inhibiting the growth of cancer cells, inhibiting the replication of cancer cells, reducing the overall tumor burden, and improving one or more symptoms associated with the disease.

[0105] The term "inhibition" or "inhibit" refers to a reduction or cessation of any phenotypic trait, or a reduction or cessation of the occurrence, extent, or likelihood of that trait. Compared to the reference, "reduction" or "inhibit" refers to a reduction, decrease, or prevention of activity, function, and / or quantity. In some embodiments, "reduction" or "inhibit" refers to the ability to cause an overall reduction of 20% or greater. In another embodiment, "reduction" or "inhibit" refers to the ability to cause an overall reduction of 50% or greater. In yet another embodiment, "reduction" or "inhibit" refers to the ability to cause an overall reduction of 75%, 85%, 90%, 95%, or greater.

[0106] As used herein, “reference” means any sample, standard, or level used for comparative purposes. References may be obtained from healthy and / or disease-free samples. In some instances, references may be obtained from untreated samples. In some instances, references may be obtained from disease-free or untreated samples of an individual. In some instances, references may be obtained from one or more healthy individuals who are not that person or patient.

[0107] As used herein, “delaying the development of a disease” means postponing, hindering, slowing, stabilizing, inhibiting, and / or delaying the development of a disease (e.g., cancer). This delay can vary in length, depending on the history of the disease and / or the individual being treated. It will be apparent to those skilled in the art that a sufficient or significant delay can effectively cover prevention, as the individual has not developed the disease. For example, it may delay the development of advanced cancer (e.g., metastasis).

[0108] As used in this article, “prevention” includes providing prevention of the onset or recurrence of the disease in individuals who may be susceptible to the disease but have not yet been diagnosed with it.

[0109] As used herein, "inhibition" function or activity is a reduction in function or activity compared to the same condition other than the condition or parameter of interest, or alternatively compared to another condition. For example, an antibody that inhibits tumor growth reduces the rate of tumor growth compared to the rate of tumor growth in the absence of the antibody.

[0110] The terms “subject,” “individual,” and “patient” are used interchangeably herein and refer to mammals, including but not limited to humans, cattle, horses, cats, dogs, rodents, or primates. In some embodiments, an individual is a human.

[0111] The “effective dose” of a drug refers to the amount that effectively achieves the desired therapeutic outcome within the necessary dosage and time period. A specific dose may vary depending on one or more of the following: the specific drug chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to be imaged, and the physical delivery system that carries it.

[0112] The "therapeutic effective amount" of the substance / molecule, agonist, or antagonist of this application may vary depending on factors such as disease state, age, sex, individual weight, and the ability of the substance / molecule, agonist, or antagonist to elicit a desired response in an individual. Therapeutic effective amount is also the amount at which any toxic or harmful effects of the substance / molecule, agonist, or antagonist are offset by the beneficial therapeutic effects. Therapeutic effective amount may be delivered by single or multiple administrations.

[0113] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a formulation in a form that allows for the bioactivity of one or more active ingredients and does not contain other components that would have unacceptable toxicity to the individual to whom the formulation will be administered. Such formulations may be sterile.

[0114] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation aid, or carrier that is commonly used in the art to be used together with therapeutic agents to form a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dose and concentration used and is compatible with other components of the formulation. A pharmaceutically acceptable carrier is suitable for use in the formulation.

[0115] "Sterile" preparations are sterilized or contain virtually no live microorganisms or their spores.

[0116] Administering in combination with one or more other therapeutic agents includes simultaneous (parallel) and sequential administration or administration in any order.

[0117] The term “parallel” is used herein to refer to the administration of two or more therapeutic agents, wherein at least part of the administration is time-overlapping or wherein one therapeutic agent is administered within a short period of time during which one therapeutic agent is administered. For example, two or more therapeutic agents are administered at intervals not exceeding about 60 minutes (e.g., not exceeding any one of about 30, 15, 10, 5, or 1 minute).

[0118] The term “in sequence” is used in this document to refer to the administration of two or more therapeutic agents, wherein the administration of one or more agents continues after the administration of one or more other agents has been discontinued. For example, the administration of two or more therapeutic agents at intervals of more than about 15 minutes, such as about 20, 30, 40, 50 or 60 minutes, 1 day, 2 days, 3 days, 1 week, 2 weeks or 1 month or longer.

[0119] As used in this article, "in combination with" means applying another treatment in addition to one treatment. Similarly, "in combination with" means applying another treatment before, during, or after applying one treatment to an individual.

[0120] The term "packaging insert" refers to the instruction leaflet typically included in the commercial packaging of a therapeutic product, which contains information about the indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings for using such a therapeutic product.

[0121] "Article of manufacture" is any article (e.g., packaging or container) or kit containing at least one reagent, such as a medicine for treating a disease or disorder (e.g., cancer) or a probe for specifically detecting a biomarker described herein. In some embodiments, the article or kit is promoted, distributed, or sold as a unit for performing the methods described herein.

[0122] It should be understood that the embodiments described herein include “consisting of embodiments” and / or “substantially consisting of embodiments”.

[0123] References to “about” values ​​or parameters in this document include (and describe) variations of that value or parameter itself. For example, a description involving “about X” includes a description of “X”.

[0124] As used in this article, references to "not" values ​​or parameters generally mean and describe something that is "different" from that value or parameter. For example, "the method is not for treating type X cancer" means that the method is for treating cancers other than type X.

[0125] The term “about XY” used in this article has the same meaning as “about X to about Y”.

[0126] When used in this document and the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly indicates otherwise.

[0127] II. Fusion protein and TGFβR extracellular domain peptide

[0128] This application provides fusion proteins comprising one or more TGFβ superfamily receptor extracellular domains (as described herein) and a second portion. In some embodiments, the fusion protein further comprises an anti-PD-L1 antibody portion (such as a full-length anti-PD-L1 antibody). In some embodiments, the fusion protein further comprises a half-life extension portion.

[0129] A fusion protein containing the extracellular domain of the TGFβ superfamily receptor and the anti-PD-L1 antibody moiety.

[0130] This application provides, in one aspect, fusion proteins comprising an extracellular domain of a TGFβ superfamily receptor (any of the TGFβ superfamily receptor extracellular domains described herein) and an anti-PD-L1 antibody moiety (any of the anti-PD-L1 antibody moiety described herein).

[0131] In some embodiments, a fusion protein is provided comprising a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V). H Furthermore, each of the two light chains contains a light chain variable region (V). L b) TGFβ superfamily receptor extracellular domain; wherein the TGFβ superfamily receptor extracellular domain is fused to at least one or both of the heavy or light chains of the anti-PD-L1 antibody. In some embodiments, the full-length antibody comprises an Fc fragment derived from human IgG1. In some embodiments, the Fc fragment derived from human IgG1 comprises a K214R mutation and an A297N mutation. In some embodiments, the TGFβ superfamily receptor extracellular domain comprises a TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the TGFβ receptor type II extracellular domain or a variant thereof comprises a human TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type II extracellular domain or a variant thereof comprises a human TGFβ receptor type II extracellular domain variant having at least about 90% sequence identity with SEQ ID NO:2, wherein the variant contains one or more amino acid substitutions at positions N47, N71, and / or N131 according to the numbers in SEQ ID NO:2. In some embodiments, the variant comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the TGFβ superfamily receptor extracellular domain comprises the TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the TGFβ receptor type I extracellular domain or a variant thereof comprises the human TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type I extracellular domain or a variant thereof comprises the amino acid sequence of SEQ ID NO:1 or a variant having at least about 90% sequence identity. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to at least one of the heavy or light chains of an anti-PD-L1 antibody via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is about 2 to about 25 amino acids in length (e.g., about 2 to about 10 amino acids). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:56-68.

[0132] In some embodiments, a fusion protein is provided comprising a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V). H Furthermore, each of the two light chains contains a light chain variable region (V). La) TGFβ superfamily receptor extracellular domain; wherein the TGFβ superfamily receptor extracellular domain is fused to at least one or both of the light chains of the anti-PD-L1 antibody. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to the C-terminus of at least one or both of the two light chains. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to the N-terminus of at least one or both of the two light chains. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to both the N-terminus and C-terminus of at least one or both of the two light chains. In some embodiments, the full-length antibody comprises an Fc fragment derived from human IgG1. In some embodiments, the Fc fragment derived from human IgG1 comprises a K214R mutation and an A297N mutation. In some embodiments, the TGFβ superfamily receptor extracellular domain comprises a TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the TGFβ receptor type II extracellular domain or a variant thereof comprises a human TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type II extracellular domain or a variant thereof comprises a human TGFβ receptor type II extracellular domain variant having at least about 90% sequence identity with SEQ ID NO:2, wherein the variant contains one or more amino acid substitutions at positions N47, N71, and / or N131 according to the numbering of SEQ ID NO:2. In some embodiments, the variant comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the TGFβ superfamily receptor extracellular domain comprises the TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the TGFβ receptor type I extracellular domain or a variant thereof comprises the human TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type I extracellular domain or a variant thereof comprises the amino acid sequence of SEQ ID NO:1 or a variant having at least about 90% sequence identity. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to at least one of the heavy or light chains of the anti-PD-L1 antibody via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is about 2 to about 25 amino acids in length (e.g., about 2 to about 10 amino acids). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:56-68.

[0133] In some embodiments, a fusion protein is provided comprising a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V). H Furthermore, each of the two light chains contains a light chain variable region (V). La) TGFβ superfamily receptor extracellular domain; wherein the TGFβ superfamily receptor extracellular domain is fused to at least one or both of the heavy chains of the anti-PD-L1 antibody. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to the C-terminus of at least one or both of the two heavy chains. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to the N-terminus of at least one or both of the two heavy chains. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to both the N-terminus and C-terminus of at least one or both of the two heavy chains. In some embodiments, the full-length antibody comprises an Fc fragment derived from human IgG1. In some embodiments, the Fc fragment derived from human IgG1 comprises a K214R mutation and an A297N mutation. In some embodiments, the TGFβ superfamily receptor extracellular domain comprises a TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the TGFβ receptor type II extracellular domain or a variant thereof comprises a human TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type II extracellular domain or a variant thereof comprises a human TGFβ receptor type II extracellular domain variant having at least about 90% sequence identity with SEQ ID NO:2, wherein the variant contains one or more amino acid substitutions at positions N47, N71, and / or N131 according to the numbering of SEQ ID NO:2. In some embodiments, the variant comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the TGFβ superfamily receptor extracellular domain comprises the TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the TGFβ receptor type I extracellular domain or a variant thereof comprises the human TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type I extracellular domain or a variant thereof comprises the amino acid sequence of SEQ ID NO:1 or a variant having at least about 90% sequence identity. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to at least one of the heavy or light chains of the anti-PD-L1 antibody via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is about 2 to about 25 amino acids in length (e.g., about 2 to about 10 amino acids). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:56-68.

[0134] In some embodiments, a fusion protein is provided comprising a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V). H Furthermore, each of the two light chains contains a light chain variable region (V). Lb) The TGFβ superfamily receptor extracellular domain; wherein the TGFβ superfamily receptor extracellular domain is fused with: i) at least one or two of the light chains of the anti-PD-L1 antibody, and ii) at least one or two of the heavy chains of the anti-PD-L1 antibody. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused with the C-terminus of i) the two heavy chains and ii) the two light chains. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused with the N-terminus of i) the two heavy chains and ii) the two light chains. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused with: i) the N-terminus of the two heavy chains, and ii) the C-terminus of the two light chains. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused with: i) the C-terminus of the two heavy chains, and ii) the N-terminus of the two light chains. In some embodiments, the full-length antibody comprises an Fc fragment derived from human IgG1. In some embodiments, the Fc fragment derived from human IgG1 comprises a K214R mutation and an A297N mutation. In some embodiments, the TGFβ superfamily receptor extracellular domain comprises the TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the TGFβ receptor type II extracellular domain or a variant thereof comprises the human TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type II extracellular domain or a variant thereof comprises a human TGFβ receptor type II extracellular domain variant having at least about 90% sequence identity with SEQ ID NO:2, wherein the variant contains one or more amino acid substitutions at positions N47, N71, and / or N131 according to the numbering of SEQ ID NO:2. In some embodiments, the variant comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the TGFβ superfamily receptor extracellular domain comprises the TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the TGFβ receptor type I extracellular domain or a variant thereof comprises the human TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type I extracellular domain or a variant thereof comprises the amino acid sequence of SEQ ID NO:1 or a variant having at least about 90% sequence identity. In some embodiments, the extracellular domain of the TGFβ superfamily receptor is fused to at least one of the heavy or light chains of the anti-PD-L1 antibody via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is about 2 to about 25 amino acids in length (e.g., about 2 to about 10 amino acids). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 56-68.

[0135] In some embodiments, a fusion protein is provided comprising a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V). HFurthermore, each of the two light chains contains a light chain variable region (V). La) a first TGFβ superfamily receptor extracellular domain, the first TGFβ superfamily receptor extracellular domain comprising a TGFβ receptor type I extracellular domain or a variant thereof; and c) a second TGFβ superfamily receptor extracellular domain, the second TGFβ superfamily receptor extracellular domain comprising a TGFβ receptor type II extracellular domain or a variant thereof, wherein the first and second TGFβ superfamily receptor extracellular domains are fused to one or both of the two heavy chains of the anti-PD-L1 antibody. In some embodiments, the first and second TGFβ superfamily receptor extracellular domains are fused to the N-terminus of one or both of the two heavy chains of the anti-PD-L1 antibody. In some embodiments, the first and second TGFβ superfamily receptor extracellular domains are fused to the C-terminus of one or both of the two heavy chains of the anti-PD-L1 antibody. In some embodiments, the first and second TGFβ superfamily receptor extracellular domains are connected to each other via a linker (such as any of the linkers described herein, such as a linker comprising the amino acid sequence of any one of SEQ ID NO: 56-68). In some embodiments, one of the first and second TGFβ superfamily receptor extracellular domains is fused to the N-terminus of one or both of the two heavy chains of the anti-PD-L1 antibody, and the other of the first and second TGFβ superfamily receptor extracellular domains is fused to the C-terminus of one or both of the two heavy chains of the anti-PD-L1 antibody. In some embodiments, the TGFβ receptor type I extracellular domain or a variant thereof is fused to the N-terminus of one or both of the two heavy chains of the anti-PD-L1 antibody; and the TGFβ receptor type II extracellular domain or a variant thereof is fused to the C-terminus of one or both of the two heavy chains of the anti-PD-L1 antibody. In some embodiments, the TGFβ receptor type I extracellular domain or a variant thereof is fused to the C-terminus of one or both of the two heavy chains of the anti-PD-L1 antibody; and the TGFβ receptor type II extracellular domain or a variant thereof is fused to the N-terminus of one or both of the two heavy chains of the anti-PD-L1 antibody. In some embodiments, the full-length antibody comprises an Fc fragment derived from human IgG1. In some embodiments, the Fc fragment derived from human IgG1 comprises a K214R mutation and an A297N mutation. In some embodiments, the TGFβ superfamily receptor extracellular domain comprises the TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the TGFβ receptor type II extracellular domain or a variant thereof comprises the human TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type II extracellular domain or a variant thereof comprises a human TGFβ receptor type II extracellular domain variant having at least about 90% sequence identity with SEQ ID NO:2, wherein the variant contains one or more amino acid substitutions at positions N47, N71, and / or N131 according to the numbers in SEQ ID NO:2. In some embodiments, the variant comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the TGFβ superfamily receptor extracellular domain comprises the TGFβ receptor type I extracellular domain or a variant thereof.In some embodiments, the TGFβ receptor type I extracellular domain or a variant thereof comprises the human TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type I extracellular domain or a variant thereof comprises the amino acid sequence of SEQ ID NO:1 or a variant having at least about 90% sequence identity. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to at least one of the heavy or light chains of the anti-PD-L1 antibody via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is about 2 to about 25 amino acids in length (e.g., about 2 to about 10 amino acids). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:56-68.

[0136] In some embodiments, a fusion protein is provided comprising a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V). H Furthermore, each of the two light chains contains a light chain variable region (V). La) a first TGFβ superfamily receptor extracellular domain, the first TGFβ superfamily receptor extracellular domain comprising a TGFβ receptor type I extracellular domain or a variant thereof; and c) a second TGFβ superfamily receptor extracellular domain, the second TGFβ superfamily receptor extracellular domain comprising a TGFβ receptor type II extracellular domain or a variant thereof, wherein the first and second TGFβ superfamily receptor extracellular domains are fused to one or both of the two light chains of the anti-PD-L1 antibody. In some embodiments, the first and second TGFβ superfamily receptor extracellular domains are fused to the N-terminus of one or both of the two light chains of the anti-PD-L1 antibody. In some embodiments, the first and second TGFβ superfamily receptor extracellular domains are fused to the C-terminus of one or both of the two light chains of the anti-PD-L1 antibody. In some embodiments, the first and second TGFβ superfamily receptor extracellular domains are connected to each other via a linker (such as any of the linkers described herein, such as a linker comprising the amino acid sequence of any one of SEQ ID NO: 56-68). In some embodiments, one of the first and second TGFβ superfamily receptor extracellular domains is fused to the N-terminus of one or both of the two light chains of the anti-PD-L1 antibody, and the other of the first and second TGFβ superfamily receptor extracellular domains is fused to the C-terminus of one or both of the two light chains of the anti-PD-L1 antibody. In some embodiments, the TGFβ receptor type I extracellular domain or a variant thereof is fused to the N-terminus of one or both of the two light chains of the anti-PD-L1 antibody; and the TGFβ receptor type II extracellular domain or a variant thereof is fused to the C-terminus of one or both of the two light chains of the anti-PD-L1 antibody. In some embodiments, the TGFβ receptor type I extracellular domain or a variant thereof is fused to the C-terminus of one or both of the two light chains of the anti-PD-L1 antibody; and the TGFβ receptor type II extracellular domain or a variant thereof is fused to the N-terminus of one or both of the two light chains of the anti-PD-L1 antibody. In some embodiments, the full-length antibody comprises an Fc fragment derived from human IgG1. In some embodiments, the Fc fragment derived from human IgG1 comprises a K214R mutation and an A297N mutation. In some embodiments, the TGFβ superfamily receptor extracellular domain comprises the TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the TGFβ receptor type II extracellular domain or a variant thereof comprises the human TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type II extracellular domain or a variant thereof comprises a human TGFβ receptor type II extracellular domain variant having at least about 90% sequence identity with SEQ ID NO:2, wherein the variant contains one or more amino acid substitutions at positions N47, N71, and / or N131 according to the numbers in SEQ ID NO:2. In some embodiments, the variant comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the TGFβ superfamily receptor extracellular domain comprises the TGFβ receptor type I extracellular domain or a variant thereof.In some embodiments, the TGFβ receptor type I extracellular domain or a variant thereof comprises the human TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type I extracellular domain or a variant thereof comprises the amino acid sequence of SEQ ID NO:1 or a variant having at least about 90% sequence identity. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to at least one of the heavy or light chains of the anti-PD-L1 antibody via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is about 2 to about 25 amino acids in length (e.g., about 2 to about 10 amino acids). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:56-68.

[0137] In some embodiments, a fusion protein is provided comprising a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V). H Furthermore, each of the two light chains contains a light chain variable region (V). La) a first TGFβ superfamily receptor extracellular domain; and c) a second TGFβ superfamily receptor extracellular domain, wherein the first TGFβ superfamily receptor extracellular domain is fused to one or both of the two light chains of the anti-PD-L1 antibody, and wherein the second TGFβ superfamily receptor extracellular domain is fused to one or both of the two heavy chains of the anti-PD-L1 antibody. In some embodiments, the first TGFβ superfamily receptor extracellular domain is fused to the N-terminus of one or both of the two light chains of the anti-PD-L1 antibody, and the second TGFβ superfamily receptor extracellular domain is fused to the N-terminus of one or both of the two heavy chains of the anti-PD-L1 antibody. In some embodiments, the first TGFβ superfamily receptor extracellular domain is fused to the C-terminus of one or both of the two light chains of the anti-PD-L1 antibody, and the second TGFβ superfamily receptor extracellular domain is fused to the C-terminus of one or both of the two heavy chains of the anti-PD-L1 antibody. In some embodiments, the extracellular domain of a first TGFβ superfamily receptor is fused to the N-terminus of one or both of the two light chains of the anti-PD-L1 antibody, and the extracellular domain of a second TGFβ superfamily receptor is fused to the C-terminus of one or both of the two heavy chains of the anti-PD-L1 antibody. In some embodiments, the extracellular domain of a first TGFβ superfamily receptor is fused to the C-terminus of one or both of the two light chains of the anti-PD-L1 antibody, and the extracellular domain of a second TGFβ superfamily receptor is fused to the N-terminus of one or both of the two heavy chains of the anti-PD-L1 antibody. In some embodiments, the first TGFβ receptor extracellular domain comprises a TGFβ receptor type I extracellular domain or a variant thereof, and the second TGFβ receptor extracellular domain comprises a TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the first TGFβ receptor extracellular domain comprises a TGFβ receptor type II extracellular domain or a variant thereof, and the second TGFβ receptor extracellular domain comprises a TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the full-length antibody comprises an Fc fragment derived from human IgG1. In some embodiments, the Fc fragment derived from human IgG1 comprises the K214R mutation and the A297N mutation. In some embodiments, the TGFβ receptor type II extracellular domain or a variant thereof comprises the human TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type II extracellular domain or a variant thereof comprises a human TGFβ receptor type II extracellular domain variant having at least about 90% sequence identity with SEQ ID NO:2, wherein the variant contains one or more amino acid substitutions at positions N47, N71, and / or N131 according to the numbering of SEQ ID NO:2. In some embodiments, the variant comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the TGFβ superfamily receptor extracellular domain comprises the TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the TGFβ receptor type I extracellular domain or a variant thereof comprises the human TGFβ receptor type I extracellular domain or a variant thereof.In some embodiments, the human TGFβ receptor type I extracellular domain or a variant thereof comprises the amino acid sequence of SEQ ID NO:1 or a variant having at least about 90% sequence identity. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to at least one of the heavy or light chains of an anti-PD-L1 antibody via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is about 2 to about 25 amino acids in length (e.g., about 2 to about 10 amino acids). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:56-68.

[0138] In some embodiments, a fusion protein is provided comprising a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V). H Furthermore, each of the two light chains contains a light chain variable region (V). L b) A TGFβ superfamily receptor extracellular domain comprising a TGFβ receptor type II extracellular domain or a variant thereof; wherein the TGFβ superfamily receptor extracellular domain is fused to the C-terminus of two of the light chains of an anti-PD-L1 antibody. In some embodiments, the fusion protein has a lower TGFβ2 binding affinity than a reference fusion protein (e.g., M7824). In some embodiments, the TGFβ receptor type II extracellular domain or a variant thereof comprises a human TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type II extracellular domain or a variant thereof comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, the human TGFβ receptor type II extracellular domain or a variant thereof comprises a human TGFβ receptor type II extracellular domain variant having at least about 90% sequence identity with SEQ ID NO:2, wherein the variant contains one or more amino acid substitutions at positions N47, N71, and / or N131 according to the numbers in SEQ ID NO:2. In some embodiments, the variant comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the full-length anti-PD-L1 antibody comprises an Fc fragment derived from human IgG1. In some embodiments, the Fc fragment derived from human IgG1 comprises a K214R mutation and an A297N mutation. In some embodiments, the extracellular domain of the TGFβ superfamily receptor is fused to the light chain of the anti-PD-L1 antibody via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is about 2 to about 25 amino acids in length (e.g., about 2 to about 10 amino acids). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:56-68.

[0139] In some embodiments, a fusion protein is provided comprising a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V). H Furthermore, each of the two light chains contains a light chain variable region (V). L b) A TGFβ superfamily receptor extracellular domain comprising a TGFβ receptor type I extracellular domain or a variant thereof; wherein the TGFβ superfamily receptor extracellular domain is fused to the C-terminus of two lines of the light chain of an anti-PD-L1 antibody. In some embodiments, the fusion protein has a lower TGFβ2 binding affinity than a reference fusion protein (e.g., M7824). In some embodiments, the TGFβ receptor type I extracellular domain or a variant thereof comprises a human TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type I extracellular domain or a variant thereof comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, the full-length anti-PD-L1 antibody comprises an Fc fragment derived from human IgG1. In some embodiments, the Fc fragment derived from human IgG1 comprises a K214R mutation and an A297N mutation. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to the light chain of the anti-PD-L1 antibody via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is about 2 to about 25 amino acids long (e.g., about 2 to about 10 amino acids). In some embodiments, the adapter comprises the amino acid sequence of SEQ ID NO:56-68.

[0140] In some embodiments, a fusion protein is provided comprising a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V). H Furthermore, each of the two light chains contains a light chain variable region (V). L b) The TGFβ superfamily receptor extracellular domain comprising the amino acid sequence of any one of SEQ ID NO: 1-3; wherein the TGFβ superfamily receptor extracellular domain is fused to the C-terminus of two lines of the light chain of the anti-PD-L1 antibody. In some embodiments, the full-length anti-PD-L1 antibody comprises an Fc fragment derived from human IgG1. In some embodiments, the Fc fragment derived from human IgG1 comprises a K214R mutation and an A297N mutation. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to the light chain of the anti-PD-L1 antibody via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is about 2 to about 25 amino acids long (e.g., about 2 to about 10 amino acids). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 56-68.

[0141] In some embodiments, a fusion protein is provided comprising a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V). H Furthermore, each of the two light chains contains a light chain variable region (V). L (a) The TGFβ superfamily receptor extracellular domain comprises the TGFβ receptor type II extracellular domain or a variant thereof; wherein the TGFβ superfamily receptor extracellular domain is fused to the C-terminus of two lines of the heavy chain of the anti-PD-L1 antibody. In some embodiments, the TGFβ receptor type II extracellular domain or a variant thereof comprises the human TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type II extracellular domain or a variant thereof comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, the human TGFβ receptor type II extracellular domain or a variant thereof comprises a human TGFβ receptor type II extracellular domain variant having at least about 90% sequence identity with SEQ ID NO:2, wherein the variant comprises one or more amino acid substitutions at positions N47, N71, and / or N131 according to the numbering of SEQ ID NO:2. In some embodiments, the variant comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the full-length anti-PD-L1 antibody comprises an Fc fragment derived from human IgG1. In some embodiments, the Fc fragment derived from human IgG1 contains K214R and A297N mutations. In some embodiments, the extracellular domain of the TGFβ superfamily receptor is fused to the heavy chain of the anti-PD-L1 antibody via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is about 2 to about 25 amino acids in length (e.g., about 2 to about 10 amino acids). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 56-68.

[0142] In some embodiments, a fusion protein is provided comprising a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V). H Furthermore, each of the two light chains contains a light chain variable region (V). Lb) The TGFβ superfamily receptor extracellular domain comprising the TGFβ receptor type I extracellular domain or a variant thereof; wherein the TGFβ superfamily receptor extracellular domain is fused to the C-terminus of two lines of the heavy chain of the anti-PD-L1 antibody. In some embodiments, the TGFβ receptor type I extracellular domain or a variant thereof comprises the human TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type I extracellular domain or a variant thereof comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, the full-length anti-PD-L1 antibody comprises an Fc fragment derived from human IgG1. In some embodiments, the Fc fragment derived from human IgG1 comprises a K214R mutation and an A297N mutation. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to the heavy chain of the anti-PD-L1 antibody via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is about 2 to about 25 amino acids long (e.g., about 2 to about 10 amino acids). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:56-68.

[0143] In some embodiments, a fusion protein is provided comprising a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V). H Furthermore, each of the two light chains contains a light chain variable region (V). L a) The TGFβ superfamily receptor extracellular domain, comprising the amino acid sequence of any one of SEQ ID NO: 1-3; wherein the TGFβ superfamily receptor extracellular domain is fused to the C-terminus of two lines of the heavy chain of the anti-PD-L1 antibody. In some embodiments, the full-length anti-PD-L1 antibody comprises an Fc fragment derived from human IgG1. In some embodiments, the Fc fragment derived from human IgG1 comprises a K214R mutation and an A297N mutation. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to the heavy chain of the anti-PD-L1 antibody via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is about 2 to about 25 amino acids long (e.g., about 2 to about 10 amino acids). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 56-68.

[0144] In some embodiments, a fusion protein is provided comprising a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V). H Furthermore, each of the two light chains contains a light chain variable region (V). Lb) a first TGFβ superfamily receptor extracellular domain, the first TGFβ superfamily receptor extracellular domain comprising a TGFβ receptor type I extracellular domain; and c) a second TGFβ superfamily receptor extracellular domain, the second TGFβ superfamily receptor extracellular domain comprising a TGFβ receptor type II extracellular domain, wherein the first TGFβ superfamily receptor extracellular domain is optionally fused to the C-terminus of two light chains of an anti-PD-L1 antibody via a first linker, and wherein the second TGFβ superfamily receptor extracellular domain is optionally fused to the C-terminus of the first TGFβ superfamily receptor extracellular domain via a second linker. In some embodiments, a fusion protein is provided comprising a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V). H Furthermore, each of the two light chains contains a light chain variable region (V). L a) a first TGFβ superfamily receptor extracellular domain comprising a TGFβ receptor type II extracellular domain; and c) a second TGFβ superfamily receptor extracellular domain comprising a TGFβ receptor type I extracellular domain, wherein the first TGFβ superfamily receptor extracellular domain is optionally fused to the C-terminus of two light chains of an anti-PD-L1 antibody via a first linker, and wherein the second TGFβ superfamily receptor extracellular domain is optionally fused to the C-terminus of the first TGFβ superfamily receptor extracellular domain via a second linker. In some embodiments, the fusion protein has a lower TGFβ2 binding affinity than a reference fusion protein (e.g., M7824). In some embodiments, the TGFβ receptor type II extracellular domain or a variant thereof comprises a human TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type II extracellular domain or a variant thereof comprises the amino acid sequence of SEQ ID NO:2 or a variant having at least about 90% sequence identity with SEQ ID NO:2, wherein the variant contains one or more amino acid substitutions at positions N47, N71, and / or N131 according to the numbers in SEQ ID NO:2. In some embodiments, the variant comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the TGFβ superfamily receptor extracellular domain comprises the TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the TGFβ receptor type I extracellular domain or a variant thereof comprises the human TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type I extracellular domain or a variant thereof comprises the amino acid sequence of SEQ ID NO:1 or a variant having at least about 90% sequence identity. In some embodiments, the first and / or second linker is a peptide linker. In some embodiments, the peptide linker is about 2 to about 25 amino acids in length (e.g., about 2 to about 10 amino acids). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:56-68.

[0145] In some embodiments, a fusion protein is provided comprising a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V). H Furthermore, each of the two light chains contains a light chain variable region (V). L b) a first TGFβ superfamily receptor extracellular domain, the first TGFβ superfamily receptor extracellular domain comprising a TGFβ receptor type I extracellular domain; and c) a second TGFβ superfamily receptor extracellular domain, the second TGFβ superfamily receptor extracellular domain comprising a TGFβ receptor type II extracellular domain, wherein the first TGFβ superfamily receptor extracellular domain is optionally fused to the C-terminus of the two heavy chains of an anti-PD-L1 antibody via a first linker, and wherein the second TGFβ superfamily receptor extracellular domain is optionally fused to the C-terminus of the first TGFβ superfamily receptor extracellular domain via a second linker. In some embodiments, a fusion protein is provided comprising a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V). H Furthermore, each of the two light chains contains a light chain variable region (V). La) a first TGFβ superfamily receptor extracellular domain comprising a TGFβ receptor type II extracellular domain; and c) a second TGFβ superfamily receptor extracellular domain comprising a TGFβ receptor type I extracellular domain, wherein the first TGFβ superfamily receptor extracellular domain is optionally fused to the C-terminus of both heavy chains of an anti-PD-L1 antibody via a first linker, and wherein the second TGFβ superfamily receptor extracellular domain is optionally fused to the C-terminus of the first TGFβ superfamily receptor extracellular domain via a second linker. In some embodiments, the fusion protein has a lower TGFβ2 binding affinity than a reference fusion protein (e.g., M7824). In some embodiments, the TGFβ receptor type II extracellular domain or a variant thereof comprises a human TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type II extracellular domain or a variant thereof comprises the amino acid sequence of SEQ ID NO:2 or a variant having at least about 90% sequence identity with SEQ ID NO:2, wherein the variant contains one or more amino acid substitutions at positions N47, N71, and / or N131 according to the numbers in SEQ ID NO:2. In some embodiments, the variant comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the TGFβ superfamily receptor extracellular domain comprises the TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the TGFβ receptor type I extracellular domain or a variant thereof comprises the human TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type I extracellular domain or a variant thereof comprises the amino acid sequence of SEQ ID NO:1 or a variant having at least about 90% sequence identity. In some embodiments, the first and / or second linker is a peptide linker. In some embodiments, the peptide linker is about 2 to about 25 amino acids in length (e.g., about 2 to about 10 amino acids). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:56-68.

[0146] In some embodiments, a fusion protein is provided comprising a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V). H Furthermore, each of the two light chains contains a light chain variable region (V). La) a first TGFβ superfamily receptor extracellular domain, the first TGFβ superfamily receptor extracellular domain comprising a TGFβ receptor type I extracellular domain; and c) a second TGFβ superfamily receptor extracellular domain, the second TGFβ superfamily receptor extracellular domain comprising a TGFβ receptor type II extracellular domain, wherein the first TGFβ superfamily receptor extracellular domain is optionally fused to the C-terminus of two heavy chains of an anti-PD-L1 antibody via a first linker, and wherein the second TGFβ superfamily receptor extracellular domain is optionally fused to the C-terminus of two light chains of an anti-PD-L1 antibody via a second linker. In some embodiments, a fusion protein is provided comprising a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V). H Furthermore, each of the two light chains contains a light chain variable region (V). L a) a first TGFβ superfamily receptor extracellular domain comprising a TGFβ receptor type II extracellular domain; and c) a second TGFβ superfamily receptor extracellular domain comprising a TGFβ receptor type I extracellular domain, wherein the first TGFβ superfamily receptor extracellular domain is optionally fused to the C-terminus of the two heavy chains of an anti-PD-L1 antibody via a first linker, and wherein the second TGFβ superfamily receptor extracellular domain is optionally fused to the C-terminus of the two light chains of an anti-PD-L1 antibody via a second linker. In some embodiments, the fusion protein has a lower TGFβ2 binding affinity than a reference fusion protein (e.g., M7824). In some embodiments, the TGFβ receptor type II extracellular domain or a variant thereof comprises the human TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type II extracellular domain or a variant thereof comprises the amino acid sequence of SEQ ID NO:2 or a variant having at least about 90% sequence identity with SEQ ID NO:2, wherein the variant contains one or more amino acid substitutions at positions N47, N71, and / or N131 according to the numbers in SEQ ID NO:2. In some embodiments, the variant comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the TGFβ superfamily receptor extracellular domain comprises the TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the TGFβ receptor type I extracellular domain or a variant thereof comprises the human TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type I extracellular domain or a variant thereof comprises the amino acid sequence of SEQ ID NO:1 or a variant having at least about 90% sequence identity. In some embodiments, the first and / or second linker is a peptide linker. In some embodiments, the peptide linker is about 2 to about 25 amino acids in length (e.g., about 2 to about 10 amino acids). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:56-68.

[0147] In some embodiments, a fusion protein is provided comprising a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V). H Furthermore, each of the two light chains contains a light chain variable region (V). L b) The TGFβ superfamily receptor extracellular domain comprising the amino acid sequence of any one of SEQ ID NO:1-3 or a variant having at least about 90% sequence identity, wherein the TGFβ superfamily receptor extracellular domain is fused to the C-terminus of both heavy chains and both light chains of the anti-PD-L1 antibody.

[0148] In some embodiments, the fusion protein has a lower TGFβ2 binding affinity than a reference fusion protein (such as M7824, AVID200, or SHR-1701). In some embodiments, the K+ binding affinity between the fusion protein and TGFβ2 is... D The K binding between the reference fusion protein (e.g., M7824) and TGFβ2 is greater than that between the reference fusion protein and TGFβ2. D The EC50 of the fusion protein binding to TGFβ2 is at least about 50%, 60%, 70%, 80%, 90%, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10 times higher than that of a reference fusion protein binding to TGFβ2 (such as M7824).

[0149] In some embodiments, the fusion protein has comparable TGFβ1 binding affinity to a reference fusion protein (such as M7824, AVID200, or SHR-1701). For example, in some embodiments, the K+ binding affinity between the fusion protein and TGFβ1 is... D K binding between reference fusion proteins (such as M7824) and TGFβ1 D The EC50 of the binding between the fusion protein and TGFβ1 is within about 50% to about 200% (e.g., about 50% to about 150%, about 75% to about 125%). In some embodiments, the EC50 of the binding between the fusion protein and TGFβ1 is within about 50% to about 200% (e.g., about 50% to about 150%, about 75% to about 125%) of the EC50 of the binding between a reference fusion protein (e.g., M7824) and TGFβ1.

[0150] In some embodiments, the fusion protein has comparable TGFβ3 binding affinity to a reference fusion protein (such as M7824, AVID200, or SHR-1701). For example, in some embodiments, the fusion protein binds to K+ β3. D K binding between reference fusion proteins (such as M7824) and TGFβ3 D The EC50 of the binding between the fusion protein and TGFβ3 is within about 50% to about 200% (e.g., about 50% to about 150%, about 75% to about 125%). In some embodiments, the EC50 of the binding between the fusion protein and TGFβ3 is within about 50% to about 200% (e.g., about 50% to about 150%, about 75% to about 125%) of the EC50 of the binding between a reference fusion protein (e.g., M7824) and TGFβ3.

[0151] In some embodiments, the fusion protein has comparable TGFβ1 and / or TGFβ3 binding affinity to a reference fusion protein (such as M7824), but lower TGFβ2 binding affinity than the reference fusion protein.

[0152] TGFβ superfamily receptor extracellular domain

[0153] Transforming growth factor β (TGF-β or TGF-beta) is a multifunctional cytokine composed of three distinct mammalian isoforms (including TGF-β1, TGF-β2, and TGF-β3). TGF-β proteins are produced by many cell types. It is expressed as a latent molecule comprising a latency-related peptide (LAP) and transforming growth factor β (TGF-β). Once activated upon release of LAP via plasmin-mediated cleavage, active TGF-β binds to TGF-β receptors (TGFβR), including TGFβR1 and TGFβR2, triggering the translocation of SMAD proteins from the cytoplasm to the nucleus, subsequently leading to the production of specific mRNAs and related protein expression to regulate growth and differentiation across various cell types. Therefore, TGF-β signaling plays a crucial role in a wide range of processes, including normal mammary gland development and immune responses.

[0154] TGF-β ligands bind to three isoforms of the TGF-β receptor (TGFβR1, TGFβR2, and TGFβR3) with varying affinities. Since TGF-β1 and TGF-β3 have higher affinities for TGFβR2 than TGFβR1, TGF-β and TGFβR2 first form a complex, and then TGFβR1 is recruited into a complex consisting of one dimerized TGF-β molecule, two TGFβR1 molecules, and two TGFβR3 molecules, forming a symmetrical 2:2:2 complex. Additionally, the three TGF-β isoforms exhibit different binding affinities to the TGF-β type II receptor (TGFβR2). In most cells, TGF-β1 and TGF-β3 have significantly higher affinities for TGFβR2 than TGF-β2. High levels of TGF-β1 and TGF-β3 have been found in many cancers, such as breast, gastric, and lung cancer, and blocking their interaction with the TGF-β receptor inhibits tumor growth and metastasis. In contrast, TGF-β2 plays a physiological role in normal cardiac function and early hematopoiesis, and blocking TGF-β2 leads to cardiotoxicity, as confirmed in clinical trials by small-molecule pan-TGF-β pathway inhibitors. Therefore, an ideal TGF-β blocker should selectively block both TGF-β1 and TGF-β3 signaling pathways to minimize TGF-β2 neutralization.

[0155] TGFβ suppresses local anti-tumor immunity in the tumor microenvironment, thus limiting the effectiveness of immune checkpoint inhibitors. To overcome resistance to immune checkpoint inhibitors, this study designed a bifunctional construct by linking the TGFβ receptor to an anti-PD-L1 mAb that can enhance the efficacy of immunotherapy. More importantly, the antibody fusion protein was optimized to minimize the side effect of TGF-β2 neutralization.

[0156] Single-strand recombination traps targeting TGF-β superfamily growth factors were designed from the extracellular portions of their native receptors. Based on the structural classifications of SCOP (Andreeva et al., 2008, Nucl. Acid Res. 36: D419) and Pfam (Finn et al., 2006, Nucl. Acid Res. 34: D247), the extracellular fragments of all these TGF-β superfamily receptors contain a single-structured domain belonging to the snake venom family. The complete extracellular portions of these receptors typically include unstructured segments flanking their folded ligand-binding domains. Based on experimentally determined 3D structures available from the PDB database, these unstructured extracellular regions are evident (Berman et al., 2000, Nucl. Acid Res. 28:235), for example, the crystal structures of the following extracellular domains: type II TGF-β receptor extracellular domain (Hart et al., 2002 Nat. Struct. Biol. 9:203; Boesen et al., 2002, Structure 10:913; Groppe et al., 2008, Mol. Cell 29:157), type I TGF-β receptor extracellular domain (Groppe et al., 2008, Mol. Cell 29:157), type IIa activin receptor extracellular domain (Allendorph et al., 2006, Proc. Natl. Acad. Sci. USA 103:7643), type IIb activin receptor extracellular domain (Thompson et al., 2003, EMBO). J.22:1555; Greenwald et al., 2004, Mol.Cell 15:485), the extracellular domain of type I BMP receptor (Kirsch et al., 2000, Nat. Struct. Biol. 7:492), or the NMR structure of the extracellular domain of type II TGF-β receptor (Deep et al., 2003, Biochemistry 42:10126).

[0157] A.TGFβ superfamily receptor type I extracellular domain

[0158] In some embodiments, the extracellular domain of the TGFβ superfamily receptor includes the extracellular domain of TGFβ superfamily receptor type I or a variant thereof.

[0159] In some embodiments, the extracellular domain of TGFβ receptor type I or a variant thereof comprises the extracellular domain of human TGFβ receptor type I or a variant thereof. In some embodiments, the extracellular domain of human TGFβ receptor type I or a variant thereof comprises a variant of the extracellular domain of human TGFβ receptor type I that has at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:1.

[0160] In some embodiments, the TGFβ receptor type I extracellular domain or a variant thereof comprises a truncated form of the TGFβ receptor type I extracellular domain. Exemplary TGFβ receptor type I extracellular domains include those described, for example, in WO 2017037634, which is incorporated herein by reference in its entirety. In some embodiments, the truncated form comprises the amino acid sequence of SEQ ID NO:89 or 90 or a variant thereof.

[0161] extracellular domain of B.TGFβ superfamily receptor type II

[0162] In some embodiments, the extracellular domain of the TGFβ superfamily receptor includes the extracellular domain of the TGFβ superfamily receptor type II or a variant thereof.

[0163] In some embodiments, the TGFβ receptor type II extracellular domain or a variant thereof comprises the human TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type II extracellular domain comprises a human TGFβ receptor type II extracellular domain variant having at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:2, wherein the variant contains one or more amino acid substitutions at positions N47, N71, and / or N131 according to the numbers numbered in SEQ ID NO:2. In some embodiments, the variant comprises the N47Q mutation according to the numbers numbered in SEQ ID NO:2. In some embodiments, the variant comprises the N71Q mutation according to the numbers numbered in SEQ ID NO:2. In some embodiments, the variant comprises the N131Q mutation according to the numbers numbered in SEQ ID NO:2. In some embodiments, the variant comprises the amino acid sequence of SEQ ID NO:3.

[0164] In some embodiments, the TGFβ receptor type II extracellular domain or variants thereof comprises a truncated form of the TGFβ receptor type II extracellular domain (e.g., SEQ ID NO: 2 or 3). Exemplary TGFβ receptor type II extracellular domains (such as truncated forms) are described, for example, in WO2018205985 and WO 2017037634, which are incorporated herein by reference in their entirety. In some embodiments, the truncated form omits the first 1-26 (e.g., the first 14-21) amino acids at the N-terminus of the TGFβ receptor type II extracellular domain (e.g., SEQ ID NO: 2 or 3). In some embodiments, the truncated form omits the first 14 amino acids at the N-terminus of the TGFβ receptor type II extracellular domain (e.g., SEQ ID NO: 2 or 3). In some embodiments, the truncated form omits the first 19 amino acids at the N-terminus of the TGFβ receptor type II extracellular domain (e.g., SEQ ID NO: 2 or 3). In some embodiments, the truncated form lacks the first 21 amino acids at the N-terminus of the TGFβ receptor type II extracellular domain (e.g., SEQ ID NO: 2 or 3). In some embodiments, the truncated form lacks the first 26 amino acids at the N-terminus of the TGFβ receptor type II extracellular domain (e.g., SEQ ID NO: 2 or 3). In some embodiments, the truncated form comprises the amino acid sequence of any one of SEQ ID NO: 91-93 or a variant thereof.

[0165] TGFβR extracellular domain peptides and fusion proteins containing such peptides

[0166] This application also provides a non-naturally occurring polypeptide comprising a variant of the TGFβ receptor type II extracellular domain. In some embodiments, the polypeptide comprises a variant of the human TGFβ receptor type II extracellular domain having at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:2, wherein the variant contains one or more amino acid substitutions at positions N47, N71, and / or N131 according to SEQ ID NO:2. In some embodiments, the variant comprises the N47Q mutation according to SEQ ID NO:2. In some embodiments, the variant comprises the N71Q mutation according to SEQ ID NO:2. In some embodiments, the variant comprises the N131Q mutation according to SEQ ID NO:2.

[0167] In some embodiments, the variant comprises the amino acid sequence of SEQ ID NO:3.

[0168] In some embodiments, the TGFβ receptor type II extracellular domain variant comprises a truncated form of the TGFβ receptor type II extracellular domain. In some embodiments, the truncated form comprises the amino acid sequence of SEQ ID NO:91 or a variant thereof. In some embodiments, the truncated form comprises the amino acid sequence of SEQ ID NO:92. In some embodiments, the truncated form comprises the amino acid sequence of SEQ ID NO:93.

[0169] This application also provides fusion proteins comprising any of the polypeptides described above. In some embodiments, the fusion protein further comprises a second portion. In some embodiments, the second portion comprises a half-life extension domain.

[0170] In some embodiments, a fusion protein is provided comprising a) a TGFβ superfamily receptor extracellular domain containing a variant of the human TGFβ receptor type II extracellular domain, the human TGFβ receptor type II extracellular domain variant having at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:2, wherein the variant contains one or more amino acid substitutions at positions N47, N71, and / or N131 according to the numbering of SEQ ID NO:2, and b) a second portion (e.g., a half-life extension portion). In some embodiments, the second portion is fused to the C-terminus of the TGFβ superfamily receptor extracellular domain. In some embodiments, the second portion is fused to the N-terminus of the TGFβ superfamily receptor extracellular domain. In some embodiments, the second portion is fused to the TGFβ superfamily receptor extracellular domain via a linker (as described herein).

[0171] In some embodiments, a fusion protein is provided comprising a) a truncated form of the human TGFβ receptor type II extracellular domain, the human TGFβ receptor type II extracellular domain comprising the amino acid sequence of any one of SEQ ID NO: 91-93 or a variant having at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and b) a second portion (e.g., a half-life extension portion). In some embodiments, the second portion is fused to the C-terminus of the TGFβ superfamily receptor extracellular domain. In some embodiments, the second portion is fused to the N-terminus of the TGFβ superfamily receptor extracellular domain. In some embodiments, the second portion is fused to the TGFβ superfamily receptor extracellular domain via a linker (as described herein).

[0172] Extended half-life portion

[0173] In some embodiments, the extended half-life portion is an Fc fragment. In some embodiments, the extended half-life portion is an albumin-binding portion (e.g., an albumin-binding antibody portion).

[0174] In some embodiments, the extended half-life portion is an Fc fragment (any of the Fc fragments or variants thereof as described herein). The terms “Fc region,” “Fc domain,” or “Fc” refer to the C-terminal non-antigen-binding region of an immunoglobulin heavy chain, which comprises at least a portion of a constant region. This term includes native Fc regions and variant Fc regions. In some embodiments, the human IgG heavy chain Fc region extends from Cys226 to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present without affecting the structure or stability of the Fc region. Unless otherwise specified herein, the amino acid residues in the IgG or Fc region are numbered according to the EU numbering system for antibodies (also known as the EU index), as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0175] In some embodiments, the Fc fragment is derived from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE, IgM, and combinations thereof and their hybrids. In some embodiments, the Fc fragment is derived from an immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3, IgG4, and combinations thereof and their hybrids.

[0176] In some embodiments, the Fc fragment has reduced effector function compared to the corresponding wild-type Fc fragment (e.g., a reduction in effector function of at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95%, as measured by antibody-dependent cytotoxicity (ADCC) levels).

[0177] In some embodiments, the Fc fragment is an IgG1 Fc fragment. In some embodiments, the IgG1 Fc fragment contains an L234A mutation and / or an L235A mutation. In some embodiments, the Fc fragment is an IgG2 or IgG4 Fc fragment. In some embodiments, the Fc fragment is an IgG4 Fc fragment containing S228P, F234A, and / or L235A mutations. In some embodiments, the Fc fragment contains an N297A mutation. In some embodiments, the Fc fragment contains an N297G mutation.

[0178] In some embodiments, the extracellular domain and half-life extension portion of the TGFβ superfamily receptor are connected via a linker (such as any of the linkers described in the “Linkers” section).

[0179] Anti-PD-L1 antibody portion

[0180] The anti-PD-L1 antibody portions (such as full-length antibodies) described in this application include any antibody portions that specifically bind to PD-L1, such as those described in WO 2019129211, which is incorporated herein by reference in its entirety.

[0181] In some embodiments, the anti-PD-L1 antibody portion comprises a full-length antibody containing two heavy chains and two light chains. In some embodiments, the full-length antibody has an Fc fragment selected from the group consisting of Fc fragments and combinations thereof from IgG, IgA, IgD, IgE, and IgM. In some embodiments, the Fc fragment is selected from the group consisting of Fc fragments and combinations thereof from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fc fragment is an IgG1 or IgG4 Fc fragment.

[0182] In some embodiments, PD-L1 is human PD-L1.

[0183] In some embodiments, the anti-PD-L1 antibody comprises a full-length antibody containing a heavy chain variable region (VL1). H ) and light chain variable region (V L The antibody partially competitively binds to PD-L1; where a) the V H Contains: i) HC-CDR1 containing the amino acid sequence of SEQ ID NO:6; ii) HC-CDR2 containing the amino acid sequence of SEQ ID NO:7; and iii) HC-CDR3 containing the amino acid sequence of SEQ ID NO:8; and b) the V L It includes: i) LC-CDR1 containing the amino acid sequence of SEQ ID NO:9; ii) LC-CDR2 containing the amino acid sequence of SEQ ID NO:10; and iii) LC-CDR3 containing the amino acid sequence of SEQ ID NO:11.

[0184] In some embodiments, the anti-PD-L1 antibody comprises a full-length antibody containing a heavy chain variable region (VL1). H ) and light chain variable region (V L ); where a) the V HContains: i) HC-CDR1 containing the amino acid sequence of SEQ ID NO:6, or its variants containing up to about 3 amino acid substitutions (such as any one of about 1, 2, or 3); ii) HC-CDR2 containing the amino acid sequence of SEQ ID NO:7, or its variants containing up to about 3 amino acid substitutions (such as any one of about 1, 2, or 3); and iii) HC-CDR3 containing the amino acid sequence of SEQ ID NO:8, or its variants containing up to about 3 amino acid substitutions (such as any one of about 1, 2, or 3); and b) the V L It includes: i) LC-CDR1 containing the amino acid sequence of SEQ ID NO:9, or its variants containing up to about 3 amino acid substitutions (such as any one of about 1, 2, or 3); ii) LC-CDR2 containing the amino acid sequence of SEQ ID NO:10, or its variants containing up to about 3 amino acid substitutions (such as any one of about 1, 2, or 3); and iii) LC-CDR3 containing the amino acid sequence of SEQ ID NO:11, or its variants containing up to about 3 amino acid substitutions (such as any one of about 1, 2, or 3).

[0185] In some embodiments, the anti-PD-L1 antibody comprises a full-length antibody containing a heavy chain variable region (VL1). H ) and light chain variable region (V L ); where a) the V H It includes HC-CDR1, HC-CDR2, and HC-CDR3, each containing a recurrent variable region (V) having the sequence shown in SEQ ID No:12. H The amino acid sequences of CDR1, CDR2, and CDR3 within SEQ ID NO:12, or variants thereof having at least about 80% (including, for example, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the sequence shown in SEQ ID NO:12; and b) the V L It comprises LC-CDR1, LC-CDR2, and LC-CDR3, each containing a light chain variable region (V) having the sequence shown in SEQ ID NO:13. L The amino acid sequences of CDR1, CDR2, and CDR3 within SEQ ID NO:13, or its variants having at least about 80% (including, for example, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the sequence shown in SEQ ID NO:13.

[0186] In some embodiments, the anti-PD-L1 antibody comprises a heavy chain variable region (V H ) and light chain variable region (V LThe full-length antibody of ) in which V H Contains the sequence shown in SEQ ID No:12, or a variant thereof having at least about 80% (including, for example, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the sequence shown in SEQ ID NO:12; and / or the V L Includes the sequence shown in SEQ ID NO:13, or a variant thereof having at least about 80% (including, for example, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the sequence shown in SEQ ID NO:13.

[0187] In some embodiments, the amino acid substitutions described above are limited to the “Exemplary Substitutions” shown in Table 2 of this application. In some embodiments, the amino acid substitutions are limited to the “Preferred Substitutions” shown in Table 2 of this application.

[0188] Antibody portion (e.g., anti-PD-L1 antibody portion)

[0189] The antibody portion described herein (such as the anti-PD-L1 antibody portion) may have any one or more of the features described below.

[0190] In some embodiments, the antibody portion comprises an Fc fragment. In some embodiments, the antibody portion comprises an scFv. In some embodiments, the antibody portion comprises an scFv fused to an Fc fragment. In some embodiments, the antibody portion comprises an scFv fused to an Fc fragment via a peptide linker. In some embodiments, the Fc fragment is a human IgG1 Fc fragment. In some embodiments, the Fc fragment contains one or more mutations to increase clearance or reduce half-life.

[0191] In some embodiments, the Fc fragment comprises a constant region of the immunoglobulin IgG heavy chain, which includes a hinge region (starting from Cys226), an IgG CH2 domain, and a CH3 domain. As used herein, the terms "hinge region" or "hinge sequence" refer to the amino acid sequence located between the linker and the CH2 domain. In some embodiments, the fusion protein comprises an Fc fragment containing a hinge region. In some embodiments, the Fc fragment of the fusion protein begins at the hinge region and extends to the C-terminus of the IgG heavy chain. In some embodiments, the fusion protein comprises an Fc fragment that does not contain a hinge region.

[0192] In some embodiments, the antibody portion comprises an Fc fragment selected from the group consisting of Fc fragments derived from IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof. In some embodiments, the Fc fragment is derived from human IgG. In some embodiments, the Fc fragment comprises the Fc region of human IgG1, IgG2, IgG3, IgG4, or a combination or hybrid of IgG. In some embodiments, the Fc fragment is an IgG1 Fc fragment. In some embodiments, the Fc fragment comprises the CH2 and CH3 domains of IgG1. In some embodiments, the Fc fragment is an IgG4 Fc fragment. In some embodiments, the Fc fragment comprises the CH2 and CH3 domains of IgG4. IgG4 Fc is known to have lower effector activity than IgG1 Fc, and may therefore be desirable for certain applications. In some embodiments, the Fc fragment is derived from mouse immunoglobulin.

[0193] In some embodiments, the CH2 domain of IgG begins at Ala231. In some embodiments, the CH3 domain begins at Gly341. It is understood that the C-terminal Lys residue of human IgG may optionally be absent. It should also be understood that conserved amino acid substitutions in the Fc region are considered to be within the scope of this invention without affecting the desired structure and / or stability of the Fc.

[0194] Heterodimerization of different peptides in the Fc fragment of an antibody moiety can be facilitated by methods known in the art, including but not limited to heterodimerization via a knock-in-hole technique. Structures and assembly methods for the knock-in-hole technique can be found, for example, in US 5,821,333, US 7,642,228, US 2011 / 0287009 and PCT / US 2012 / 059810, the entire contents of which are incorporated herein by reference. This technique is carried out by introducing a "knob" (or protrusion) in the CH3 domain of one Fc by replacing a small amino acid residue with a large amino acid residue, and introducing a "hole" (or cavity) in the CH3 domain of another Fc by replacing one or more large amino acid residues with smaller amino acid residues. In some embodiments, one chain of the Fc fragment in the fusion protein contains a knob, and a second chain of the Fc fragment contains a hole.

[0195] Preferred residues for forming the pestle are typically naturally occurring amino acid residues, and are preferably selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Tryptophan and tyrosine are most preferred. In one embodiment, the original residues for forming the pestle have small side chain volumes, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine. Exemplary amino acid substitutions in the CH3 domain for forming the pestle include, but are not limited to, T366W, T366Y, or F405W substitutions.

[0196] Preferred residues for forming the mortar are typically naturally occurring amino acid residues, and are preferably selected from alanine (A), serine (S), threonine (T), and valine (V). In one embodiment, the original residues for forming the mortar have a large side chain volume, such as tyrosine, arginine, phenylalanine, or tryptophan. Exemplary amino acid substitutions in the CH3 domain for generating the mortar include, but are not limited to, T366S, L368A, F405A, Y407A, Y407T, and Y407V substitutions. In some embodiments, the pestle contains a T366W substitution, and the mortar contains a T366S / L368A / Y407V substitution. It should be understood that other modifications to the Fc region known in the art that facilitate heterodimerization are also considered and included in this application.

[0197] Other antibody variants were considered, including any of the variants described herein (e.g., Fc variants, effector functional variants, glycosylated variants, cysteine-engineered variants), or combinations thereof.

[0198] a) Antibody affinity

[0199] The binding specificity of the antibody moiety can be experimentally determined using methods known in the art. These methods include, but are not limited to, Western blotting, ELISA, RIA, ECL, IRMA, EIA, and BIACORE. TM - Inspection and peptide scanning.

[0200] In some embodiments, the antibody portion binds to the target antigen (e.g., PD-L1) via a K-type antigen. D For about 10 -7 M to approximately 10 -12 M, approximately 10 -7 M to approximately 10 -8 M, approximately 10 -8 M to approximately 10 -9 M, approximately 10 -9 M to approximately 10 -10 M, approximately 10 -10 M to approximately 10 -11 M, approximately 10 - 11 M to approximately 10-12 M, approximately 10 -7 M to approximately 10 -12 M, approximately 10 -8 M to approximately 10 -12 M, approximately 10 -9 M to approximately 10 -12 M, approximately 10 -10 M to approximately 10 - 12 M, approximately 10 -7 M to approximately 10 -11 M, approximately 10 -8 M to approximately 10 -11 M, approximately 10 -9 M to approximately 10 -11 M, approximately 10 -7 M to approximately 10 -10 M, approximately 10 -8 M to approximately 10 -10 M, or approximately 10 -7 M to approximately 10 -9 M. In some embodiments, the antibody portion binds to the target antigen (e.g., PD-L1) via K. D about 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 Any of M is strong. In some embodiments, the target antigen (e.g., PD-L1) is a human antigen.

[0201] In some embodiments, the antibody portion binds to the target antigen (e.g., PD-L1) via a K-type antigen. on For about 10 3 M -1 s -1 To about 10 8 M -1 s -1 Approximately 10 3 M -1 s -1 To about 10 4 M -1 s -1 Approximately 10 4 M -1 s -1 To about 10 5 M -1 s -1 Approximately 10 5 M -1 s -1 To about 10 6 M -1 s -1Approximately 10 6 M -1 s -1 To about 10 7 M -1 s -1 or about 10 7 M -1 s -1 To about 10 8 M -1 s -1 In some embodiments, the antibody portion binds to the target antigen (e.g., PD-L1) via a K-type antigen. on For about 10 3 M -1 s -1 To about 10 5 M -1 s -1 Approximately 10 4 M -1 s -1 To about 10 6 M -1 s -1 Approximately 10 5 M -1 s -1 To about 10 7 M -1 s -1 Approximately 10 6 M -1 s -1 To about 10 8 M -1 s -1 Approximately 10 4 M -1 s -1 To about 10 7 M -1 s -1 or about 10 5 M -1 s -1 To about 10 8 M - 1 s -1 In some embodiments, the antibody portion binds to the target antigen (e.g., PD-L1) via a K-type antigen. on No more than approximately 10 3 M -1 s -1 10 4 M -1 s -1 10 5 M -1 s -1 10 6 M -1 s-1 10 7 M -1 s -1 Or 10 8 M -1 s -1 Any of them. In some embodiments, the target antigen (e.g., PD-L1) is a human antigen.

[0202] In some embodiments, the antibody portion binds to the target antigen (e.g., PD-L1) via a K-type antigen. off For about 1 second -1 To about 10 -6 s -1 Approximately 1 second -1 To about 10 -2 s -1 Approximately 10 -2 s -1 To about 10 -3 s -1 Approximately 10 -3 s -1 To about 10 -4 s -1 Approximately 10 -4 s -1 To about 10 -5 s -1 Approximately 10 -5 s -1 To about 10 -6 s -1 Approximately 1 second -1 To about 10 -5 s -1 Approximately 10 -2 s -1 To about 10 -6 s -1 Approximately 10 -3 s -1 To about 10 -6 s -1 Approximately 10 -4 s -1 To about 10 -6 s -1 Approximately 10 -2 s -1 To about 10 -5 s -1 or about 10 -3 s -1 To about 10 -5 s -1 In some embodiments, the antibody portion binds to the target antigen (e.g., PD-L1) via a K-type antigen. off For at least about 1 second -1 10 -2 s -110 -3 s -1 10 -4 s -1 10 -5 s -1 Or 10 -6 s -1 Any of them. In some embodiments, the target antigen (e.g., PD-L1) is a human antigen.

[0203] b) Chimeric or humanized antibodies

[0204] In some embodiments, the antibody portion is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In some embodiments, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse) and a human constant region. In some embodiments, a chimeric antibody is a “class-switching” antibody in which the class or subclass has been changed from that of the parent antibody. A chimeric antibody includes its antigen-binding fragment.

[0205] In some embodiments, the chimeric antibody is a humanized antibody. Typically, nonhuman antibodies are humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the parental nonhuman antibody. Typically, humanized antibodies contain one or more variable domains, wherein the HVR, such as the CDR, (or a portion thereof) is derived from the nonhuman antibody, and the FR (or a portion thereof) is derived from the human antibody sequence. Humanized antibodies may also optionally contain at least a portion of the human constant region. In some embodiments, some FR residues in the humanized antibody are substituted with corresponding residues from the nonhuman antibody (e.g., antibodies derived from HVR residues) to restore or improve antibody specificity or affinity, for example.

[0206] Humanized antibodies and their manufacturing methods are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describes SDR(a-CDR) transplantation); Padlan, Mol. Immunol. 28:489-498 (1991) (describes “reworked surfaces”); Dall'Acqua et al., Methods 36:43-60 (2005) (describes “FR reorganization”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describes the “guided selection” approach to FR reorganization).

[0207] Human frame regions that can be used for humanization include, but are not limited to: frame regions selected using a “best fit” method (see, for example, Sims et al., J. Immunol. 151:2296 (1993)); frame regions of the common sequence of human antibodies derived from specific subgroups of the light chain or heavy chain variable region (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:2623 (1993)). ); human mature (somatic mutation) frame regions or hominid frame regions (see, for example, Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and frame regions derived from screening FR libraries (see, for example, Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

[0208] c) Human antibodies

[0209] In some embodiments, the antibody portion is a human antibody (referred to as a human domain antibody, or human DAb). Human antibodies can be generated using various techniques known in the art. Human antibodies are generally described in the following: van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001), Lonberg, Curr. Opin. Immunol. 20:450-459 (2008), and Chen, Mol. Immunol. 47(4):912-21 (2010). Transgenic mice or rats capable of generating fully human single-domain antibodies (or DAbs) are known in the art. See, for example, US 20090307787 A1, US Patent No. 8,754,287, US 20150289489 A1, US 20100122358 A1, and WO 2004049794.

[0210] Human antibodies (e.g., human DAb) can be prepared by administering immunogens to transgenic animals modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin loci, which replace endogenous immunoglobulin loci, or are present extrachromosomally or randomly integrated into the animal's chromosome. In such transgenic mice, endogenous immunoglobulin loci are typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, a description of XENOMOUSE. TM The technology is described in U.S. Patent Nos. 6,075,181 and 6,150,584. US Patent No. 5,770,429; Description KM U.S. Patent No. 7,041,870, and description of the technology The technology is described in U.S. Patent Application Publication No. US 2007 / 0061900. The human variable region of an intact antibody produced from such animals can be further modified (e.g., by binding to a different human constant region).

[0211] Human antibodies (e.g., human DAbs) can also be prepared using hybridoma-based methods. Human myeloma and mouse-human heterologous myeloma cell lines used to produce human monoclonal antibodies have been described (see, for example, Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991)). Human antibodies produced via human B-cell hybridoma technology have also been described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Other methods include, for example, those described in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridoma). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0212] Human antibodies (e.g., human DAbs) can also be generated by isolating variable domain sequences of Fv clones selected from human phage display libraries. These variable domain sequences can then be bound to the desired human constant domain. The technique for selecting human antibodies from an antibody library is described below.

[0213] d) Library-derived antibodies

[0214] Antibody moieties can be isolated by screening combinatorial libraries of antibodies with desired one or more activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with desired binding characteristics. Such methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., editor, Human Press, Totowa, NJ, 2001), and further described, for example, in: McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, editor, Human Press, Human Press, Totowa, NJ, 2001). Press, Totowa, NJ, 2003; Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004). Methods for constructing single-domain antibody libraries have been described, for example, see U.S. Patent No. 7371849.

[0215] In some phage display methods, V H and V LGene libraries are cloned separately using polymerase chain reaction (PCR) and randomly recombined in phage libraries, which can then be screened for antigen-binding phages, as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phages typically display antibody fragments as scFv or Fab fragments. Libraries derived from immune sources provide high-affinity antibodies against immunogens without the need to construct hybridomas. Alternatively, original libraries can be cloned (e.g., from humans) to provide a single source of antibodies against a variety of non-self antigens as well as self antigens without any immunization, as described in Griffiths et al., EMBO J, 12:725-734 (1993). Finally, the original library can also be synthesized by cloning the unrearranged V gene fragment from stem cells, encoding the highly variable CDR3 region using PCR primers containing random sequences, and performing in vitro rearrangement, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373 and U.S. Patent Publications Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0216] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments in this paper.

[0217] e) Substitution, insertion, deletion, and variants

[0218] In some embodiments, antibody variants with one or more amino acid substitutions are provided. Target sites for substitution mutagenesis include HVR (or CDR) and FR. Conserved substitutions are shown in Table 2, titled “Preferred Substitutions.” More substantial variations are provided in Table 2, titled “Exemplary Substitutions,” and are further described below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into the target antibody, and products can be screened for desired activities (e.g., preserved / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

[0219] Table 2. Amino acid substitutions

[0220] Original residues Exemplary replacement Preferred replacement Ala(A) Val; Leu; Ile Val Arg(R) Lys;Gln;Asn Lys Asn(N) Gln; His; Asp, Lys; Arg Gln Asp(D) Glu;Asn Glu Cys(C) Ser;Ala Ser Gln(Q) Asn; Glu Asn Glu(E) Asp; Gln Asp Gly(G) Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe; Leucine Leu Leu(L) Leucine; Ile; Val; Met; Ala; Phe Ile Lys(K) Arg;Gln;Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala; Leucine Leu

[0221] Amino acids can be grouped according to their common side chain characteristics: (1) hydrophobic: leucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0222] Non-conservative replacement would require swapping members of one of these categories with members of another category.

[0223] One type of substitution variant involves replacing one or more hypervariable residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variants selected for further research will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody and / or will substantially retain certain biological properties of the parent antibody. Exemplary substitution variants are affinity-matured antibodies, which can be conveniently generated, for example, using phage display-based affinity maturation techniques (such as those described herein). In short, one or more HVR residues are mutated and the variant antibody is displayed on a phage and screened for specific biological activities (e.g., binding affinity).

[0224] Modifications (e.g., substitutions) can be made in the HVR, for example, to improve antibody affinity. Such modifications can be made in HVR “hotspots,” which are residues encoded by codons that undergo frequent mutations during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or SDR (a-CDR), where the tested variant V H or V LThe binding affinity is described by constructing and reselecting from a secondary library, as illustrated in, for example, Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., editor, Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variant gene selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variant with the desired affinity. Another method for introducing diversity involves an HVR-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scan mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are frequently targeted.

[0225] In some embodiments, substitution, insertion, or deletion can occur within one or more HVRs, as long as such changes do not substantially reduce the antibody's ability to bind to the antigen. For example, conserved changes that do not significantly reduce binding affinity (e.g., conserved substitutions as provided herein) can be made in the HVR. Such changes can occur outside of the HVR "hotspot" or CDR. In the variants V provided above... H In some embodiments of the sequence, each HVR is unchanged or contains no more than one, two, or three amino acid substitutions.

[0226] As described by Cunningham and Wells (1989) Science, 244:1081-1085, a useful method for identifying residues or regions of an antigen that can be targeted for mutagenesis is called "alanine scan mutagenesis." In this method, a target residue or group of residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction between the antibody and the antigen is affected. Additional substitutions can be introduced at the amino acid positions to demonstrate functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine whether they contain the desired properties.

[0227] Amino acid sequence insertions include fusion of the N-terminus and / or C-terminus, ranging in length from one residue to a polypeptide containing one hundred or more residues, and insertions within a sequence of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusing the N-terminus or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or polypeptide that increases the serum half-life of the antibody.

[0228] f) Glycosylation variants

[0229] In some embodiments, the antibody moiety is altered to increase or decrease the degree of glycosylation of the antibody moiety. Adding or deleting glycosylation sites in an antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0230] When an antibody contains an Fc region, the carbohydrates to which they are attached can be altered. Naturally occurring antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, which are usually attached to the C-terminal region of the Fc region via N-bonds. H Asn297 of the 2-domain structure. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibody moiety can be modified to produce antibody variants with certain improved properties.

[0231] In some embodiments, the antibody portion has a carbohydrate structure lacking (directly or indirectly) fucose attached to the Fc region. For example, the fucose content in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the Asn297 glycan relative to the sum of all sugar structures (e.g., complex, hybrid, and high-mannose structures) attached to Asn 297 as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue (EU number of Fc region residues) located at approximately position 297 in the Fc region; however, due to minor sequence variations in the antibody, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucoidylated variants can possess improved ADCC function. See, for example, U.S. Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (KyowaHakko Kogyo Co., Ltd.). Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO 2002 / 031140; Okazaki et al. J.Mol.Biol.336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng.87:614 (2004).Examples of cell lines capable of producing defucosylation antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application No. US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11), and knockout cell lines, such as α-1,6-fucosylation gene FUT8, knockout CHO cells (see, for example, Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al. Biotechnol. Bioeng. 94(4):680-688 (2006); and WO 2003 / 085107).

[0232] In some embodiments, the antibody portion has a bipartite oligosaccharide, for example, wherein the bitendril oligosaccharide attached to the Fc region of the antibody is bipartitely divided by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue attached to the Fc region on the oligosaccharide are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0233] g) Fc region variant

[0234] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody moiety to generate an Fc region variant. The Fc region variant may contain a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0235] In some embodiments, the Fc fragment possesses some (but not all) effector functions that make the antibody variant an ideal candidate for applications where the in vivo half-life of the antibody moiety is important, but certain effector functions (e.g., complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm a reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding capacity (and therefore may lack ADCC activity), but may retain FcRn binding capacity. Primary NK cells used to mediate ADCC express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of target molecules are described in the following: U.S. Patent Nos. 5,500,362 (see, for example, Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be used (e.g., see ACTI for flow cytometry). TM Non-radioactive cytotoxicity assays (Cell Technology, Inc., Mountain View, CA; and CytoTox) Non-radioactive cytotoxicity assays (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the target molecule can be assessed in vivo, for example in animal models, as disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody cannot bind C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, CDC assays can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0236] Antibodies with reduced effector function include antibodies having substitutions for one or more of the Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056). Such Fc mutants include Fc mutants that substitute two or more of the amino acid positions 265, 269, 270, 297, and 327, including the Fc mutant known as “DANA” (US Patent No. 7,332,581) where residues 265 and 297 are substituted with alanine.

[0237] Certain antibody variants that enhance or reduce binding to FcR are described (see, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604(2001)).

[0238] In some embodiments, the Fc fragment is an IgG1 Fc fragment. In some embodiments, the IgG1 Fc fragment contains an L234A mutation and / or an L235A mutation. In some embodiments, the Fc fragment is an IgG2 or IgG4 Fc fragment. In some embodiments, the Fc fragment is an IgG4 Fc fragment containing an S228P, F234A, and / or L235A mutation.

[0239] In some embodiments, the antibody portion comprises an Fc region having one or more amino acid substitutions, which (e.g., substitutions at positions 298, 333, and / or 334 within the Fc region (EU numbers of residues)) improve ADCC.

[0240] In some embodiments, alterations occur within the Fc region that result in changes (i.e., increases or decreases) in C1q binding and / or complement-dependent cytotoxicity (CDC), for example, as described in U.S. Patent Nos. 6,194,551, WO99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0241] In some embodiments, antibody variants include a variant Fc region containing one or more amino acid substitutions that alter the half-life and / or the binding to the neonatal Fc receptor (FcRn). Antibodies with extended half-lives and improved binding to the neonatal Fc receptor (FcRn), which are responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) are described in US 2005 / 0014934 A1 (Hinton et al.). Those antibodies contain an Fc region with one or more amino acid substitutions that alter the binding of the Fc region to the FcRn. Such Fc variants include those with substitutions (e.g., substitution of Fc region residue 434) on one or more Fc region residues (US Patent No. 7,371,826).

[0242] See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351 concerning other examples of Fc region variants.

[0243] h) Cysteine-engineered antibody variants

[0244] In some embodiments, it may be necessary to produce cysteine-engineered antibody moieties, such as "thioMAb," in which one or more residues of the antibody are replaced by cysteine ​​residues. In a particular embodiment, the substituted residues are located at accessible sites on the antibody. By replacing those residues with cysteine, a reactive thiol group is thereby located at an accessible site on the antibody and can be used to conjugate the antibody to other moieties, such as pharmaceutical moieties or linker-pharmaceutical moieties, to produce immunoconjugates, as further described herein. In some embodiments, any one or more of the following residues may be replaced by cysteine: A118 (EU number) of the heavy chain; and S400 (EU number) of the Fc region of the heavy chain. Cysteine-engineered antibody moieties can be produced as described, for example, in U.S. Patent No. 7,521,541.

[0245] i) Antibody derivatives

[0246] In some embodiments, the antibody portion described herein may be further modified to include other non-protein portions known in the art and readily available. Suitable antibody-derived portions include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers) and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethyleneized polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. Polymers may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. Generally, the quantity and / or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, and whether the antibody derivative will be used for a given diagnostic condition.

[0247] In some embodiments, the antibody portion may be further modified to include one or more bioactive proteins, peptides, or fragments thereof. As used interchangeably herein, “bioactive” or “having biological activity” means exhibiting biological activity in vivo to perform a specific function. For example, it may mean binding to a specific biomolecule (e.g., protein, DNA, etc.) and then promoting or inhibiting the activity of that biomolecule. In some embodiments, bioactive proteins or fragments thereof include: proteins and peptides administered to patients as active pharmaceutical substances for the prevention or treatment of a disease or condition; proteins and peptides for diagnostic purposes (e.g., enzymes used in diagnostic tests or in vitro assays); and proteins and peptides administered to patients for the prevention of disease (e.g., vaccines).

[0248] connector

[0249] In some embodiments, the fusion protein described herein includes one or more linkers between two parts (e.g., the TGFβ superfamily receptor extracellular domain and the anti-PD-L1 antibody part described above). The length, flexibility, and / or other characteristics of the one or more linkers used in the fusion protein may have some influence on the properties, including but not limited to affinity, specificity, or affinity for one or more specific antigens or epitopes. For example, a longer linker may be selected to ensure that two adjacent domains do not spatially interfere with each other. In some embodiments, the linker (e.g., a peptide linker) includes flexible residues (e.g., glycine and serine) such that adjacent domains are free to move relative to each other. For example, a glycine-serine duplex may be a suitable peptide linker. In some embodiments, the linker is a non-peptide linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is an inclevable linker. In some embodiments, the linker is a clevable linker.

[0250] Other connector considerations include the impact on the physical or pharmacokinetic properties of the resulting compound, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (more stable or less stable and planned degradation), rigidity, flexibility, immunogenicity, regulation of antibody binding, and the ability to be incorporated into micelles or liposomes.

[0251] In some embodiments, the anti-PD-L1 antibody portion comprises a full-length antibody. In some embodiments, the extracellular domain of the TGFβ superfamily receptor is fused to the full-length antibody via a linker.

[0252] In some embodiments, the linker is a peptide linker as described below. In some embodiments, the length of the peptide linker is about one to about fifty, about two to about forty, about three to about thirty, or about four to about twenty amino acids.

[0253] In some embodiments, the connector is a cuttable connector. In some embodiments, the connector is a non-cuttable connector.

[0254] In some embodiments, the connector is a GS connector.

[0255] In some embodiments, the adapter comprises the amino acid sequence of any one of SEQ ID NO:72-80 and 90-96. In some embodiments, the adapter comprises the amino acid sequence of any one of SEQ ID NO:72-80. In some embodiments, the adapter comprises the amino acid sequence of any one of SEQ ID NO:72-77.

[0256] peptide linkers

[0257] Peptide linkers can have naturally occurring or non-natural sequences. For example, sequences derived from the hinge region of antibodies containing only the heavy chain can be used as linkers. See, for example, WO 1996 / 34103.

[0258] The peptide linker can have any suitable length. In some embodiments, the length of the peptide linker is at least about any one of the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acids. In some embodiments, the length of the peptide linker is no more than about any one of the following: 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or fewer amino acids. In some embodiments, the length of the peptide linker is any one of the following: about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids.

[0259] The fundamental technical feature of this peptide linker is that it does not contain any polymerization activity. The characteristics of peptide linkers (including the absence of secondary structure promoting activity) are known in the art and described, for example, by Dall'Acqua et al. (Biochem. (1998) 37, 9266-9273), Cheadle et al. (Mol Immunol (1992) 29, 21-30), and Raag and Whitlow (FASEB (1995) 9(1), 73-80). For the purpose of "peptide linker," a particularly preferred amino acid is Gly. Furthermore, peptide linkers that do not promote any secondary structure are preferred. The linkage between the domains can be provided, for example, through genetic engineering. Methods for preparing fusion proteins and peptides and expressing them in mammalian cells or bacteria are well known in the art (e.g., WO 99 / 54440, Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY 1989 and 1994 or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

[0260] Peptide linkers can be stable linkers that cannot be cleaved by proteases, especially matrix metalloproteinases (MMPs).

[0261] The connector can also be a flexible connector. An example flexible connector includes a glycine polymer (G). n (SEQ ID NO:85), glycine-serine polymers (including, for example, (GS)). n (SEQ ID NO:86), (GSGGS) n (SEQ ID NO:87), (GGGGS) n (SEQ ID NO:82) and (GGGS) n(SEQ ID NO:88), where n is an integer of at least one, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and therefore may be able to serve as neutral linkers between components. Glycine has significantly more phi-psi space than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11:173-142 (1992)). Those skilled in the art will recognize that the design of antibody fusion proteins can include linkers that are wholly or partially flexible, such that the linker can include a flexible linker portion and one or more portions that impart a smaller flexible structure to provide the desired antibody fusion protein structure. In some embodiments, the linker is a GS linker.

[0262] In some embodiments, the peptide linker comprises a hinge region of IgG, such as the hinge region of human IgG1. In some embodiments, the peptide linker comprises a hinge region of IgG, such as the hinge region of human IgG1. In some embodiments, the peptide linker comprises a modified sequence derived from the IgG hinge region. In some embodiments, the linker comprises an amino acid sequence of any one of SEQ ID NO: 57-58 and 63-68.

[0263] In some embodiments, the TGFβ superfamily receptor extracellular domain and the anti-PD-L1 antibody portion are linked together by a sufficiently long linker to allow the TGFβ superfamily receptor extracellular domain and the anti-PD-L1 antigen-binding domain to fold in a manner that allows binding to TGFβ (such as TGFβ1 or TGFβ3) and PD-L1. In some embodiments, the linker has (GGGGS) n The amino acid sequence of (SEQ ID NO:82), where n is an integer between 1 and 8, for example (GGGGS)6 (SEQ ID NO:83; hereinafter referred to as "(G4S)6" or "GS6"). In some embodiments, the peptide linker comprises (GSTSGSGKPGSGEGS). n The amino acid sequence of (SEQ ID NO:84), where n is an integer between 1 and 3.

[0264] In some embodiments, the adapter has an amino acid sequence selected from the group consisting of SEQ ID NO: 56-68 and 82-88. In some embodiments, the adapter has an amino acid sequence selected from the group consisting of SEQ ID NO: 56-68.

[0265] Non-peptide linkers

[0266] The coupling of two parts can be accomplished by any chemical reaction that binds the two molecules together, provided that both components retain their respective activity, for example, binding to TGFβ (e.g., TGFβ1 and / or TGFβ3) and PD-L1, respectively. This linking can include a variety of chemical mechanisms, such as covalent binding, affinity binding, insertion, coordination binding, and complexation. In some embodiments, the binding is covalent. Covalent binding can be achieved through the direct condensation of existing side chains or through the incorporation of external bridging molecules. In this case, many divalent or multivalent linkers can be used to couple protein molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimidides, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to exhaust all known coupling agents in the art, but rather to provide examples of more common coupling agents (see Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987)).

[0267] The linkers that can be used in this application are described in the literature (see, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleiminobenzoyl-N-hydroxysuccinimide). In some embodiments, the non-peptide linkers used herein include: (i) EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride); (ii) SMPT (4-succinimide-oxycarbonyl-α-methyl-α-(2-pyridyl-dithio)-toluene (Pierce Chem. Co., catalog number (21558G)); (iii) SPDP (succinimide-6-[3-(2-pyridyl-dithio)propamido]hexanoate (Pierce Chem. Co., catalog number (21558G)). (iv) sulfonyl-LC-SPDP (sulfosuccinimide-6-[3-(2-pyridyldithio)propionamide]hexanoate) (Pierce Chem.Co. catalog 2165-G); and (v) sulfonyl-NHS (N-hydroxysulfosuccinimide: Pierce Chem.Co. catalog 24510) conjugated to EDC.

[0268] The aforementioned linkers contain components with different properties, thus potentially leading to fusion proteins with varying physicochemical characteristics. For example, sulfonated NHS esters of alkyl carboxylates are more stable than those of aromatic carboxylates. Linkers containing NHS esters are less soluble than those containing sulfonated NHS esters. Furthermore, the linker SMPT contains sterically hindered disulfide bonds and can form antibody fusion proteins with enhanced stability. Disulfide bonds are generally less stable than other bonds because they are cleaved in vitro, resulting in fewer usable antibody fusion proteins. In particular, sulfonated NHS can enhance the stability of carbodiimide coupling. When used in conjunction with sulfonated NHS, carbodiimide coupling (e.g., EDC) forms esters that are more resistant to hydrolysis than carbodiimide coupling reactions alone.

[0269] Nucleic acid

[0270] Nucleic acid molecules encoding the various fusion proteins, peptides, or antibody motifs described herein are also considered. In some embodiments, nucleic acids (or a group of nucleic acids) encoding one or more of the fusion proteins, peptides, or antibody motifs or portions thereof described herein are provided.

[0271] This article also considers isolated host cells containing fusion proteins or peptides, isolated nucleic acids encoding peptide components of the peptide or fusion protein, or vectors containing nucleic acids encoding peptide components of the peptide or fusion protein described herein.

[0272] This application also includes variants of these nucleic acid sequences. For example, variants include nucleotide sequences that hybridize with nucleic acid sequences encoding polypeptides or fusion proteins described herein under at least moderately stringent hybridization conditions.

[0273] This application also provides a vector in which the nucleic acid of this application is inserted.

[0274] Using standard gene delivery protocols, the nucleic acids of this invention can also be used for nucleic acid immunotherapy and gene therapy. Methods for gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety. In some embodiments, the invention provides gene therapy vectors.

[0275] Nucleic acids can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors, including but not limited to plasmids, phage particles, phage derivatives, animal viruses, and entrapments. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0276] In addition, expression vectors can be delivered to cells in the form of viral vectors. Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Typically, a suitable vector contains an origin of replication that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more optional markers (see, for example, WO01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0277] III. Preparation Method

[0278] In some embodiments, methods are provided for preparing the fusion proteins (e.g., fusion proteins containing the extracellular domain of the TGFβ superfamily receptor bound to PD-L1) or peptides described herein. In some embodiments, methods are provided for preparing compositions, such as polynucleotides, nucleic acid constructs, vectors, host cells, or culture media, generated during the preparation of the fusion protein or peptide. The fusion proteins, peptides, or compositions described herein can be prepared by a variety of methods, as generally described below and more specifically described in the examples.

[0279] In some embodiments, a method is provided for generating any of the fusion proteins or peptides described herein, the method comprising: a) culturing host cells expressing the fusion protein or a portion thereof or the peptide under conditions for efficient expression of the fusion protein or a portion thereof or the peptide; and b) obtaining the expressed fusion protein or a portion thereof or the peptide from the host cells. In some embodiments, the method further comprises purifying the expressed fusion protein or a portion thereof or the peptide.

[0280] Antibody expression and production

[0281] The antibody components described herein (including anti-PD-L1 antibodies) can be prepared using any method known in the art, including the methods described below and in examples.

[0282] Monoclonal antibodies

[0283] Monoclonal antibodies are obtained from a substantially homogeneous population of antibodies, meaning that the individual antibodies comprising the population are identical, except for possibly small amounts of naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation). Therefore, the modifier “monoclonal” indicates that the antibody is not a mixture of discrete antibodies. For example, monoclonal antibodies can be prepared using a hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or by a recombinant DNA method (US Patent No. 4,816,567). In the hybridoma method, mice or other suitable host animals, such as hamsters or llamas, are immunized as described above to induce lymphocytes that produce or are capable of producing antibodies that will specifically bind to proteins used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent (e.g., polyethylene glycol) to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). See also Camel Immunization in Example 1.

[0284] Immunosuppressants typically include an antigen protein or a fusion variant thereof. Peripheral blood lymphocytes (“PBLs”) are usually used if human-derived cells are required, or spleen cells or lymph node cells are used if non-human mammalian-derived cells are required. The lymphocytes are then fused with an immortalized cell line using a suitable fusion agent (such as polyethylene glycol) to form hybridoma cells. (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59–103.)

[0285] Immortalized cell lines are typically transformed mammalian cells, particularly rodent, bovine, and human myeloma cells. Rat or mouse myeloma cell lines are commonly used. The hybridoma cells thus prepared are seeded and grown in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma culture medium typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), substances that inhibit the growth of HGPRT-deficient cells.

[0286] Preferred immortalized myeloma cells are those that are efficiently fused, support the stable high-level antibody production of selected antibody-producing cells, and are sensitive to culture media (such as HAT medium). Among these, murine myeloma cell lines are preferred, for example, those derived from MOPC-21 and MPC-11 mouse tumors obtained from the Salk Institute Cell Distribution Center in San Diego, California, and SP-2 cells (and their derivatives, such as X63-Ag8-653) obtained from the American Type Culture Collection in Manassas, Virginia. The use of human myeloma and mouse-human heterologous myeloma cell lines for the production of human monoclonal antibodies has also been described (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0287] The production of monoclonal antibodies against antigens in the culture medium in which hybridoma cells are grown is measured. Preferably, the binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or in vitro binding assays (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)).

[0288] The presence of monoclonal antibodies against a desired antigen in the culture medium for hybridoma cells can be determined. Preferably, the binding affinity and specificity of the monoclonal antibody can be determined by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked assay (ELISA). Such techniques and assays are known in the art. For example, binding affinity can be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0289] Once hybridoma cells are identified that can produce antibodies with the desired specificity, affinity, and / or activity, the clones can be subcloned using a limiting dilution procedure and cultured using standard methods (Goding, ibid.). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 media. Additionally, hybridoma cells can grow in vivo as tumors in mammals.

[0290] Monoclonal antibodies secreted by subclones can be isolated from culture medium, ascites, or serum using conventional immunoglobulin purification procedures such as protein A-agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0291] Monoclonal antibodies can also be prepared using recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567 and as stated above. DNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional procedures, e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of murine antibodies. Hybridoma cells are a preferred source of this DNA. Once isolated, the DNA can be placed in expression vectors, which are then transfected into host cells that do not additionally produce immunoglobulins (e.g., *E. coli* cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells) to synthesize monoclonal antibodies in these recombinant host cells. Commentary articles on the recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., *Curr. Opinion in Immunol.*, 5:256-262 (1993) and Plückthun, *Immunol. Revs.*, 130:151-188 (1992).

[0292] In another embodiment, antibodies can be isolated from the generated antibody phage library using the technique described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of mouse and human antibodies using phage libraries, respectively. Subsequent publications describe strategies for generating high-affinity (nM range) human antibodies by chain tamponade (Marks et al., Bio / Technology, 10:779-783 (1992)) and by combining infection and in vivo recombination as strategies for constructing ultra-large phage libraries (Waterhouse et al., Nucl. Acids Res., 21:2265-2266 (1993)). Therefore, these technologies are viable alternatives to traditional monoclonal antibody hybridoma technologies used for isolating monoclonal antibodies.

[0293] DNA can also be modified, for example, by replacing homologous mouse sequences with coding sequences of constant domains of the human heavy and light chains (US Patent No. 4,816,567; Morrison et al., Proc. Natl. Sci. USA, 81:6851 (1984)), or by covalently binding all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. Typically, such non-immunoglobulin polypeptides replace constant domains of antibodies, or they replace variable domains of an antigen-binding site of an antibody, to produce chimeric bivalent antibodies containing one antigen-specific antigen-binding site and another antigen-binding site specific to a different antigen.

[0294] The monoclonal antibodies described herein can be monovalent, and their preparation is well known in the art. For example, one method involves the recombinant expression of an immunoglobulin light chain and a modified heavy chain. Typically, the heavy chain is truncated at any point in the Fc region to prevent heavy chain cross-linking. Alternatively, the associated cysteine ​​residue can be substituted with another amino acid residue or deleted to prevent cross-linking. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of the antibody to produce its fragments (particularly Fab fragments) can be performed using conventional techniques known in the art.

[0295] Chimeric or hybrid antibodies can also be prepared in vitro using known methods in synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiolates and methyl-4-mercaptobutyrimidate.

[0296] Nucleic acid molecules encoding fusion proteins or polypeptides

[0297] In some embodiments, a polynucleotide encoding any one of the fusion proteins (or portions thereof) or polypeptides described herein is provided. In some embodiments, a polynucleotide prepared using any of the methods described herein is provided. In some embodiments, the nucleic acid molecule comprises a heavy chain or light chain of a polynucleotide encoding an antibody moiety (e.g., an anti-PD-L1 antibody moiety, e.g., a full-length anti-PD-L1 antibody). In some embodiments, the nucleic acid molecule comprises both a heavy chain of a polynucleotide encoding an antibody moiety (e.g., an anti-PD-L1 antibody moiety, e.g., a full-length anti-PD-L1 antibody) and a light chain thereof. In some embodiments, a first nucleic acid molecule comprises a first polynucleotide encoding the heavy chain, and a second nucleic acid molecule comprises a second polynucleotide encoding the light chain.

[0298] In some embodiments, the heavy and light chains are expressed from a single nucleic acid molecule or from two separate nucleic acid molecules as two separate polypeptides. In some embodiments, a single polynucleotide encodes a single polypeptide comprising both a heavy and light chain linked together.

[0299] In some embodiments, the polynucleotide encoding the heavy or light chain of an antibody moiety (e.g., an anti-PD-L1 antibody moiety, such as a full-length anti-PD-L1 antibody) includes a nucleotide sequence encoding a leader sequence located at the N-terminus of the heavy or light chain during translation. As described above, the leader sequence can be a natural heavy or light chain leader sequence or another heterologous leader sequence.

[0300] In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding a TGFβR extracellular domain polypeptide (such as any of the polypeptides described herein).

[0301] In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is RNA. In some embodiments, the RNA is mRNA. In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least about 80% (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with any one of the nucleic acid sequences of SEQ ID Nos: 19-21 and 38-53.

[0302] Nucleic acid molecules can be constructed using recombinant DNA techniques conventional in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell.

[0303] Nucleic acid constructs

[0304] In some embodiments, nucleic acid constructs comprising any of the polynucleotides described herein are provided. In some embodiments, nucleic acid constructs prepared using any of the methods described herein are provided.

[0305] In some embodiments, the nucleic acid construct further includes a promoter operatively linked to a polynucleotide. In some embodiments, the polynucleotide corresponds to a gene, wherein the promoter is a wild-type promoter of the gene.

[0306] carrier

[0307] In some embodiments, a vector comprising any polynucleotide encoding any of the antibody moieties or fusion proteins described herein, or any nucleic acid construct described herein, is provided. In some embodiments, a vector prepared using any of the methods described herein is provided. A vector comprising a polynucleotide encoding any of the fusion proteins or polypeptides described herein is also provided. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In some embodiments, the vector comprises a) a first polynucleotide sequence encoding a heavy or light chain of a full-length anti-PD-L1 antibody fused to the extracellular domain of a TBFb superfamily receptor, and b) a second polynucleotide sequence encoding a light or heavy chain of a full-length anti-PD-L1 antibody paired with the first nucleotide. In some embodiments, the first and second vectors are transfected into host cells in similar amounts (e.g., similar molar amounts or similar masses). In some embodiments, the first and second vectors are transfected into host cells in a molar ratio or mass ratio between 5:1 and 1:5. In some embodiments, a mass ratio between 1:1 and 1:5 is used for vectors encoding heavy chains and vectors encoding light chains. In some embodiments, a 1:2 mass ratio is used for the carrier encoding the heavy chain and the carrier encoding the light chain.

[0308] In some embodiments, a vector optimized for expression of the peptide in CHO or CHO-derived cells (e.g., CHO-3E7 cells) or NSO cells is selected. Exemplary such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

[0309] host cells

[0310] In some embodiments, a host cell comprising any of the polypeptides, nucleic acid constructs, and / or vectors described herein is provided. In some embodiments, a host cell prepared using any of the methods described herein is provided. In some embodiments, the host cell is capable of producing any of the antibody moieties or fusion proteins described herein under fermentation conditions.

[0311] In some embodiments, the fusion proteins and peptides described herein may be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells (e.g., fungal cells (e.g., yeast), plant cells, insect cells, and mammalian cells). Such expression may be performed, for example, according to procedures known in the art. Exemplary eukaryotic cells that may be used to express peptides include, but are not limited to, COS cells (including COS 7 cells); 293 cells (including 293-6E cells); and CHO cells (including CHO-S, CHO-3E7, DG44, Lec13 CHO cells, and FUT8 CHO cells). Crucells; and NSO cells. In some embodiments, the antibody moiety and fusion protein described herein may be expressed in yeast. See, for example, U.S. Publication No. US 2006 / 0270045 A1. In some embodiments, specific eukaryotic host cells are selected based on the ability to perform desired post-translational modifications on the heavy and / or light chains of the antibody moiety. For example, in some embodiments, peptides produced by CHO cells have a higher level of sialylation than the same peptides produced by 293 cells.

[0312] The introduction of one or more nucleic acids into a desired host cell can be accomplished by any method, including but not limited to calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, etc. Non-limiting exemplary methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, p. 3. rd Published by Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be transiently or stably transfected in the desired host cells using any suitable method.

[0313] This invention also provides host cells comprising any of the polynucleotides or vectors described herein. In some embodiments, the invention provides host cells comprising a fusion protein or polypeptide or a portion thereof described herein. Any host cell capable of overexpressing heterologous DNA can be used for the purpose of isolating genes encoding a target antibody, polypeptide, or protein. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa, and CHO cells. See also PCT Publication WO 87 / 04462. Suitable non-mammalian host cells include prokaryotes (such as Escherichia coli or Bacillus subtilis) and yeasts (e.g., Saccharomyces cerevisiae, S. pombe; or Kluyveromyces lactis).

[0314] culture medium

[0315] In some embodiments, a culture medium comprising any antibody moiety, polynucleotide, fusion protein, nucleic acid construct, vector, and / or host cell described herein is provided. In some embodiments, a culture medium prepared using any of the methods described herein is provided.

[0316] In some embodiments, the culture medium contains hypoxanthine, aminopterin, and / or thymidine (e.g., HAT medium). In some embodiments, the culture medium does not contain serum. In some embodiments, the culture medium contains serum. In some embodiments, the culture medium is D-MEM or RPMI-1640 medium.

[0317] Purification of fusion proteins or peptides

[0318] The fusion proteins or peptides described herein can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include ROR1 ECD and ligands that bind to the constant region of an antibody. For example, protein A, protein G, protein A / G, or antibody affinity columns can be used to bind to the constant region and purify fusion proteins containing the Fc fragment. Hydrophobic interaction chromatography, such as butyl or phenyl columns, can also be used to purify certain peptides, such as antibodies. Ion exchange chromatography (e.g., anion exchange chromatography and / or cation exchange chromatography) can also be used to purify certain peptides, such as antibodies. Mixed-mode chromatography (e.g., reversed-phase / anion exchange, reversed-phase / cation exchange, hydrophilic interaction / anion exchange, hydrophilic interaction / cation exchange, etc.) can also be used to purify certain peptides, such as antibodies. Many methods for purifying peptides are known in the art.

[0319] V. Treatment Methods

[0320] This article also provides methods for treating individual diseases or conditions. These methods involve administering the fusion protein or peptide described herein to an individual (e.g., a mammal, such as a human).

[0321] In some embodiments, a method of treating an individual disease or condition (e.g., cancer) is provided, the method comprising administering to the individual an effective amount of a fusion protein comprising a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V... H Furthermore, each of the two light chains contains a light chain variable region (V). Lb) TGFβ superfamily receptor extracellular domain; wherein the TGFβ superfamily receptor extracellular domain is fused to at least one or both of the heavy or light chains of the anti-PD-L1 antibody. In some embodiments, the full-length antibody comprises an Fc fragment derived from human IgG1. In some embodiments, the Fc fragment derived from human IgG1 comprises a K214R mutation and an A297N mutation. In some embodiments, the TGFβ superfamily receptor extracellular domain comprises a TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the TGFβ receptor type II extracellular domain or a variant thereof comprises a human TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type II extracellular domain or a variant thereof comprises a human TGFβ receptor type II extracellular domain variant having at least about 90% sequence identity with SEQ ID NO:2, wherein the variant contains one or more amino acid substitutions at positions N47, N71, and / or N131 according to the numbering of SEQ ID NO:2. In some embodiments, the variant comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the TGFβ superfamily receptor extracellular domain comprises the TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the TGFβ receptor type I extracellular domain or a variant thereof comprises the human TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type I extracellular domain or a variant thereof comprises the amino acid sequence of SEQ ID NO:1 or a variant having at least about 90% sequence identity. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to at least one of the heavy or light chains of an anti-PD-L1 antibody via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is about 2 to about 25 amino acids in length (e.g., about 2 to about 10 amino acids). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:56-68.

[0322] In some embodiments, a method of treating an individual disease or condition (e.g., cancer) is provided, the method comprising administering to the individual an effective amount of a fusion protein comprising a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V... H Furthermore, each of the two light chains contains a light chain variable region (V). La) TGFβ superfamily receptor extracellular domain; wherein the TGFβ superfamily receptor extracellular domain is fused to at least one or both of the light chains of the anti-PD-L1 antibody. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to the C-terminus of at least one or both of the two light chains. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to the N-terminus of at least one or both of the two light chains. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to both the N-terminus and C-terminus of at least one or both of the two light chains. In some embodiments, the full-length antibody comprises an Fc fragment derived from human IgG1. In some embodiments, the Fc fragment derived from human IgG1 comprises a K214R mutation and an A297N mutation. In some embodiments, the TGFβ superfamily receptor extracellular domain comprises a TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the TGFβ receptor type II extracellular domain or a variant thereof comprises a human TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type II extracellular domain or a variant thereof comprises a human TGFβ receptor type II extracellular domain variant having at least about 90% sequence identity with SEQ ID NO:2, wherein the variant contains one or more amino acid substitutions at positions N47, N71, and / or N131 according to the numbering of SEQ ID NO:2. In some embodiments, the variant comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the TGFβ superfamily receptor extracellular domain comprises the TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the TGFβ receptor type I extracellular domain or a variant thereof comprises the human TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type I extracellular domain or a variant thereof comprises the amino acid sequence of SEQ ID NO:1 or a variant having at least about 90% sequence identity. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to at least one of the heavy or light chains of the anti-PD-L1 antibody via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is about 2 to about 25 amino acids in length (e.g., about 2 to about 10 amino acids). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:56-68.

[0323] In some embodiments, a method of treating an individual disease or condition (e.g., cancer) is provided, the method comprising administering to the individual an effective amount of a fusion protein comprising a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V... H Furthermore, each of the two light chains contains a light chain variable region (V). La) TGFβ superfamily receptor extracellular domain; wherein the TGFβ superfamily receptor extracellular domain is fused to at least one or both of the heavy chains of the anti-PD-L1 antibody. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to the C-terminus of at least one or both of the two heavy chains. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to the N-terminus of at least one or both of the two heavy chains. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to both the N-terminus and C-terminus of at least one or both of the two heavy chains. In some embodiments, the full-length antibody comprises an Fc fragment derived from human IgG1. In some embodiments, the Fc fragment derived from human IgG1 comprises a K214R mutation and an A297N mutation. In some embodiments, the TGFβ superfamily receptor extracellular domain comprises a TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the TGFβ receptor type II extracellular domain or a variant thereof comprises a human TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type II extracellular domain or a variant thereof comprises a human TGFβ receptor type II extracellular domain variant having at least about 90% sequence identity with SEQ ID NO:2, wherein the variant contains one or more amino acid substitutions at positions N47, N71, and / or N131 according to the numbering of SEQ ID NO:2. In some embodiments, the variant comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the TGFβ superfamily receptor extracellular domain comprises the TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the TGFβ receptor type I extracellular domain or a variant thereof comprises the human TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type I extracellular domain or a variant thereof comprises the amino acid sequence of SEQ ID NO:1 or a variant having at least about 90% sequence identity. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to at least one of the heavy or light chains of the anti-PD-L1 antibody via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is about 2 to about 25 amino acids in length (e.g., about 2 to about 10 amino acids). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:56-68.

[0324] In some embodiments, a method of treating an individual disease or condition (e.g., cancer) is provided, the method comprising administering to the individual an effective amount of a fusion protein comprising a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V... H Furthermore, each of the two light chains contains a light chain variable region (V). Lb) The TGFβ superfamily receptor extracellular domain; wherein the TGFβ superfamily receptor extracellular domain is fused with: i) at least one or two of the light chains of the anti-PD-L1 antibody, and ii) at least one or two of the heavy chains of the anti-PD-L1 antibody. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused with the C-terminus of i) the two heavy chains and ii) the two light chains. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused with the N-terminus of i) the two heavy chains and ii) the two light chains. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused with: i) the N-terminus of the two heavy chains, and ii) the C-terminus of the two light chains. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused with: i) the C-terminus of the two heavy chains, and ii) the N-terminus of the two light chains. In some embodiments, the full-length antibody comprises an Fc fragment derived from human IgG1. In some embodiments, the Fc fragment derived from human IgG1 comprises a K214R mutation and an A297N mutation. In some embodiments, the TGFβ superfamily receptor extracellular domain comprises the TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the TGFβ receptor type II extracellular domain or a variant thereof comprises the human TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type II extracellular domain or a variant thereof comprises a human TGFβ receptor type II extracellular domain variant having at least about 90% sequence identity with SEQ ID NO:2, wherein the variant contains one or more amino acid substitutions at positions N47, N71, and / or N131 according to the numbering of SEQ ID NO:2. In some embodiments, the variant comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the TGFβ superfamily receptor extracellular domain comprises the TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the TGFβ receptor type I extracellular domain or a variant thereof comprises the human TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type I extracellular domain or a variant thereof comprises the amino acid sequence of SEQ ID NO:1 or a variant having at least about 90% sequence identity. In some embodiments, the extracellular domain of the TGFβ superfamily receptor is fused to at least one of the heavy or light chains of the anti-PD-L1 antibody via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is about 2 to about 25 amino acids in length (e.g., about 2 to about 10 amino acids). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 56-68.

[0325] In some embodiments, a method of treating an individual disease or condition (e.g., cancer) is provided, the method comprising administering to the individual an effective amount of a fusion protein comprising a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V... H Furthermore, each of the two light chains contains a light chain variable region (V). La) a first TGFβ superfamily receptor extracellular domain, the first TGFβ superfamily receptor extracellular domain comprising a TGFβ receptor type I extracellular domain or a variant thereof; and c) a second TGFβ superfamily receptor extracellular domain, the second TGFβ superfamily receptor extracellular domain comprising a TGFβ receptor type II extracellular domain or a variant thereof, wherein the first and second TGFβ superfamily receptor extracellular domains are fused to one or both of the two heavy chains of the anti-PD-L1 antibody. In some embodiments, the first and second TGFβ superfamily receptor extracellular domains are fused to the N-terminus of one or both of the two heavy chains of the anti-PD-L1 antibody. In some embodiments, the first and second TGFβ superfamily receptor extracellular domains are fused to the C-terminus of one or both of the two heavy chains of the anti-PD-L1 antibody. In some embodiments, the first and second TGFβ superfamily receptor extracellular domains are connected to each other via a linker (such as any of the linkers described herein, such as a linker comprising the amino acid sequence of any one of SEQ ID NO: 56-68). In some embodiments, one of the first and second TGFβ superfamily receptor extracellular domains is fused to the N-terminus of one or both of the two heavy chains of the anti-PD-L1 antibody, and the other of the first and second TGFβ superfamily receptor extracellular domains is fused to the C-terminus of one or both of the two heavy chains of the anti-PD-L1 antibody. In some embodiments, the TGFβ receptor type I extracellular domain or a variant thereof is fused to the N-terminus of one or both of the two heavy chains of the anti-PD-L1 antibody; and the TGFβ receptor type II extracellular domain or a variant thereof is fused to the C-terminus of one or both of the two heavy chains of the anti-PD-L1 antibody. In some embodiments, the TGFβ receptor type I extracellular domain or a variant thereof is fused to the C-terminus of one or both of the two heavy chains of the anti-PD-L1 antibody; and the TGFβ receptor type II extracellular domain or a variant thereof is fused to the N-terminus of one or both of the two heavy chains of the anti-PD-L1 antibody. In some embodiments, the full-length antibody comprises an Fc fragment derived from human IgG1. In some embodiments, the Fc fragment derived from human IgG1 comprises a K214R mutation and an A297N mutation. In some embodiments, the TGFβ superfamily receptor extracellular domain comprises the TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the TGFβ receptor type II extracellular domain or a variant thereof comprises the human TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type II extracellular domain or a variant thereof comprises a human TGFβ receptor type II extracellular domain variant having at least about 90% sequence identity with SEQ ID NO:2, wherein the variant contains one or more amino acid substitutions at positions N47, N71, and / or N131 according to the numbers in SEQ ID NO:2. In some embodiments, the variant comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the TGFβ superfamily receptor extracellular domain comprises the TGFβ receptor type I extracellular domain or a variant thereof.In some embodiments, the TGFβ receptor type I extracellular domain or a variant thereof comprises the human TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type I extracellular domain or a variant thereof comprises the amino acid sequence of SEQ ID NO:1 or a variant having at least about 90% sequence identity. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to at least one of the heavy or light chains of the anti-PD-L1 antibody via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is about 2 to about 25 amino acids in length (e.g., about 2 to about 10 amino acids). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:56-68.

[0326] In some embodiments, a method of treating an individual disease or condition (e.g., cancer) is provided, the method comprising administering to the individual an effective amount of a fusion protein comprising a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V... H Furthermore, each of the two light chains contains a light chain variable region (V). La) a first TGFβ superfamily receptor extracellular domain, the first TGFβ superfamily receptor extracellular domain comprising a TGFβ receptor type I extracellular domain or a variant thereof; and c) a second TGFβ superfamily receptor extracellular domain, the second TGFβ superfamily receptor extracellular domain comprising a TGFβ receptor type II extracellular domain or a variant thereof, wherein the first and second TGFβ superfamily receptor extracellular domains are fused to one or both of the two light chains of the anti-PD-L1 antibody. In some embodiments, the first and second TGFβ superfamily receptor extracellular domains are fused to the N-terminus of one or both of the two light chains of the anti-PD-L1 antibody. In some embodiments, the first and second TGFβ superfamily receptor extracellular domains are fused to the C-terminus of one or both of the two light chains of the anti-PD-L1 antibody. In some embodiments, the first and second TGFβ superfamily receptor extracellular domains are connected to each other via a linker (such as any of the linkers described herein, such as a linker comprising the amino acid sequence of any one of SEQ ID NO: 56-68). In some embodiments, one of the first and second TGFβ superfamily receptor extracellular domains is fused to the N-terminus of one or both of the two light chains of the anti-PD-L1 antibody, and the other of the first and second TGFβ superfamily receptor extracellular domains is fused to the C-terminus of one or both of the two light chains of the anti-PD-L1 antibody. In some embodiments, the TGFβ receptor type I extracellular domain or a variant thereof is fused to the N-terminus of one or both of the two light chains of the anti-PD-L1 antibody; and the TGFβ receptor type II extracellular domain or a variant thereof is fused to the C-terminus of one or both of the two light chains of the anti-PD-L1 antibody. In some embodiments, the TGFβ receptor type I extracellular domain or a variant thereof is fused to the C-terminus of one or both of the two light chains of the anti-PD-L1 antibody; and the TGFβ receptor type II extracellular domain or a variant thereof is fused to the N-terminus of one or both of the two light chains of the anti-PD-L1 antibody. In some embodiments, the full-length antibody comprises an Fc fragment derived from human IgG1. In some embodiments, the Fc fragment derived from human IgG1 comprises a K214R mutation and an A297N mutation. In some embodiments, the TGFβ superfamily receptor extracellular domain comprises the TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the TGFβ receptor type II extracellular domain or a variant thereof comprises the human TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type II extracellular domain or a variant thereof comprises a human TGFβ receptor type II extracellular domain variant having at least about 90% sequence identity with SEQ ID NO:2, wherein the variant contains one or more amino acid substitutions at positions N47, N71, and / or N131 according to the numbers in SEQ ID NO:2. In some embodiments, the variant comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the TGFβ superfamily receptor extracellular domain comprises the TGFβ receptor type I extracellular domain or a variant thereof.In some embodiments, the TGFβ receptor type I extracellular domain or a variant thereof comprises the human TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type I extracellular domain or a variant thereof comprises the amino acid sequence of SEQ ID NO:1 or a variant having at least about 90% sequence identity. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to at least one of the heavy or light chains of the anti-PD-L1 antibody via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is about 2 to about 25 amino acids in length (e.g., about 2 to about 10 amino acids). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:56-68.

[0327] In some embodiments, a method of treating an individual disease or condition (e.g., cancer) is provided, the method comprising administering to the individual an effective amount of a fusion protein comprising a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V... H Furthermore, each of the two light chains contains a light chain variable region (V). La) a first TGFβ superfamily receptor extracellular domain; and c) a second TGFβ superfamily receptor extracellular domain, wherein the first TGFβ superfamily receptor extracellular domain is fused to one or both of the two light chains of the anti-PD-L1 antibody, and wherein the second TGFβ superfamily receptor extracellular domain is fused to one or both of the two heavy chains of the anti-PD-L1 antibody. In some embodiments, the first TGFβ superfamily receptor extracellular domain is fused to the N-terminus of one or both of the two light chains of the anti-PD-L1 antibody, and the second TGFβ superfamily receptor extracellular domain is fused to the N-terminus of one or both of the two heavy chains of the anti-PD-L1 antibody. In some embodiments, the first TGFβ superfamily receptor extracellular domain is fused to the C-terminus of one or both of the two light chains of the anti-PD-L1 antibody, and the second TGFβ superfamily receptor extracellular domain is fused to the C-terminus of one or both of the two heavy chains of the anti-PD-L1 antibody. In some embodiments, the extracellular domain of a first TGFβ superfamily receptor is fused to the N-terminus of one or both of the two light chains of the anti-PD-L1 antibody, and the extracellular domain of a second TGFβ superfamily receptor is fused to the C-terminus of one or both of the two heavy chains of the anti-PD-L1 antibody. In some embodiments, the extracellular domain of a first TGFβ superfamily receptor is fused to the C-terminus of one or both of the two light chains of the anti-PD-L1 antibody, and the extracellular domain of a second TGFβ superfamily receptor is fused to the N-terminus of one or both of the two heavy chains of the anti-PD-L1 antibody. In some embodiments, the first TGFβ receptor extracellular domain comprises a TGFβ receptor type I extracellular domain or a variant thereof, and the second TGFβ receptor extracellular domain comprises a TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the first TGFβ receptor extracellular domain comprises a TGFβ receptor type II extracellular domain or a variant thereof, and the second TGFβ receptor extracellular domain comprises a TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the full-length antibody comprises an Fc fragment derived from human IgG1. In some embodiments, the Fc fragment derived from human IgG1 contains the K214R mutation and the A297N mutation. In some embodiments, the TGFβ superfamily receptor extracellular domain contains the TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the TGFβ receptor type II extracellular domain or a variant thereof contains the human TGFβ receptor type II extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type II extracellular domain or a variant thereof contains a human TGFβ receptor type II extracellular domain variant having at least about 90% sequence identity with SEQ ID NO:2, wherein the variant contains one or more amino acid substitutions at positions N47, N71, and / or N131 according to the numbers in SEQ ID NO:2. In some embodiments, the variant contains the amino acid sequence of SEQ ID NO:3. In some embodiments, the TGFβ superfamily receptor extracellular domain contains the TGFβ receptor type I extracellular domain or a variant thereof.In some embodiments, the TGFβ receptor type I extracellular domain or a variant thereof comprises the human TGFβ receptor type I extracellular domain or a variant thereof. In some embodiments, the human TGFβ receptor type I extracellular domain or a variant thereof comprises the amino acid sequence of SEQ ID NO:1 or a variant having at least about 90% sequence identity. In some embodiments, the TGFβ superfamily receptor extracellular domain is fused to at least one of the heavy or light chains of the anti-PD-L1 antibody via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is about 2 to about 25 amino acids in length (e.g., about 2 to about 10 amino acids). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:56-68.

[0328] In some embodiments, methods for overcoming or reducing resistance to immune checkpoint inhibitors (such as anti-PD-L1 antibodies) are also provided, the methods comprising administering any of the fusion proteins or peptides described herein.

[0329] As described herein, the fusion protein of this application exhibits a lower TGFβ2 binding affinity, which may result in a better safety profile (e.g., milder toxicity associated with TGFβ2 neutralization) compared to methods involving reference fusion proteins (such as M7824). In some embodiments, the methods described herein are used in cardiac patients to minimize treatment-induced cardiotoxicity.

[0330] In some embodiments, the severity of TGFβ2 neutralization-related side effects is less than that of corresponding treatments involving a reference fusion protein (such as M7824). In some embodiments, TGFβ2 neutralization-related side effects include cardiotoxicity associated with TGFβ2 neutralization. In some embodiments, cardiotoxicity can be measured by assessing before and after treatment a) chest pain, b) changes in heart rate, c) fatigue, d) shortness of breath, e) weight gain, and / or f) swelling.

[0331] Disease or ailment

[0332] The fusion proteins or peptides described herein can be used to treat any disease or condition. In some embodiments, the disease or condition is cancer. In some embodiments, the disease or condition is or originates from an infectious disease (such as a viral infection).

[0333] In some embodiments, the fusion protein or peptide is used in methods for treating cancer. Cancers that can be treated using any of the methods described herein include unvascularized or not yet adequately vascularized tumors, as well as vascularized tumors. As described in this application, the types of cancer treated with the fusion protein include, but are not limited to, epithelial carcinoma, blastoma, sarcoma, benign and malignant tumors, and malignant tumors (e.g., sarcoma, epithelial carcinoma, and melanoma). Adult tumors / cancers and pediatric tumors / cancers are also included. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematologic malignancy.

[0334] Examples of cancers treatable by the methods described in this application include, but are not limited to, anal cancer, astrocytoma (e.g., cerebellum and brain), basal cell carcinoma, bladder cancer, bone cancer (osteosarcoma and malignant fibrous histiocytoma), brain tumors (e.g., glioma, brainstem glioma, cerebellar or brain astrocytoma (e.g., astrocytoma, malignant glioma, medulloblastoma, and glioblastoma), breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer (e.g., uterine cancer), esophageal cancer, eye cancer (e.g., intraocular melanoma and retinoblastoma), gastric (stomach) cancer, gastrointestinal stromal tumor (GIST), head and neck cancer (e.g., head and neck squamous cell carcinoma), hepatocellular carcinoma (liver) cancer (e.g., Hepatocellular carcinoma (HCC), liver cancer, biliary tract cancer (such as bile duct cancer and / or gallbladder cancer), prostate cancer, lung cancer (such as small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma), medulloblastoma, melanoma, mesothelioma, myelodysplastic syndrome, nasopharyngeal carcinoma, neuroblastoma, ovarian cancer, pancreatic cancer, parathyroid carcinoma, peritoneal cancer, pituitary adenoma, rectal cancer, kidney cancer, renal pelvis cancer, and ureteral cancer (transitional cell carcinoma), rhabdomyosarcoma, skin cancer (such as non-melanoma (such as squamous cell carcinoma), melanoma, and Merkel cell carcinoma), small bowel cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, and tuberous sclerosis. Other examples of cancer may be found in the following: The Merck Manual of Diagnosis and Therapy, 19th Edition, § on Hematology and Oncology, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); The Merck Manual of Diagnosis and Therapy, 20th Edition, § on Hematology and Oncology, published by Merck Sharp & Dohme Corp., 2018 (ISBN 978-0-911-91042-1) (digital online version available at the Merck Manuals website in 2018); and SEER Program Coding and Staging Manual 2016, all of which are incorporated herein by reference in their full text for all purposes.

[0335] In some embodiments, the cancer is non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), cervical cancer, oropharyngeal cancer, anal cancer, vaginal or penile cancer, bile duct cancer, cholangiocarcinoma, gallbladder cancer, head and neck cancer, pancreatic cancer, prostate cancer, urothelial carcinoma, bladder cancer, urinary system tumors, urogenital tract cancer, gastric cancer, breast cancer, or colorectal cancer.

[0336] In some embodiments, the cancer is Her2-positive breast cancer. In some embodiments, the cancer is triple-negative breast cancer (such as metastatic triple-negative breast cancer). In some embodiments, the cancer is metastatic hormone receptor-positive, Her2-negative breast cancer.

[0337] In some embodiments, cancer is associated with human papillomavirus (HPV).

[0338] In various embodiments, cancer is early-stage cancer, non-metastatic cancer, primary cancer, advanced cancer, locally advanced cancer, metastatic cancer, cancer in remission, recurrent cancer, cancer in adjuvant therapy, cancer in neoadjuvant therapy, or cancer that is essentially difficult to treat.

[0339] In some embodiments, the disease or condition is or arises from an infectious disease (such as a viral infection). In some embodiments, the disease or condition is recurrent respiratory papillomavirus. In one embodiment, the virus is human papillomavirus (HPV).

[0340] In some embodiments, PD-L1 expression is abnormal in diseased tissue. For example, PD-L1 expression in diseased tissue is at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 300%, 400%, or 500% higher than the PD-L1 expression level in reference tissue. In some embodiments, the reference tissue is a corresponding tissue derived from an individual without disease or symptom. In some embodiments, the PD-L1 expression level in the reference tissue is measured by calculating the average PD-L1 expression level in the reference tissue across a group of individuals without disease or symptom.

[0341] Administration and application methods

[0342] The dosage of the fusion protein or peptide described herein for treating the disease or disorder described herein, administered to an individual, may vary depending on the specific fusion protein or peptide, the method of administration, and the type of disease or condition being treated. In some embodiments, the type of disease or condition is cancer. In some embodiments, the effective amount of the fusion protein or peptide is the amount that effectively results in an objective response (e.g., a partial or complete response). In some embodiments, the effective amount of the fusion protein or peptide is sufficient to result in a complete response in an individual. In some embodiments, the effective amount of the fusion protein or peptide is sufficient to result in a partial response in an individual. In some embodiments, the effective amount of the fusion protein or peptide is sufficient to produce an overall response rate greater than any one of about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 64%, 65%, 70%, 75%, 80%, 85%, or 90% in a population of individuals treated with the fusion protein or peptide. An individual's response to treatment with the methods described herein may be determined, for example, based on RECIST levels.

[0343] In some embodiments, the effective amount of the fusion protein or peptide is an amount sufficient to prolong an individual's progression-free survival. In some embodiments, the effective amount of the fusion protein or peptide is an amount sufficient to prolong an individual's overall survival. In some embodiments, the effective amount of the fusion protein or peptide is an amount sufficient to produce a clinical benefit greater than about 50%, 60%, 70%, or 77% in a population of individuals treated with the fusion protein or peptide.

[0344] In some embodiments, the effective amount of the fusion protein or peptide, alone or in combination with the second, third, and / or fourth agents, is an amount sufficient to reduce tumor size, cancer cell number, or tumor growth rate by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% compared to the corresponding tumor size, cancer cell number, or tumor growth rate in the same subject before treatment, or compared to the corresponding activity in other untreated subjects (e.g., receiving placebo). The magnitude of the effect can be measured using standard methods, such as in vitro assays using purified enzymes, cell-based assays, animal models, or human tests.

[0345] In some embodiments, the effective amount of the fusion protein or peptide is an amount below the level that induces a toxicological effect (i.e., an effect above the level of clinically acceptable toxicity) or an amount that is at a level where the potential side effects can be controlled or tolerated when the composition is administered to an individual.

[0346] In some embodiments, the effective amount of the fusion protein or peptide is an amount close to the maximum tolerated dose (MTD) of the composition administered according to the same dosing regimen. In some embodiments, the effective amount of the fusion protein or peptide is greater than any one of about 80%, 90%, 95%, or 98% of the MTD.

[0347] In some embodiments, the effective amount of the fusion protein or polypeptide is the amount that slows or inhibits the progression of the disease or condition (e.g., slows or inhibits at least about 5%, 10%, 15%, 20%, 30%, 40%, or 50%) compared to an untreated individual.

[0348] In some embodiments, the effective amount of the fusion protein or peptide is the amount that reduces the side effects (autoimmune response) of the disease (e.g., transplantation) compared to an untreated individual (e.g., by at least about 5%, 10%, 15%, 20%, 30%, 40%, or 50%).

[0349] In some embodiments of any of the foregoing aspects, the effective amount of the fusion protein or polypeptide is in the range of about 0.001 μg / kg to about 100 mg / kg of total weight, for example, about 0.005 μg / kg to about 50 mg / kg, about 0.01 μg / kg to about 10 mg / kg, or about 0.01 μg / kg to about 1 mg / kg.

[0350] In some embodiments, treatment includes more than one administration of any of the fusion protein or peptide (e.g., about two, three, four, five, six, seven, eight, nine, or ten administrations of the fusion protein or peptide). In some embodiments, two administrations are performed within about one week. In some embodiments, a second administration is performed at least 1, 2, 3, 4, 5, 6, or 7 days after the first administration.

[0351] Fusion proteins can be administered to individuals (e.g., humans) via various routes, including, for example, intravenous, intra-arterial, intraperitoneal, intrapulmonary, oral, inhalation, intracapsular, intramuscular, intratracheal, subcutaneous, intraocular, intrathecal, transmucosal, and transdermal. In some embodiments, the fusion protein is included in a pharmaceutical composition when administered to an individual. In some embodiments, a continuously releasing formulation of the composition can be used. In some embodiments, the composition is administered intravenously. In some embodiments, the composition is administered intraperitoneally. In some embodiments, the composition is administered intravenously. In some embodiments, the composition is administered intraperitoneally. In some embodiments, the composition is administered intramuscularly. In some embodiments, the composition is administered subcutaneously. In some embodiments, the composition is administered intravenously. In some embodiments, the composition is administered orally.

[0352] Combination therapy

[0353] This application also provides a method of administering any one of the fusion proteins or peptides to an individual to treat a disease or condition (such as cancer), wherein the method further includes administering a second agent or therapy. In some embodiments, the second agent or therapy is a standard or commonly used agent or therapy for treating a disease or condition. In some embodiments, the second agent or therapy comprises a chemotherapy agent. In some embodiments, the second agent or therapy comprises surgery. In some embodiments, the second agent or therapy comprises radiation therapy. In some embodiments, the second agent or therapy comprises immunotherapy. In some embodiments, the second agent or therapy comprises hormone therapy. In some embodiments, the second agent or therapy comprises a tyrosine kinase inhibitor.

[0354] In some embodiments, the fusion protein or peptide is administered simultaneously with a second agent or therapy. In some embodiments, the fusion protein or peptide is administered in parallel with a second agent or therapy. In some embodiments, the fusion protein or peptide is administered sequentially with a second agent or therapy. In some embodiments, the fusion protein or peptide is administered in the same unit dosage form as the second agent or therapy. In some embodiments, the fusion protein or peptide is administered in a different unit dosage form than the second agent or therapy.

[0355] VI. Compositions, kits, and manufactured articles

[0356] This document also provides compositions (such as formulations) comprising any one of the fusion proteins or polypeptides described herein, a nucleic acid encoding any one of the fusion proteins or portions thereof, a vector comprising a nucleic acid encoding one of the fusion proteins, or a host cell comprising the nucleic acid or vector.

[0357] Suitable fusion protein or peptide formulations described herein can be obtained by mixing a fusion protein of desired purity with an optional pharmaceutically acceptable carrier, excipient, or stabilizer in the form of a lyophilized formulation or an aqueous solution (Remington's Pharmaceutical Sciences, 16th edition, Osol, A.Ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations used. Lyophilized formulations suitable for subcutaneous administration are described in WO 97 / 04801. Such lyophilized formulations can be reconstituted to high protein concentrations using suitable diluents, and the reconstituted formulation can be subcutaneously administered to an individual for imaging, diagnosis, or treatment at this site.

[0358] Preparations intended for internal administration must be sterile. This can be easily achieved, for example, by filtration through a sterile filter membrane.

[0359] Kits containing any of the fusion proteins or peptides described herein are also provided. These kits can be used with any of the treatment methods described herein.

[0360] In some embodiments, the kit further includes a device capable of delivering either the fusion protein or the peptide to an individual. One type of device used, for example, in parenteral delivery applications, is a syringe for injecting the composition into the subject. Inhalation devices may also be used for certain applications.

[0361] In some embodiments, the kit further includes a therapeutic agent for treating a disease or condition (such as cancer).

[0362] The kit of this application is packaged in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed polyester film or plastic bags), etc. The kit may optionally include other components, such as buffer solutions and explanatory information.

[0363] Therefore, this application also provides an article of manufacture. The article of manufacture may include a container and a label or packaging insert on or associated with the container. Suitable containers include vials (such as sealed vials), bottles, jars, flexible packaging, etc. Typically, the container contains the composition and may have a sterile inlet (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle).

[0364] Those skilled in the art will recognize that various embodiments are possible within the scope and spirit of the invention. The invention will now be described in more detail with reference to the following non-limiting examples. These examples further illustrate the invention, but should not, of course, be construed as limiting its scope in any way.

[0365] Exemplary embodiments

[0366] Example 1. A fusion protein comprising: a) a full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises a heavy chain variable region (V... H Furthermore, each of the two light chains contains a light chain variable region (V). L b) TGFβ superfamily receptor extracellular domain; wherein the TGFβ superfamily receptor extracellular domain is fused to at least one of the heavy or light chains of the anti-PD-L1 antibody.

[0367] Example 2. A fusion protein as described in Example 1, wherein the extracellular domain of the TGFβ superfamily receptor is fused to at least one or both of the two light chains.

[0368] Example 3. A fusion protein as described in Example 2, wherein the extracellular domain of the TGFβ superfamily receptor is fused to the C-terminus of at least one or both of the two light chains.

[0369] Example 4. A fusion protein as described in Example 2 or Example 3, wherein the extracellular domain of the TGFβ superfamily receptor is fused to the N-terminus of at least one or both of the two light chains.

[0370] Example 5. A fusion protein as described in any one of Examples 1-4, wherein the extracellular domain of the TGFβ superfamily receptor is fused to at least one or both of the two heavy chains.

[0371] Example 6. A fusion protein as described in Example 5, wherein the extracellular domain of the TGFβ superfamily receptor is fused to the C-terminus of at least one or both of the two heavy chains.

[0372] Example 7. A fusion protein as described in Example 5 or Example 6, wherein the extracellular domain of the TGFβ superfamily receptor is fused to the N-terminus of at least one or both of the two heavy chains.

[0373] Example 8. A fusion protein as described in any one of Examples 1-7, wherein the TGFβ superfamily receptor extracellular domain comprises the TGFβ receptor type II extracellular domain or a variant thereof.

[0374] Example 9. A fusion protein as described in Example 8, wherein the TGFβ receptor type II extracellular domain or a variant thereof comprises the human TGFβ receptor type II extracellular domain or a variant thereof.

[0375] Example 10. A fusion protein as described in Example 9, wherein the human TGFβ receptor type II extracellular domain or a variant thereof comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:2, wherein the variant comprises one or more amino acid substitutions at positions N47, N71, and / or N131 according to the numbering of SEQ ID NO:2.

[0376] Example 11. A fusion protein as described in Example 10, wherein the variant comprises the N47Q mutation numbered according to SEQ ID NO:2.

[0377] Example 12. A fusion protein as described in Example 10 or Example 11, wherein the variant comprises the N71Q mutation numbered according to SEQ ID NO:2.

[0378] Example 13. A fusion protein as described in any one of Examples 10-12, wherein the variant comprises the N131Q mutation numbered according to SEQ ID NO:2.

[0379] Example 14. A fusion protein as described in any one of Examples 10-13, wherein the variant comprises the amino acid sequence of SEQ ID NO:3.

[0380] Example 15. A fusion protein as described in any one of Examples 1-14, wherein the TGFβ superfamily receptor extracellular domain comprises the TGFβ receptor type I extracellular domain or a variant thereof.

[0381] Example 16. A fusion protein as described in Example 15, wherein the TGFβ receptor type I extracellular domain or a variant thereof comprises the human TGFβ receptor type I extracellular domain or a variant thereof.

[0382] Example 17. A fusion protein as described in any one of Examples 1-16, wherein: a) the V H Contains: i) HC-CDR1 containing the amino acid sequence of SEQ ID NO:6; ii) HC-CDR2 containing the amino acid sequence of SEQ ID NO:7; and iii) HC-CDR3 containing the amino acid sequence of SEQ ID NO:8; and b) the V L It includes: i) LC-CDR1 containing the amino acid sequence of SEQ ID NO:9; ii) LC-CDR2 containing the amino acid sequence of SEQ ID NO:10; and iii) LC-CDR3 containing the amino acid sequence of SEQ ID NO:11.

[0383] Example 18. A fusion protein as described in any one of Examples 1-17, wherein the extracellular domain of the TGFβ superfamily receptor is fused to at least one of the heavy or light chains of the anti-PD-L1 antibody via a linker.

[0384] Example 19. A fusion protein as described in Example 18, wherein the linker is a peptide linker.

[0385] Example 20. A fusion protein as described in Example 19, wherein the length of the peptide linker is about 2 to about 25 amino acids.

[0386] Example 21. A fusion protein as described in Example 20, wherein the length of the peptide linker is about 2 to about 10 amino acids.

[0387] Example 22. A fusion protein as described in any one of Examples 18-21, wherein the adapter is a GS adapter or comprises a modified sequence derived from the IgG hinge region.

[0388] Example 23. A fusion protein as described in any one of Examples 18-22, wherein the adapter comprises the amino acid sequence of SEQ ID NO:56-68.

[0389] Example 24. A fusion protein as described in any one of Examples 1-23, wherein the V H Contains the amino acid sequence of SEQ ID NO:12 or a variant thereof having at least about 80% sequence identity, and wherein the VL The amino acid sequence containing SEQ ID NO:13 or a variant thereof having at least about 80% sequence identity.

[0390] Example 25. A fusion protein as described in any one of Examples 1-24, wherein the heavy chain comprises the amino acid sequence of any one of SEQ ID NO:14-17 or a variant thereof having at least about 80% sequence identity, and wherein the light chain comprises the amino acid sequence of SEQ ID NO:18 or a variant thereof having at least about 80% sequence identity.

[0391] Example 26. The fusion protein as described in Example 25, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:16 or 17, and wherein the light chain comprises the amino acid sequence of SEQ ID NO:18.

[0392] Example 27. A fusion protein as described in any one of Examples 2-26, wherein the extracellular domain or polypeptide of the TGFβ superfamily receptor fused to at least one or both of the two light chains comprises the amino acid sequence of any one of SEQ ID NO: 27-38 or a variant thereof having at least about 80% sequence identity.

[0393] Example 28. A fusion protein as described in any one of Examples 5-27, wherein the extracellular domain of the TGFβ superfamily receptor fused to at least one or both of the two heavy chains comprises an amino acid sequence of any one of SEQ ID NO: 22-26 or a variant thereof having at least about 80% sequence identity.

[0394] Example 29. A fusion protein as described in Example 27 or Example 28, wherein: a) the TGFβ superfamily receptor extracellular domain is fused to both heavy chains, comprising the amino acid sequence of SEQ ID NO: 22, and the two light chains comprise the amino acid sequence of SEQ ID NO: 18; b) the TGFβ superfamily receptor extracellular domain is fused to both heavy chains, comprising the amino acid sequence of SEQ ID NO: 23, and the two light chains comprise the amino acid sequence of SEQ ID NO: 18; c) the TGFβ superfamily receptor extracellular domain is fused to both light chains, comprising the amino acid sequence of SEQ ID NO: 27, and both heavy chains comprise the amino acid sequence of SEQ ID NO: 16 or 17; d) the TGFβ superfamily receptor extracellular domain is fused to both light chains, comprising the amino acid sequence of SEQ ID NO: 28, and both heavy chains comprise the amino acid sequence of SEQ ID NO: 16 or 17; e) the TGFβ superfamily receptor extracellular domain is fused to both heavy chains, comprising the amino acid sequence of SEQ ID NO: 22, and the two light chains comprise the amino acid sequence of SEQ ID NO: 18. The amino acid sequence of NO:22, and the amino acid sequence containing SEQ ID NO:28, in which the extracellular domain of the TGFβ superfamily receptor is also fused to both light chains; f) the amino acid sequence containing SEQ ID NO:23, in which the extracellular domain of the TGFβ superfamily receptor is fused to both heavy chains, and the amino acid sequence containing SEQ ID NO:27, in which the extracellular domain of the TGFβ superfamily receptor is fused to both heavy chains; g) the amino acid sequence containing SEQ ID NO:22, in which the extracellular domain of the TGFβ superfamily receptor is fused to both heavy chains, and the amino acid sequence containing SEQ ID NO:27, in which the extracellular domain of the TGFβ superfamily receptor is fused to both heavy chains; h) the amino acid sequence containing SEQ ID NO:23, in which the extracellular domain of the TGFβ superfamily receptor is fused to both heavy chains, and the amino acid sequence containing SEQ ID NO:28, in which the extracellular domain of the TGFβ superfamily receptor is fused to both light chains; i) the amino acid sequence containing SEQ ID NO:28, in which the polypeptide is fused to both heavy chains. The amino acid sequence of SEQ ID NO:24, and both light chains contain the amino acid sequence of SEQ ID NO:18; j) the TGFβ superfamily receptor extracellular domain fused to both heavy chains, containing the amino acid sequence of SEQ ID NO:25, and both light chains contain the amino acid sequence of SEQ ID NO:18; k) the TGFβ superfamily receptor extracellular domain fused to both heavy chains, containing the amino acid sequence of SEQ ID NO:26, and both light chains contain the amino acid sequence of SEQ ID NO:18; l) the TGFβ superfamily receptor extracellular domain fused to both light chains, containing the amino acid sequence of SEQ ID NO:29, and both heavy chains contain the amino acid sequence of SEQ ID NO:16 or 17;m) The TGFβ superfamily receptor extracellular domain fused to both light chains, containing the amino acid sequence of SEQ ID NO:30, and both heavy chains containing the amino acid sequence of SEQ ID NO:16 or 17; n) The TGFβ superfamily receptor extracellular domain fused to both light chains, containing the amino acid sequence of SEQ ID NO:31, and both heavy chains containing the amino acid sequence of SEQ ID NO:16 or 17; o) The TGFβ superfamily receptor extracellular domain fused to both light chains, containing the amino acid sequence of SEQ ID NO:32, and both heavy chains containing the amino acid sequence of SEQ ID NO:16 or 17; p) The TGFβ superfamily receptor extracellular domain fused to both light chains, containing the amino acid sequence of SEQ ID NO:33, and both heavy chains containing the amino acid sequence of SEQ ID NO:16 or 17; q) The TGFβ superfamily receptor extracellular domain fused to both light chains, containing the amino acid sequence of SEQ ID NO:34, and both heavy chains containing the amino acid sequence of SEQ ID NO:16 or 17. The amino acid sequence NO:16 or 17; r) the amino acid sequence containing SEQ ID NO:35 fused to both light chains of the TGFβ superfamily receptor extracellular domain, and both heavy chains containing SEQ ID NO:16 or 17; s) the amino acid sequence containing SEQ ID NO:36 fused to both light chains of the TGFβ superfamily receptor extracellular domain, and both heavy chains containing SEQ ID NO:16 or 17; or t) the amino acid sequence containing SEQ ID NO:37 fused to both light chains of the TGFβ superfamily receptor extracellular domain, and both heavy chains containing SEQ ID NO:16 or 17.

[0395] Example 30. A polypeptide comprising a human TGFβ receptor type II extracellular domain variant having at least about 90% sequence identity with SEQ ID NO:2, wherein the variant contains one or more amino acid substitutions at positions N47, N71, and / or N131 according to the numbering of SEQ ID NO:2.

[0396] Example 31. A polypeptide as described in Example 30, wherein the variant comprises the N47Q mutation according to SEQ ID NO:2.

[0397] Example 32. A polypeptide as described in Example 30 or Example 31, wherein the variant comprises the N71Q mutation according to SEQ ID NO:2.

[0398] Example 33. A polypeptide as described in any one of Examples 30-32, wherein the variant comprises the N131Q mutation according to SEQ ID NO:2.

[0399] Example 34. A polypeptide as described in any one of Examples 30-33, wherein the variant comprises the amino acid sequence of SEQ ID NO:3.

[0400] Example 35. A fusion protein comprising a) a polypeptide as described in any one of Examples 30-34; and b) a second part.

[0401] Example 36. A fusion protein as described in Example 35, wherein the second portion comprises a half-life extension domain.

[0402] Example 37. A fusion protein as described in Example 36, wherein the second part comprises an Fc fragment.

[0403] Example 38. A fusion protein as described in Example 37, wherein the second part comprises an immune checkpoint inhibitor.

[0404] Example 39. The fusion protein as described in Example 38, wherein the immune checkpoint inhibitor comprises an anti-PD-L1 antibody portion.

[0405] Example 40. A pharmaceutical composition comprising a fusion protein or polypeptide as described in any one of Examples 1-39.

[0406] Example 41. A nucleic acid encoding a fusion protein or a portion thereof, or a polypeptide as described in any one of Examples 1-39.

[0407] Example 42. A nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:19-21 and 38-53.

[0408] Example 43. A vector comprising nucleic acid as described in Example 41 or Example 42.

[0409] Example 44. A host cell comprising nucleic acid as described in Example 41 or Example 42, or a vector as described in Example 43.

[0410] Example 45. A method for producing a fusion protein or polypeptide as described in any one of Examples 1-39, the method comprising: a) culturing a host cell as described in Example 44 under conditions that effectively express the fusion protein or polypeptide; and b) obtaining the expressed fusion protein or polypeptide from the host cell.

[0411] Example 46. A method for treating an individual's disease or condition, the method comprising administering to the individual an effective amount of a fusion protein as described in any one of Examples 1-29 and 35-39, or a pharmaceutical composition as described in Example 40.

[0412] Example 47. The method as described in Example 46, wherein the disease or condition is cancer.

[0413] Example 48. The method as described in Example 47, wherein the cancer is non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), cervical cancer, oropharyngeal cancer, anal cancer, vaginal or penile cancer, bile duct cancer, cholangiocarcinoma, gallbladder cancer, head and neck cancer, pancreatic cancer, prostate cancer, urothelial carcinoma, bladder cancer, urinary system tumor, urogenital tract cancer, gastric cancer, breast cancer, or colorectal cancer.

[0414] Example 49. The method as described in Example 47 or Example 48, wherein the cancer is an advanced or metastatic cancer.

[0415] Example 50. The method as described in any one of Examples 46-49, wherein the method further comprises administering a second agent or therapy.

[0416] Example 51. The method as described in any one of Examples 46-50, wherein the fusion protein or pharmaceutical composition is administered parenterally to the individual.

[0417] Example 52. The method as described in any one of Examples 46-51, wherein the individual is a person.

[0418] Example 53. A kit for treating a disease or condition, comprising a pharmaceutical composition as described in Example 40 and instructions for use.

[0419] Example

[0420] The following examples are merely illustrative of this application and should not be construed as limiting this application in any way. The following examples and detailed descriptions are provided for illustration and not for limitation.

[0421] Example 1. Construction and expression of PDL1-TGFβ receptor fusion protein.

[0422] A series of PDL1-TGFβ receptor fusion proteins were designed using an anti-PD-L1 monoclonal antibody (mAb), the extracellular domain of TGFβ receptor I (TBRI-ECD), and / or the extracellular domain of TGFβ receptor II (TBRII-ECD). To construct these fusion proteins, TBRI-ECD and / or TBRII-ECD were fused with the light / heavy chain of the anti-PD-L1 mAb.

[0423] Specifically, the exemplary TGFβ receptor extracellular domain is located at the C-terminus of either the heavy or light chain of the anti-PD-L1 mAb, and has different types of linkers (E-linker: EPKSSDKTHTSPPSP, E2-linker: ERKSSVESPPSP, G21-linker: GGGGSGGGGSGGGGGSGGGG, G15-linker: (G4S)3, G9-linker: GGSGGGGSG, G4-linker: GGGS, G2-linker: GS, E4-linker: SPPS, E5-linker: SPPSP, E5a-linker: GPPGP, E8-linker: DKTHTSPP, E10-linker: DKTHTSPPSP, and E12-linker: ESKYGPPSPPSP) for fusion. Some fusion protein constructs consist of one fusion polypeptide chain and one native polypeptide chain, while others consist of two fusion polypeptide chains. The DNA sequence expressing each polypeptide chain was inserted into the pTT5 vector between the EcoRI and HindIII restriction sites. Each plasmid also included a secretion signal sequence for the protein secreted into the growth medium. The M7824 fusion protein was used as a reference protein. The fusion proteins are shown in Table 3 below.

[0424] Table 3.

[0425]

[0426]

[0427]

[0428] CHO-3E7 cells transfected with the expression plasmid were cultured at 37°C and 100 rpm for 6 days. The supernatant was collected by centrifugation, and the fusion protein was purified by protein A column purification.

[0429] As mentioned above, the TGFβ receptor extracellular domain, including both type I and type II extracellular domains, was used for fusion protein construction. The anti-PD-L1 mAb consists of a heavy chain called H0 and a light chain called L0. Modifying the heavy chain H0 at the Fc region with site mutations of K214R and A297N generates a new polypeptide called H1. Combining the new heavy chain H1 with the native light chain of L0 produces a modified anti-PD-L1 mAb called ADD19-15-00, and a series of fusion proteins were designed by fusing the TGFβ receptor extracellular domain with ADD19-15-00 using different linkers. Fusing TBRII-ECD with the C-terminus of the heavy chain of H1 via a G21-linker generates a new polypeptide called H2. Similarly, a new polypeptide called H3 is generated using TBRII-ECD. Peptide H2 was engineered within the TBRII-ECD using mutations at sites N47Q, N71Q, and N131Q to generate a new peptide called H4. The TBRII-ECD was then fused to the C-terminus of H2 via a G15 linker to generate a new peptide called H5. The TBRII-ECD was fused to the C-terminus of the heavy chain of H1 via an E linker to generate a new peptide called H6. The TBRII-ECD was fused to the C-terminus of the light chain of L0 via a G21 linker to generate a new peptide called L1. The TBRII-ECD was fused to the C-terminus of the light chain of L0 via a G21 linker to generate a new peptide called L2. By linking the C-terminus of the light chain of TBRII-ECD to L0 via E / E4 / E5 / E5a / E8 / E10 / E12 / G2 / G4-linkers, nine new polypeptides named L3, L4, L5, L6, L7, L8, L9, L10, and L11 are generated.

[0430] Using these novel peptides, a series of antibody fusion proteins were generated by combining novel heavy chain fusion proteins with the native light chain of L0, or by combining novel light chain fusion proteins with the native heavy chain of H1, or by combining novel heavy chain peptides with novel light chain peptides. The combination of novel heavy chain peptides with the native light chain of L0 produced novel fusion proteins named ADD19-15-01, ADD19-15-02, ADD19-15-09, ADD19-15-10, and ADD19-15-H01-E. Combining novel light chain peptides with the natural heavy chain of H1 generates novel fusion proteins named ADD19-15-03, ADD19-15-04, ADD19-15-L03-E, ADD19-15-L03-E4, ADD19-15-L03-E5, ADD19-15-L03-E5a, ADD19-15-L03-E8, ADD19-15-L03-E10, ADD19-15-L03-E12, ADD19-15-L03-G2, and ADD19-15-L03-G4. Combining novel heavy chain peptides with novel light chain peptides generates novel fusion proteins named ADD19-15-05 and ADD19-15-08.

[0431] Example 2. Characterization of fusion proteins

[0432] A. ELISA binding assay

[0433] 96-well disposable microplates were coated with TGF-β1, TGF-β2, or TGF-β3 at 4°C for 16 hours. After washing with washing buffer, the coated plates were blocked with blocking buffer at 37°C for 2 hours. The plates were then incubated with diluted samples at 37°C for 1 hour, followed by incubation with HRP-conjugated second-generation antibodies at 37°C for 0.5 hours. After washing and incubation with TMB substrate, stop solution was added to each well, and the plates were read using a 450 nM plate reader.

[0434] In such Figure 1-3 Of the first batch of fusion proteins shown, compared with the baseline M7824, (1) ADD19-15-01 showed similar binding affinity to TGFβ1, TGFβ2 and TGFβ3; (2) ADD19-15-03 showed similar binding affinity to TGFβ1 and TGFβ3, but lower binding affinity to TGFβ2; (3) ADD19-15-07 showed higher binding affinity to all three TGFβ proteins; and (4) ADD19-15-10 showed lower binding affinity to all three TGFβ proteins.

[0435] In such Figure 6-8In the second batch of fusion proteins shown, all fusion proteins exhibited similar TGFβ1 and TGFβ3 binding affinities compared to the baseline M7824, but different TGFβ2 binding affinities. ADD19-15-03 was identified as a lower TGFβ2 binder in the first batch of fusion proteins, and some fusion proteins showed even lower TGFβ2 binding affinities in the second batch, including ADD19-15-03-E4, ADD19-15-03-E5, ADD19-15-03-E8, ADD19-15-03-E10, ADD19-15-03-E12, ADD19-15-03-G2, and ADD19-15-03-G4.

[0436] B. SPR Affinity Measurement

[0437] TGFβ ligands were covalently immobilized on a CM5 Sencor chip via an amine coupling process. Assays were performed at 25°C using HBS-EP as the run buffer. A series of analyte concentrations were sequentially injected onto the ligand surface as the association phase, followed by injection into the run buffer as the dissociation phase. The affinity and kinetics of the TGFβ ligands against the PD-L1 mAb-TGFβR fusion protein are summarized in Table 4.

[0438] Table 4: Affinity of TGF-β1, TGF-β2 and TGF-β3 to samples determined by SPR

[0439]

[0440]

[0441] For TGFβ binding assays, compared to the baseline M7824, (1) ADD19-15-01 showed similar binding affinity for TGFβ1, TGFβ2 and TGFβ3; (2) ADD19-15-03 showed similar binding affinity for TGFβ1 and TGFβ3, but lower binding affinity for TGFβ2; (3) ADD19-15-07 showed similar binding affinity for TGFβ2, but higher binding affinity for TGFβ1 and TGFβ3; and (4) ADD19-15-10 showed lower binding affinity for all three TGFβ proteins.

[0442] C.FACS combined assay

[0443] The binding pattern of the antibody-antigen binding protein overexpressed on PD-L1 in CHO-K1 cells was plotted using 3X serially diluted samples, starting at a concentration of 300 nM. Antibody-antigen binding curves were generated using geometric mean. Raw data were plotted using GraphPad Prism v6.02 software, and EC50 was analyzed using a four-parameter, best-fit program.

[0444] For PD-L1 binding, such as Figure 4 The results showed that all antibody fusion proteins exhibited similar affinity for the PD-L1 antigen compared to the parental mAb of ADD19-15-00. Therefore, protein fusion did not affect the antigen affinity of the final constructs. These constructs, such as ADD19-15-01 and ADD19-15-03, also showed almost the same PD-L1 binding affinity as the baseline M7824. Meanwhile, as... Figure 9 As shown, fusion proteins with different linkers do not affect PD-L1 binding affinity.

[0445] D. In vitro bioassay

[0446] For in vitro bioassays of anti-PD-L1 antibody fusion protein, the Promega assay kit was used for PD-1 / PD-L1 blocking bioassays.

[0447] Promega's PD-1 / PD-L1 blocking bioassay system can be used to measure the potency and stability of antibodies and other biologics designed to block PD-1 / PD-L1 interactions. The assay consists of two genetically engineered cell lines: PD-1 effector cells, which are Jurkat T cells expressing human PD-1 and luciferase reporter cells driven by NFAT response elements (NFAT-RE), and PD-L1 aAPC / CHO-K1 cells, which are CHO-K1 cells expressing human PD-L1 and engineered cell surface proteins designed to activate the homologous TCR in a non-antigen-dependent manner. When the two cell types are co-cultured, the PD-1 / PD-L1 interaction inhibits TCR signaling and NFAT-RE-mediated luminescence. Addition of anti-PD-1 or anti-PD-L1 antibodies that block PD-1 / PD-L1 interactions releases the inhibitory signal, leading to TCR activation and NFAT-RE-mediated luminescence.

[0448] For PD-1 / PD-L1 blocking bioassays, Tecentriq biosimilars were used as reference antibodies. Figure 5The results showed that the fusion proteins of ADD19-15-01 and ADD19-15-03 exhibited comparable activity compared to the parental mAb of ADD19-15-00. Furthermore, their potency was similar to that of the baseline M7824. Meanwhile, as... Figure 10 and Figure 11 As shown, fusion proteins with different linkers do not affect their activity.

[0449] sequence list

[0450]

[0451]

[0452]

[0453]

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469]

[0470] sequence list <110> Nanjing GenScript Biotech Co., Ltd. <120> Fusion proteins and their uses <130> 75989-20006.40 <140> Not specified <141> Filed together with this application <160> 93 <170> Windows version FastSEQ 4.0 <210> 1 <211> 91 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 1 Gln Cys Phe Cys His Leu Cys Thr Lys Asp Asn Phe Thr Cys Val Thr 1 5 10 15 Asp Gly Leu Cys Phe Val Ser Val Thr Glu Thr Thr Asp Lys Val Ile 20 25 30 His Asn Ser Met Cys Ile Ala Glu Ile Asp Leu Ile Pro Arg Asp Arg 35 40 45 Pro Phe Val Cys Ala Pro Ser Ser Lys Thr Gly Ser Val Thr Thr Thr 50 55 60 Tyr Cys Cys Asn Gln Asp His Cys Asn Lys Ile Glu Leu Pro Thr Thr 65 70 75 80 Val Lys Ser Ser Pro Gly Leu Gly Pro Val Glu 85 90 <210> 2 <211> 136 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 2 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 3 <211> 136 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 3 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Gln Cys 35 40 45 Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Gln Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Gln Thr Ser Asn Pro Asp 130 135 <210> 4 <211> 273 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 4 cagtgcttct gccatctgtg caccaaggac aacttcacct gcgtgaccga tggcctgtgt 60 tttgtgtccg tgaccgagac aaccgacaaa gtgatccaca actccatgtg catcgccgag 120 atcgacctga tccctagaga tcggcccttc gtgtgtgctc ctagctctaa gaccggctct 180 gtcaccacaa cctactgctg taatcaggac cactgcaaca agatcgagct gcctaccacc 240 gtgaagtcct ctccaggact gggccctgtg gaa 273 <210> 5 <211> 408 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 5 attcctcctc acgtgcagaa atccgtgaac aacgacatga tcgtgaccga caacaacggc 60 gctgtgaagt ttccacagct gtgcaagttc tgcgacgtca gattctctac ctgtgacaac 120 cagaagtcct gtatgtccaa ctgctccatc acctctatct gcgagaagcc ccaagaggtg 180 tgcgtggccg tgtggcggaa gaacgatgag aacatcaccc tggaaaccgt gtgtcatgat 240 cctaagctgc cttaccacga cttcatcctg gaagatgccg cttctcccaa gtgcatcatg 300 aaagagaaga agaagcctgg cgagacattc ttcatgtgct cttgtagctc cgacgagtgc 360 aacgacaata tcatcttctc cgaggaatac aacaccagca atcctgac 408 <210> 6 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 6 Gly Tyr Gly Ile Thr 1 5 <210> 7 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 7 Glu Ile Phe Pro Arg Arg Val Gln Thr Tyr Tyr Tyr Ser Glu Lys Phe Lys 1 5 10 15 Gly <210> 8 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 8 Asp Tyr Asp Pro Tyr Phe Ala Leu Asp Tyr 1 5 10 <210> 9 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 9 Arg Ala Ser Gln Asp Val Ser Thr Ala Val Asp 1 5 10 <210> 10 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 10 Ser Ala Ser Tyr Arg Tyr Thr 1 5 <210> 11 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 11 Gln Gln His Tyr Ser Ile Pro Phe Thr 1 5 <210> 12 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 12 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Gly Tyr 20 25 30 Gly Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Phe Pro Arg Arg Val Gln Thr Tyr Tyr Ser Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Asp Pro Tyr Phe Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 13 <211> 110 <212> PRT <213> artificial sequence <220> <223> Synthesize construction <400> 13 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Ile Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val 100 105 110 <210> 14 <211> 449 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 14 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Gly Tyr 20 25 30 Gly Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Phe Pro Arg Arg Val Gln Thr Tyr Tyr Ser Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Asp Pro Tyr Phe Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 15 <211> 449 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 15 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Gly Tyr 20 25 30 Gly Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Phe Pro Arg Arg Val Gln Thr Tyr Tyr Ser Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Asp Pro Tyr Phe Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Ala <210> 16 <211> 449 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 16 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Gly Tyr 20 25 30 Gly Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Phe Pro Arg Arg Val Gln Thr Tyr Tyr Ser Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Asp Pro Tyr Phe Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 17 <211> 449 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 17 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Gly Tyr 20 25 30 Gly Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Phe Pro Arg Arg Val Gln Thr Tyr Tyr Ser Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Asp Pro Tyr Phe Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Ala <210> 18 <211> 214 <212> PRT <213> artificial sequence <220> <223> synthetic structure <400> 18 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Ile Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 19 <211> 1347 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 19 gaagtccagc tggtgcagag cggagccgag gtgaagaaac caggcgcttc tgtgaaggtg 60 tcctgcaagg cctctgggta catcttcacc ggctacggca tcacctgggt gcggcaggct 120 cctggccagg gcctggaatg gatgggcgag atctttccca ggagagtgca gacctactac 180 tccgagaagt tcaagggcag agtgaccatg accaccgaca cctccacctc taccgcctac 240 atggaactgc ggtctctgag atccgatgac accgctgtgt actactgcgc cagagactac 300 gacccttatt tcgccctgga ttattggggc caaggcacca ccgtgacagt ctcctccgcc 360 tctaccaagg gcccttccgt gttccccctg gcccctagca gcaagtccac atcaggaggc 420 accgctgctc tgggctgcct ggtcaaggac tacttccctg aacctgtgac cgtgtcctgg 480 aactccggcg ccctgacaag tggagtgcat accttccccg ccgtgctgca gtcctctggc 540 ctgtactctc tgtctagcgt ggtcactgtg ccttcctcta gcctcggcac acagacatac 600 atctgcaacg tgaaccacaa gccttccaac accaaagtgg acaagaaggt ggaacccaag 660 tcttgcgaca aaacccacac atgtccacct tgtcctgccc ccgagctgct gggcggcccc 720 tccgtgtttc tgtttcctcc taagccgaag gatacactga tgatctcccg gacccctgag 780 gtgacctgtg tggtggtgga cgtgtctcac gaggaccccg aagtgaagtt caactggtac 840 gtggatggcg tggaagtgca caatgctaag accaagccta gagaagagca gtacgcctcc 900 acctaccggg tggtctctgt gctgaccgtc ctgcatcagg actggctgaa cggcaaagag 960 tacaagtgca aggtgtctaa caaggctctg cctgctccta tcgagaaaac catctctaag 1020 gccaagggac agcctcggga accacaagtg tacaccctgc ctccttctag agaggagatg 1080 accaagaacc aggtgagcct gacctgcctc gtgaaaggct tctacccctc tgacatcgcc 1140 gtggagtggg agtccaatgg ccagcctgag aacaactaca agaccacacc tccagttctg 1200 gactccgacg gttccttctt cctgtactcc aagctgaccg ttgataagtc cagatggcag 1260 cagggcaacg tgttctcctg ttccgtgatg cacgaggccc tgcacaacca ctacacccag 1320 aagtccctga gcttgtctcc tggcaag 1347 <210> 20 <211> 1347 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 20 gaagtccagc tggtgcagag cggagccgag gtgaagaaac caggcgcttc tgtgaaggtg 60 tcctgcaagg cctctgggta catcttcacc ggctacggca tcacctgggt gcggcaggct 120 cctggccagg gcctggaatg gatgggcgag atctttccca ggagagtgca gacctactac 180 tccgagaagt tcaagggcag agtgaccatg accaccgaca cctccacctc taccgcctac 240 atggaactgc ggtctctgag atccgatgac accgctgtgt actactgcgc cagagactac 300 gacccttatt tcgccctgga ttattggggc caaggcacca ccgtgacagt ctcctccgcc 360 tctaccaagg gcccttccgt gttccccctg gcccctagca gcaagtccac atcaggaggc 420 accgctgctc tgggctgcct ggtcaaggac tacttccctg aacctgtgac cgtgtcctgg 480 aactccggcg ccctgacaag tggagtgcat accttccccg ccgtgctgca gtcctctggc 540 ctgtactctc tgtctagcgt ggtcactgtg ccttcctcta gcctcggcac acagacatac 600 atctgcaacg tgaaccacaa gccttccaac accaaagtgg acaagagagt ggaacccaag 660 tcttgcgaca aaacccacac atgtccacct tgtcctgccc ccgagctgct gggcggcccc 720 tccgtgtttc tgtttcctcc taagccgaag gatacactga tgatctcccg gacccctgag 780 gtgacctgtg tggtggtgga cgtgtctcac gaggaccccg aagtgaagtt caactggtac 840 gtggatggcg tggaagtgca caatgctaag accaagccta gagaagagca gtacaactcc 900 acctaccggg tggtctctgt gctgaccgtc ctgcatcagg actggctgaa cggcaaagag 960 tacaagtgca aggtgtctaa caaggctctg cctgctccta tcgagaaaac catctctaag 1020 gccaagggac agcctcggga accacaagtg tacaccctgc ctccttctag agaggagatg 1080 accaagaacc aggtgagcct gacctgcctc gtgaaaggct tctacccctc tgacatcgcc 1140 gtggagtggg agtccaatgg ccagcctgag aacaactaca agaccacacc tccagttctg 1200 gactccgacg gttccttctt cctgtactcc aagctgaccg ttgataagtc cagatggcag 1260 cagggcaacg tgttctcctg ttccgtgatg cacgaggccc tgcacaacca ctacacccag 1320 aagtccctga gcttgtctcc tggcaag 1347 <210> 21 <211> 642 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 21 gatatccaga tgacccagtc tcctagcagc ctgagcgctt ctgtgggcga cagagtgaca 60 atcacctgta gagcctctca ggacgtgtcc accgccgtgg attggtacca gcagaagccc 120 ggcaaggctc ctaagctgct gatctactct gcctcctacc ggtacacagg agtccccgat 180 agattctctg gctccggctc tggaaccgac ttcaccttca ccatctcctc tctgcagcct 240 gaggacattg ccacctacta ctgccagcag cactactcca tcccttttac cttcggccag 300 ggcaccaagc tggaaatcaa gcggaccgtg gccgctccat ccgtgttcat ctttcctcct 360 tccgacgagc agctgaagtc tggcaccgct tccgtggtgt gcctgctgaa caacttctac 420 cctcgggaag ccaaggtgca gtggaaagtg gacaacgccc tgcagtccgg caatagccaa 480 gagtccgtca ccgagcaaga ctccaaggac tctacctatt ctctctccag cacactgacc 540 ctgtctaaag ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600 ctgtcttccc ccgtgacaaa gtccttcaac agaggcgagt gt 642 <210> 22 <211> 606 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 22 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Gly Tyr 20 25 30 Gly Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Phe Pro Arg Arg Val Gln Thr Tyr Tyr Ser Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Asp Pro Tyr Phe Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 450 455 460 Gly Gly Gly Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser Val Asn 465 470 475 480 Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln 485 490 495 Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys 500 505 510 Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln 515 520 525 Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu 530 535 540 Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu 545 550 555 560 Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro 565 570 575 Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp 580 585 590 Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 595 600 605 <210> 23 <211> 561 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 23 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Gly Tyr 20 25 30 Gly Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Phe Pro Arg Arg Val Gln Thr Tyr Tyr Ser Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Asp Pro Tyr Phe Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 450 455 460 Gly Gly Gly Gly Ser Gly Gln Cys Phe Cys His Leu Cys Thr Lys Asp 465 470 475 480 Asn Phe Thr Cys Val Thr Asp Gly Leu Cys Phe Val Ser Val Thr Glu 485 490 495 Thr Thr Asp Lys Val Ile His Asn Ser Met Cys Ile Ala Glu Ile Asp 500 505 510 Leu Ile Pro Arg Asp Arg Pro Phe Val Cys Ala Pro Ser Ser Lys Thr 515 520 525 Gly Ser Val Thr Thr Thr Tyr Cys Cys Asn Gln Asp His Cys Asn Lys 530 535 540 Ile Glu Leu Pro Thr Thr Val Lys Ser Ser Pro Gly Leu Gly Pro Val 545 550 555 560 Glu <210> 24 <211> 606 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 24 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Gly Tyr 20 25 30 Gly Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Phe Pro Arg Arg Val Gln Thr Tyr Tyr Ser Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Asp Pro Tyr Phe Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 450 455 460 Gly Gly Gly Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser Val Asn 465 470 475 480 Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln 485 490 495 Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys 500 505 510 Ser Cys Met Ser Gln Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln 515 520 525 Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Gln Ile Thr Leu 530 535 540 Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu 545 550 555 560 Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro 565 570 575 Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp 580 585 590 Asn Ile Ile Phe Ser Glu Glu Tyr Gln Thr Ser Asn Pro Asp 595 600 605 <210> 25 <211> 712 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 25 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Gly Tyr 20 25 30 Gly Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Phe Pro Arg Arg Val Gln Thr Tyr Tyr Ser Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Asp Pro Tyr Phe Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 450 455 460 Gly Gly Gly Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser Val Asn 465 470 475 480 Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln 485 490 495 Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys 500 505 510 Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln 515 520 525 Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu 530 535 540 Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu 545 550 555 560 Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro 565 570 575 Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp 580 585 590 Asn Ile Ile Phe Ser Glu Glu ...

Claims

1. A fusion protein, said fusion protein comprising: a) A full-length antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains, wherein each of the two heavy chains contains a heavy chain variable region (VL1). H ), and each of the two light chains contains a light chain variable region (V L ),in The V H Contains: i) HC-CDR1 with the amino acid sequence shown in SEQ ID NO: 6; ii) HC-CDR2 with the amino acid sequence shown in SEQ ID NO: 7; and iii) HC-CDR3 with the amino acid sequence shown in SEQ ID NO: 8; and, The V L It includes: i) LC-CDR1 with the amino acid sequence shown in SEQ ID NO: 9; ii) LC-CDR2 with the amino acid sequence shown in SEQ ID NO: 10; and iii) LC-CDR3 with the amino acid sequence shown in SEQ ID NO: 11; b) The extracellular domain of the TGFβ superfamily receptor, wherein the amino acid sequence of the extracellular domain of the TGFβ superfamily receptor is shown in SEQ ID NO: 2; The extracellular domain of the TGFβ superfamily receptor is fused to the C-terminus of the light chain of the anti-PD-L1 antibody via a linker, wherein the linker is a GS linker or a modified sequence derived from the hinge region of IgG, and the linker is 2 to 25 amino acids in length.

2. The fusion protein of claim 1, wherein the extracellular domain of the TGFβ superfamily receptor is fused to two of the two light chains.

3. The fusion protein of claim 1 or 2, wherein the amino acid sequence of the linker is any one of SEQ ID NO: 56-68.

4. The fusion protein of claim 1 or 2, wherein the V H Containing the amino acid sequence of SEQ ID NO: 12 or a variant thereof having at least 80% sequence identity, and wherein the V L Contains the amino acid sequence of SEQ ID NO: 13 or a variant thereof having at least 80% sequence identity.

5. The fusion protein of claim 1 or 2, wherein the heavy chain comprises the amino acid sequence of any one of SEQ ID NO: 14-17 or a variant thereof having at least 80% sequence identity, and wherein the light chain comprises the amino acid sequence of SEQ ID NO: 18 or a variant thereof having at least 80% sequence identity.

6. The fusion protein of claim 5, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 16 or 17, and wherein the light chain comprises the amino acid sequence of SEQ ID NO:

18.

7. The fusion protein of claim 1 or 2, wherein the extracellular domain of the TGFβ superfamily receptor, after fusion with the light chain, comprises an amino acid sequence of any one of SEQ ID NO: 27 and 29-37 or a variant thereof having at least 80% sequence identity.

8. The fusion protein of claim 7, wherein: a) The extracellular domain of the TGFβ superfamily receptor is fused to both light chains and the fused chains contain the amino acid sequence of SEQ ID NO:27, and both heavy chains contain the amino acid sequence of SEQ ID NO:16 or 17. b) The extracellular domain of the TGFβ superfamily receptor is fused to both of the two light chains and the fused chains contain the amino acid sequence of SEQ ID NO:29, and both heavy chains contain the amino acid sequence of SEQ ID NO:16 or 17. c) The extracellular domain of the TGFβ superfamily receptor is fused to both of the two light chains and the fused chains each contain the amino acid sequence of SEQ ID NO:30, and both heavy chains each contain the amino acid sequence of SEQ ID NO:16 or 17. d) The extracellular domain of the TGFβ superfamily receptor is fused to both light chains and the fused chains contain the amino acid sequence of SEQ ID NO:31, and both heavy chains contain the amino acid sequence of SEQ ID NO:16 or 17. e) The extracellular domain of the TGFβ superfamily receptor is fused to both of the two light chains and the fused chains each contain the amino acid sequence of SEQ ID NO:32, and both heavy chains each contain the amino acid sequence of SEQ ID NO:16 or 17. f) The extracellular domain of the TGFβ superfamily receptor is fused to both of the two light chains and the fused chains each contain the amino acid sequence of SEQ ID NO:33, and both heavy chains each contain the amino acid sequence of SEQ ID NO:16 or 17. g) The extracellular domain of the TGFβ superfamily receptor is fused to both of the two light chains and the fused chains contain the amino acid sequence of SEQ ID NO:34, and both heavy chains contain the amino acid sequence of SEQ ID NO:16 or 17. h) The extracellular domain of the TGFβ superfamily receptor is fused to both of the two light chains and the fused chains each contain the amino acid sequence of SEQ ID NO:35, and both heavy chains each contain the amino acid sequence of SEQ ID NO:16 or 17. i) The extracellular domain of the TGFβ superfamily receptor is fused to both light chains, and the fused portions each contain the amino acid sequence of SEQ ID NO:36, and both heavy chains contain the amino acid sequence of SEQ ID NO:16 or 17; or j) The extracellular domain of the TGFβ superfamily receptor is fused to both of the two light chains and the fused chains each contain the amino acid sequence of SEQ ID NO:37, and both heavy chains each contain the amino acid sequence of SEQ ID NO:16 or 17.

9. A pharmaceutical composition comprising a fusion protein as described in any one of claims 1-8.

10. A nucleic acid that encodes a fusion protein as described in any one of claims 1-8.

11. A nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 19-21, 43 and 45-53.

12. A vector comprising the nucleic acid as described in claim 10 or claim 11.

13. A host cell comprising the nucleic acid as claimed in claim 10 or claim 11, or the vector as claimed in claim 12.

14. A method for producing a fusion protein as described in any one of claims 1-8, the method comprising: a) Culture the host cells as described in claim 13 under conditions that effectively express the fusion protein; and b) Obtain the expressed fusion protein or polypeptide from the host cell.

15. Use of an effective amount of the fusion protein as described in any one of claims 1-8, or the pharmaceutical composition as described in claim 9, in the preparation of a medicament for treating an individual disease or condition, wherein said disease or condition is cancer.

16. The use as described in claim 15, wherein the cancer is non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), cervical cancer, anal cancer, biliary tract cancer, head and neck cancer, pancreatic cancer, genitourinary tract cancer, gastric cancer, breast cancer, or colorectal cancer.

17. The use as claimed in claim 15, wherein the cancer is a urinary system tumor.

18. The use as described in claim 15, wherein the cancer is urothelial carcinoma, prostate cancer, vaginal or penile cancer, bile duct cancer, gallbladder cancer, or oropharyngeal cancer.

19. The use as described in claim 15, wherein the cancer is bladder cancer.

20. The use as claimed in any one of claims 15-19, wherein the cancer is an advanced or metastatic cancer.

21. The use as claimed in any one of claims 15-19, wherein the drug is formulated to further comprise a second agent or to be administered in conjunction with a second therapy.

22. The use as described in any one of claims 15-19, wherein the fusion protein or the pharmaceutical composition is administered parenterally to the individual.

23. The use as claimed in any one of claims 15-19, wherein the individual is a person.

24. A kit for treating a disease or condition, comprising the pharmaceutical composition as claimed in claim 9 and instructions for use.

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