WO2024051796A1

WO2024051796A1 - Albumin binding proteins, fusion proteins and uses thereof

Info

Publication number: WO2024051796A1
Application number: PCT/CN2023/117628
Authority: WO
Inventors: Yunying CHEN; Xin Lin; Yi Qin; Jijie Gu; Na Ji
Original assignee: Wuxi Biologics (Shanghai) Co., Ltd.; WuXi Biologics Ireland Limited
Priority date: 2022-09-09
Filing date: 2023-09-08
Publication date: 2024-03-14

Abstract

Provided in the present disclosure are albumin binding molecules, BCMA binding molecules, fusion proteins, polypeptides and constructs that comprise the albumin binding proteins. Also provided are methods for preparing the albumin binding proteins and fusion proteins, and uses thereof.

Description

ALBUMIN BINDING PROTEINS, FUSION PROTEINS AND USES THEREOF

CROSS REFERENCE

This application claims the benefit of International application PCT/CN2022/118143, filed on September 9, 2022, which is incorporated by reference in its entirety.

SEQUENCE LISTING

The present application is filed with a Sequence Listing in electronic form. The entire contents of the Sequence Listing are hereby incorporated by reference.

FIELD

This application generally relates to albumin binding proteins, BCMA binding proteins, fusion proteins comprising the albumin binding proteins, a method for preparing the same, and the use of the albumin binding proteins and the fusion proteins.

BACKGROUND

Many therapeutic drugs in their natural state are subject to rapid turn over in vivo, meaning that they are both synthesized and degraded/excreted relatively quickly, and therefore need to be frequently injected in order to keep up with the natural processes of clearance. This has led to the development of several approaches that can be used to extend the dosing interval of drugs. One widely used approach to reduce rate of clearance is direct conjugation to a second molecule that has an inherently long serum half-life. One such method is to increase the hydrodynamic size of the protein by chemical attachment of polyethlylene glycol (PEG) , which can produce a drug with a terminal half-life in humans of up to 14 days. A second method is to express the therapeutic protein as a genetic fusion with a natural protein that has a long serum half-life; either 67 kDa serum albumin (SA) or the Fc portion of an antibody, which adds an additional 60-70 kDa in its natural dimeric form, depending on glycosylation.

The serum half-life of endogenous albumin is approximately 19 days in humans and 1.5-2.5 days in rodents, mostly due to effective recycling upon internalization mediated by neonatal Fc receptor (FcRn) . FcRn protects albumin from catabolism. Protein smaller than 60 kDa is generally filtered out by the kidneys rapidly and therefore less suitable for therapeutic purposes. Fusion of protein to an anti-albumin VHH building block (15KD) becomes more favorable for therapeutic purposes if pharmacokinetics (Albumin mediated) and tissue penetration (small size) are concerned.

On the other hand, B cell maturation antigen (BCMA, also known as TNFRSF17 and CD269) is a member of the TNF receptor family that binds the TNF family ligands BAFF and APRIL. BCMA is a type III transmembrane protein found almost exclusively on the surface of differentiated plasma cells, and their precursors, plasmablasts (Cho SF, Anderson KC, Tai YT. Targeting B Cell Maturation Antigen (BCMA) in Multiple Myeloma: Potential Uses of BCMA-Based Immunotherapy. Front Immunol. 2018 Aug 10; 9: 1821) . It therefore represents a potential target for B cell-related diseases.

In the present disclosure, novel albumin binding proteins have been disclosed and used for constructing fusion proteins/antibodies with extended half-life in vivo.

SUMMARY

These and other objectives are provided for by the present disclosure which, in a broad sense, is directed to compounds, methods, compositions and articles of manufacture that provide antigen-binding molecules with improved efficacy. The benefits provided by the present disclosure are broadly applicable in the field of therapeutic proteins and may be used in conjunction with antibodies that react with a variety of targets.

Disclosed herein are serum albumin binding molecules comprising or consisting of an immunoglobulin single variable domain, which show good thermal stability and high binding affinities to human, cyno and mouse albumin. Fusion proteins or multi-specific binding antibodies comprising the serum albumin binding proteins show significant improvement in half-life extension in vivo.

In one aspect, the present disclosure provides improved albumin binding molecules, and in particular albumin binding proteins or domains that have improved properties compared to serum albumin binders known in the art.

In some embodiments, provided herein is an albumin-binding molecule (e.g. an albumin-binding protein) comprising an immunoglobulin single variable domain, wherein the single variable domain comprises a CDR1, a CDR2 and a CDR3 same as those from a VHH as set forth in any of SEQ ID NOs: 7-12.

In some embodiments, provided herein is an albumin-binding protein comprising an immunoglobulin single variable domain that specifically binds to albumin (e.g. serum albumin) , wherein the single variable domain comprises:

a CDR1 comprising the amino acid sequence of GRAX₁SSYA, wherein X₁ is F or P;

a CDR2 comprising the amino acid sequence of VARIGDTT; and

a CDR3 comprising the amino acid sequence of AGGQTIAVYX₂TPNMYTX₃, wherein X₂ is D or S, X₃ is Y or A.

In some embodiments, the single variable domain comprises:

(A) a CDR1 comprising SEQ ID NO: 1, a CDR2 comprising SEQ ID NO: 2, a CDR3 comprising SEQ ID NO: 3;

(B) a CDR1 comprising SEQ ID NO: 1, a CDR2 comprising SEQ ID NO: 2, a CDR3 comprising SEQ ID NO: 5; or

(C) a CDR1 comprising SEQ ID NO: 4, a CDR2 comprising SEQ ID NO: 2, a CDR3 comprising SEQ ID NO: 6.

In some embodiments, the single variable domain comprises:

(A) the amino acid sequence as set forth in any one of SEQ ID NOs: 7-12;

(B) an amino acid sequence at least 85%, 90%, or 95%identical to the amino acid sequence as set forth in any one of SEQ ID NOs: 7-12; or

(C) an amino acid sequence with addition, deletion and/or substitution of one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) in the framework regions compared with the amino acid sequence as set forth in any one of SEQ ID NOs: 7-12.

In some embodiments, the single variable domain is a VHH, such as a humanized VHH domain, an affinity maturated VHH domain, and a human VHH domain. In some embodiments, the single variable domain specifically binds to at least one of human, mouse, cynomolgus, feline and canine serum albumins.

In one aspect, the present disclosure provides a fusion protein comprising the albumin-binding proteins as disclosed herein fused to heterogeneous protein. The heterogeneous protein may be an antigen-binding moiety, wherein the antigen-binding moiety is in the format of a whole antibody or an antigen-binding fragment thereof, such as a VHH, a scFv or a Fab.

In some embodiments, the fusion protein comprises the albumin-binding protein (s) as disclosed herein, and a first antigen-binding moiety and a second antigen-binding moiety that bind to antigens different from albumin. The first antigen-binding moiety and second antigen-binding moiety may bind to same or different antigens. In some embodiments, the fusion protein comprises:

(A) the first antigen-binding moiety operably linked to the albumin-binding protein, and the albumin-binding protein operably linked to the second antigen-binding moiety; or

(B) the first antigen-binding moiety operably linked to the second antigen-binding moiety, and the second antigen-binding moiety operably linked to the albumin-binding protein.

In some further embodiments, the first antigen-binding moiety is BCMA binding and the second antigen-binding moiety is CD3 binding, or vice versa.

In some embodiments, the BCMA binding moiety of the fusion protein is a VHH and comprises:

(A) a CDR1, CDR2 and CDR3 comprising SEQ ID No: 36, 37 and 38, respectively;

(B) a CDR1, CDR2 and CDR3 comprising SEQ ID No: 36, 37 and 40, respectively;

(C) a CDR1, CDR2 and CDR3 comprising SEQ ID No: 36, 37 and 41, respectively;

(D) a CDR1, CDR2 and CDR3 comprising SEQ ID No: 43, 37 and 40, respectively;

(E) a CDR1, CDR2 and CDR3 comprising SEQ ID No: 43, 37 and 41, respectively; or

(F) a CDR1, CDR2 and CDR3 comprising SEQ ID No: 43, 37 and 38, respectively.

In some embodiments, the BCMA binding moiety of the fusion protein comprises:

(A) the amino acid sequence as set forth in any one of SEQ ID NOs: 44-51;

(B) an amino acid sequence at least 85%, 90%, or 95%identical to the amino acid sequence as set forth in any one of SEQ ID NOs: 44-51; or

(C) an amino acid sequence with addition, deletion and/or substitution of one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids in the framework regions compared with the amino acid sequence as set forth in any one of SEQ ID NOs: 44-51.

In some embodiments, the CD3 binding moiety of the fusion protein is a scFv and comprises:

a HCDR1 comprising SEQ ID NO: 17, a HCDR2 comprising SEQ ID NO: 18, a HCDR3 comprising SEQ ID NO: 19; a LCDR1 comprising SEQ ID NO: 20, a LCDR2 comprising SEQ ID NO: 21, a LCDR3 comprising SEQ ID NO: 22.

In some embodiments, the CD3 binding moiety comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH comprises:

(A) the amino acid sequence as set forth in SEQ ID NO: 23;

(B) an amino acid sequence which is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%identical to SEQ ID NO: 23; or

(C) an amino acid sequence with addition, deletion and/or substitution of one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids in the framework regions compared with SEQ ID NO: 23;

and/or the VL comprises:

(A) the amino acid sequence as set forth in SEQ ID NO: 24 or 34;

(B) an amino acid sequence which is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%identical to SEQ ID NO: 24 or 34; or

(C) an amino acid sequence with addition, deletion and/or substitution of one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids in the framework regions compared with SEQ ID NO: 24 or 34.

In some embodiments, the fusion proteins as disclosed herein comprises an albumin-binding VHH, a BCMA-binding VHH and a CD3-binding scFv as disclosed above. Specifically, the fusion protein may comprise the amino acid sequence as set forth in any of SEQ ID NOs: 7-12 for the albumin-binding protein, the amino acid sequence as set forth in any one of SEQ ID NOs: 44-51 for the BCMA-binding moiety, and the amino acid sequence as set forth in SEQ ID NO: 25 or 35 for the CD3-binding moiety.

In some embodiments, the fusion protein is a single chain polypeptide comprising the amino acid sequence as set forth in any of SEQ ID NOs: 31-33.

In another aspect, the present disclosure provides BCMA binding molecules, and in particular BCMA binding proteins or domains that have improved properties compared to BCMA binders known in the art. In some embodiments, provided herein is an BCMA-binding molecule (e.g. an BCMA-binding protein) comprising an immunoglobulin single variable domain, wherein the single variable domain comprises a CDR1, a CDR2 and a CDR3 same as those from a VHH as set forth in any of SEQ ID NOs: 44-51.

In some embodiments, the single variable domain comprises:

a CDR1 comprising the amino acid sequence of GSIX₄SINA, wherein is D or W;

a CDR2 comprising the amino acid sequence of ISSGGFP (SEQ ID NO: 37) ; and

a CDR3 comprising the amino acid sequence of NAEX₅VWGGX₆IYNY, wherein X₅ is R or E, X₆ is K or R.

In some embodiments, the single variable domain comprises:

(A) a CDR1, CDR2 and CDR3 comprising SEQ ID No: 36, 37 and 38, respectively;

(B) a CDR1, CDR2 and CDR3 comprising SEQ ID No: 36, 37 and 40, respectively;

(C) a CDR1, CDR2 and CDR3 comprising SEQ ID No: 36, 37 and 41, respectively;

(D) a CDR1, CDR2 and CDR3 comprising SEQ ID No: 43, 37 and 40, respectively;

(E) a CDR1, CDR2 and CDR3 comprising SEQ ID No: 43, 37 and 41, respectively; or

(F) a CDR1, CDR2 and CDR3 comprising SEQ ID No: 43, 37 and 38, respectively.

In some embodiments, the single variable domain comprises:

(A) the amino acid sequence as set forth in any one of SEQ ID NOs: 44-51; or

(B) an amino acid sequence at least 85%, 90%, or 95%identical to the amino acid sequence as set forth in any one of SEQ ID NOs: 44-51.

In some embodiments, the single variable domain is a VHH, such as a humanized VHH domain, an affinity maturated VHH domain, and a human VHH domain.

In some embodiments, the single variable domain specifically binds to any of human, mouse and cynomolgus BCMAs.

In some embodiments, the BCMA-binding single variable domain is fused to a heterogenous protein, such as an immunoglobulin IgG constant region, including IgG1, IgG2, IgG3 or IgG4 Fc region. The heterogenous protein may also be an antigen-binding moiety in the format of an antibody or an antigen-binding fragment thereof, such as a Fab, a VHH, or a scFv.

In one aspect, provided herein is an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding the albumin-binding molecule, the BCMA-binding moelecule or the fusion protein as disclosed herein.

In one aspect, provided herein is a vector comprising the isolated nucleic acid molecule as disclosed herein.

In one aspect, provided herein is a host cell comprising the nucleic acid molecule or the vector as disclosed herein.

In one aspect, provided herein is a pharmaceutical composition comprising the albumin-binding molecule, the BCMA-binding moelecule or the fusion protein as disclosed herein, and a pharmaceutically acceptable carrier.

In one aspect, provided herein is a method for producing the albumin-binding protein or the BCMA-binding protein as disclosed herein, comprising expressing the albumin-binding protein or the BCMA-binding protein in a host cell comprising a nucleic acid sequence encoding the albumin-binding protein or the BCMA-binding protein, or culturing the host cell under suitable conditions to express the protein; and isolating the protein from the host cell.

In one aspect, provided herein is a method for producing the fusion protein as disclosed herein, comprising expressing the fusion protein in a host cell comprising a nucleic acid sequence encoding the fusion protein, or culturing the host cell under suitable conditions to express the fusion protein; and isolating the fusion protein from the host cell.

In one aspect, provided herein is a method for modulating an immune response (e.g. BCMA related) in a subject, comprising administering to the subject the BCMA-binding molecule, the fusion protein or the pharmaceutical composition as disclosed herein to the subject.

In one aspect, provided herein is a method for inhibiting growth of tumor cells (e.g. BCMA related) in a subject, comprising administering an effective amount of the BCMA-binding molecule, the fusion protein or the pharmaceutical composition as disclosed herein to the subject.

In one aspect, provided herein is a method for preventing or treating a cancer, an immune disorder, or an infection in a subject, comprising administering an effective amount of the BCMA-binding molecule, the fusion protein or the pharmaceutical composition as disclosed herein to the subject.

In some embodiments, the cancer or the immune disorder is associated with BCMA.

In some embodiments, the cancer is lymphoma selected from multiple myeloma, plasmacytoma, Hodgkins' lymphoma, follicular lymphomas, small non-cleaved cell lymphomas, endemic Burkitt's lymphoma, sporadic Burkitt's lymphoma, marginal zone lymphoma, extranodal mucosa-associated lymphoid tissue lymphoma, nodal monocytoid B cell lymphoma, splenic lymphoma, mantle cell lymphoma, large cell lymphoma, diffuse mixed cell lymphoma, immunoblastic lymphoma, primary mediastinal B cell lymphoma, pulmonary B cell angiocentric lymphoma, and small lymphocytic lymphoma.

In one aspect, provided herein is the BCMA-binding molecule or the fusion protein as disclosed herein for use in treating or preventing B cell-related disorders in a subject.

In one aspect, provided herein is a kit, comprising a container comprising the albumin-binding protein, the BCMA-binding protein, or the fusion protein of as disclosed herein.

The foregoing is a summary and thus contains, by necessity, simplifications, generalizations, and omissions of detail; consequently, those skilled in the art will appreciate that the summary is illustrative only and is not intended to be in any way limiting.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 illustrates after injecting the mixture of biotin conjugated human albumin and VHH samples over the chip, the binding signal of immobilized VHHs with biotin conjugated human Albumin as determined by SPR. Figure 1A. immobilize T6016-BMK4 on chip; Figure 1B. immobilize T6016-BMK6 on chip; Figure 1C. immobilize T6016-P1R2-6D10-Z4-R1-34G9 on chip.

Figure 2 illustrates DSF profile of T6016-P1R2-6D10 and its variants in PBS buffer.

Figures 3A-3B illustrate the binding of W3566 antibodies on NCI-H929 cells (A) and cyno BCMA transfected cells (B) , as measured by FACS.

Figure 4 illustrates pharmacokinetics for the four multispecific antibodies in mice as determined by ELISA.

Figure 5 illustrates tumor growth reduction in NCI-H929/hPBMC model treated with multispecific antibody containing T6016 variants at varying concentrations, or with a control vehicle. “WT1070” refers to WT1070-U31W2T3. D5-2. His antibody.

Figure 6 illustrates the body weight change of NCI-H929/PBMC tumor bearing mice. “WT1070” refers to WT1070-U31W2T3. D5-2. His antibody.

DETAILED DESCRIPTION

While the present disclosure may be embodied in many different forms, disclosed herein are specific illustrative embodiments thereof that exemplify the principles of the disclosure. It should be emphasized that the present disclosure is not limited to the specific embodiments illustrated. Moreover, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms “a, ” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a protein” includes a plurality of proteins; reference to “a cell” includes mixtures of cells, and the like. In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “comprising, ” as well as other forms, such as “comprises" and “comprised, ” is not limiting. In addition, ranges provided in the specification and appended claims include both end points and all points between the end points.

Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Abbas et al., Cellular and Molecular Immunology, 6^th ed., W.B. Saunders Company (2010) ; Sambrook J. &Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2000) ; Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John &Sons, Inc. (2002) ; Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1998) ; and Coligan et al., Short Protocols in Protein Science, Wiley, John &Sons, Inc. (2003) . The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

Definitions

In order to better understand the disclosure, the definitions and explanations of the relevant terms are provided as follows.

The term “antibody” or “Ab” herein is used in the broadest sense and encompasses various antibody structures including polyclonal antibodies, monospecific and multispecific antibodies, antibody fragments, antigen-binding proteins or polypeptide complexes, as long as it can specifically bind to an antigen (s) . A conventional antibody is a Y-shaped tetrameric protein comprising two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Light chains of an antibody may be classified into κ and λ light chain. Heavy chains may be classified into μ, δ, γ, α and ε, which define isotypes of an antibody as IgM, IgD, IgG, IgA and IgE, respectively. Each heavy chain consists of a heavy chain variable region (V_H) and a heavy chain constant region (C_H) . A heavy chain constant region consists of 3 domains (C_H1, C_H2 and C_H3) . Each light chain consists of a light chain variable region (V_L) and a light chain constant region (C_L) . V_H and V_L region can further be divided into hypervariable regions (called complementary determining regions (CDR) ) , which are interspaced by relatively conservative regions (called framework region (FR) ) . Each V_H and V_L consists of 3 CDRs and 4 FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from N-terminal to C-terminal. The variable region (V_H and V_L) of each heavy/light chain pair forms antigen binding sites, respectively. Antibodies may be of different antibody isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype) , IgA1, IgA2, IgD, IgE or IgM antibody. As shown in some embodiments herein, the term antibody includes albumin binding VHHs and fusion proteins comprising the albumin binding VHHs.

The term “single variable domain” or “VHH domain” or “single domain antibody” or “heavy chain only antibody variable domain” may be used interchangeably herein and refers to a single chain antigen binding domain that is capable of binding to an antigen or epitope, independently of a different variable domain. A VHH domain (e.g. variable domain of a heavy chain antibody) represents the smallest known antigen-binding unit generated by adaptive immune responses (Koch-Nolte F. et al., FASEB J. Nov; 21 (13) : 3490-8. Epub 2007 Jun 15 (2007) ) . A VHH domain may be a human domain, but also includes a single domain from other species such as rodent, nurse shark and Camelid VHH domains. Camelid VHH are immunoglobulin single variable domain polypeptides that are derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies naturally devoid of light chains. Such VHH domains may be humanized according to standard techniques available in the art and are considered as “single domain antibodies” . As used herein, VHH includes camelid VHH domains and humanized VHH domains.

In view of the above definition, the antigen-binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule) or of a Fab fragment, a F (ab') 2 fragment, an Fv fragment such as a disulphide linked Fv or a scFv fragment, or a diabody (all known in the art) derived from such conventional 4-chain antibody, would normally not be regarded as an immunoglobulin single variable domain, as, in these cases, binding to the respective epitope of an antigen would normally not occur by one (single) immunoglobulin domain but by a pair of (associating) immunoglobulin domains such as light and heavy chain variable domains, i.e. by a VH-VL pair of immunoglobulin domains, which jointly bind to an epitope of the respective antigen.

"VHH" , also known as VHH domains, VHH antibody fragments, and VHH antibodies, have originally been described as the antigen binding immunoglobulin (variable) domain of "heavy chain antibodies" (i.e. of "antibodies devoid of light chains" ; Hamers-Casterman C, et al., "Naturally occurring antibodies devoid of light chains" ; Nature 363, 446-448 (1993) ) . The term "VHH domain" has been chosen in order to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "VH domains" or "VH domains" ) and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "VL domains" or "VL domains" ) . VHH domains can specifically bind to an epitope without an additional antigen binding domain (as opposed to VH or VL domains in a conventional 4-chain antibody, in which case the epitope is recognized by a VL domain together with a VH domain) . VHH domains are small, robust and efficient antigen recognition units formed by a single immunoglobulin domain. A VHH domain may be a human domain, but also includes a single domain from other species such as rodent, nurse shark and Camelid VHH dAbs. Camelid VHH are immunoglobulin single variable domain polypeptides that are derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies naturally devoid of light chains. Such VHH domains may be humanized according to standard techniques available in the art.

A “humanized” antibody refers to an antibody (e.g. a VHH) comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain embodiments, all or substantially all of the CDRs of a humanized VHH correspond to those of a non-human VHH, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.

An "affinity-matured" albumin binding protein, in particular a VHH, has one or more alterations in one or more CDRs which result in an improved affinity for albumin, as compared to the respective parental albumin binding molecule. Affinity-matured albumin binding molecules of the invention may be prepared by methods known in the art, for example, as described by KS Johnson and RE Hawkins, "Affinity maturation of antibodies using phage display" , Oxford University Press 1996.

A protein (such as an immunoglobulin, an antibody, an immunoglobulin single variable domain) that can "bind to" or "specifically bind to" , that "has affinity to" and/or that "has specificity for" a certain epitope, antigen or protein is said to be "against' or " directed against" said epitope, antigen or protein or is a "binding" molecule with respect to such epitope, antigen or protein. In this context, an albumin binding protein may also be referred to as "albumin-neutralizing" or "anti-albumin" .

B cell maturation antigen (BCMA, also known as TNFRSF17 and CD269) , is a member of the TNF receptor family that binds the TNF family ligands BAFF and APRIL. BCMA is a type III transmembrane protein found almost exclusively on the surface of differentiated plasma cells, and their precursors, plasmablasts (Cho SF, Anderson KC, Tai YT. Targeting B Cell Maturation Antigen (BCMA) in Multiple Myeloma: Potential Uses of BCMA-Based Immunotherapy. Front Immunol. 2018 Aug 10; 9: 1821) . It therefore represents a potential target for B cell-related diseases.

CD3 (cluster of differentiation 3) is a protein complex and T cell co-receptor that is involved in activating both the cytotoxic T cell (CD8+ naive T cells) and T helper cells (CD4+naive T cells) . Anti-CD3 monoclonal antibodies are used as immunosuppressive drugs and to treat transplant rejection, and are also being investigated in the treatment of Crohn’s disease, ulcerative colitis, type 1 diabetes, and immune tolerance induction.

The term “K_D” as used herein with respect to surface plasmon resonance, is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction, which is obtained from the ratio of k_d to k_a (i.e., k_d/k_a) and is expressed as a molar concentration (M) . The term “k_a” as used herein, is intended to refer to the association rate of a particular antibody-antigen interaction, whereas the term “k_d” is intended to refer to the dissociation rate of a particular antibody-antigen interaction. K_D values for antibodies can be determined using methods well established in the art. A preferred method for determining the K_D of an antibody is by using surface plasmon resonance, preferably using a biosensor system such as a system.

The term “SPR” or “surface plasmon resonance, ” as used herein, refers to and includes an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N. J. ) . For further descriptions, see Example 5 and U., et al. (1993) Ann. Biol. Clin. 51: 19-26; U., et al. (1991) Biotechniques 11: 620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8: 125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198: 268-277.

The term “EC₅₀, ” as used herein, which is also termed as “half maximal effective concentration” refers to the concentration of a drug, antibody or toxicant which induces a response halfway between the baseline and maximum after a specified exposure time. In the context of the application, EC₅₀ is expressed in the unit of “nM” .

The term “epitope, ” as used herein, refers to a portion on antigen that an immunoglobulin or antibody specifically binds to. “Epitope” is also known as “antigenic determinant” . Epitope or antigenic determinant generally consists of chemically active surface groups of a molecule such as amino acids, carbohydrates or sugar side chains, and generally has a specific three-dimensional structure and a specific charge characteristic. For example, an epitope generally comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-consecutive amino acids in a unique steric conformation, which may be “linear” or “conformational” . See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996) . In a linear epitope, all the interaction sites between a protein and an interaction molecule (e.g., an antibody) are present linearly along the primary amino acid sequence of the protein. In a conformational epitope, the interaction sites span over amino acid residues that are separate from each other in a protein. Antibodies may be screened depending on competitiveness of binding to the same epitope by conventional techniques known by a person skilled in the art. For example, study on competition or cross-competition may be conducted to obtain antibodies that compete or cross-compete with each other for binding to antigens (e.g. RSV fusion protein) . High-throughput methods for obtaining antibodies binding to the same epitope, which are based on their cross-competition, are described in an international patent application WO 03/48731.

The term “isolated, ” as used herein, refers to a state obtained from natural state by artificial means. If a certain “isolated” substance or component is present in nature, it is possible because its natural environment changes, or the substance is isolated from natural environment, or both. For example, a certain un-isolated polynucleotide or polypeptide naturally exists in a certain living animal body, and the same polynucleotide or polypeptide with a high purity isolated from such a natural state is called isolated polynucleotide or polypeptide. The term “isolated” excludes neither the mixed artificial or synthesized substance nor other impure substances that do not affect the activity of the isolated substance.

The term “vector, ” as used herein, refers to a nucleic acid vehicle which can have a polynucleotide inserted therein. When the vector allows for the expression of the protein encoded by the polynucleotide inserted therein, the vector is called an expression vector. The vector can have the carried genetic material elements expressed in a host cell by transformation, transduction, or transfection into the host cell. Vectors are well known by a person skilled in the art, including, but not limited to plasmids, phages, cosmids, artificial chromosome such as yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC) ; phage such as λ phage or M13 phage and animal virus. The animal viruses that can be used as vectors, include, but are not limited to, retrovirus (including lentivirus) , adenovirus, adeno-associated virus, herpes virus (such as herpes simplex virus) , pox virus, baculovirus, papillomavirus, papova virus (such as SV40) . A vector may comprise multiple elements for controlling expression, including, but not limited to, a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element and a reporter gene. In addition, a vector may comprise origin of replication.

The term “host cell, ” as used herein, refers to a cellular system which can be engineered to generate proteins, protein fragments, or peptides of interest. Host cells include, without limitation, cultured cells, e.g., mammalian cultured cells derived from rodents (rats, mice, guinea pigs, or hamsters) such as CHO, BHK, NSO, SP2/0, YB2/0; or human tissues or hybridoma cells, yeast cells, and insect cells, and cells comprised within a transgenic animal or cultured tissue. The term encompasses not only the particular subject cell but also the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term “host cell. ”

The term “identity, ” as used herein, refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. “Percent identity” means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) are preferably addressed by a particular mathematical model or computer program (i.e., an “algorithm” ) . Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A. M., ed. ) , 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D. W., ed. ) , 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds. ) , 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds. ) , 1991, New York: M. Stockton Press; and Carillo et al, 1988, SIAMJ. Applied Math. 48: 1073.

The term “immunogenicity, ” as used herein, refers to ability of stimulating the formation of specific antibodies or sensitized lymphocytes in organisms. It not only refers to the property of an antigen to stimulate a specific immunocyte to activate, proliferate and differentiate so as to finally generate immunologic effector substance such as antibody and sensitized lymphocyte, but also refers to the specific immune response that antibody or sensitized T lymphocyte can be formed in immune system of an organism after stimulating the organism with an antigen. Immunogenicity is the most important property of an antigen. Whether an antigen can successfully induce the generation of an immune response in a host depends on three factors, properties of an antigen, reactivity of a host, and immunization means.

The term “subject” includes any human or nonhuman animal, preferably humans.

The term “cancer, ” as used herein, refers to any or a tumor or a malignant cell growth, proliferation or metastasis-mediated, solid tumors and non-solid tumors such as leukemia and initiate a medical condition.

The term “treatment, ” “treating” or “treated, ” as used herein in the context of treating a condition, pertains generally to treatment and therapy, whether of a human or an animal, in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, regression of the condition, amelioration of the condition, and cure of the condition. Treatment as a prophylactic measure (i.e., prophylaxis, prevention) is also included. For cancer, “treating” may refer to dampen or slow the tumor or malignant cell growth, proliferation, or metastasis, or some combination thereof. For tumors, “treatment” includes removal of all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of a tumor, or some combination thereof.

The term “an effective amount, ” as used herein, pertains to that amount of an active compound, or a material, composition or dosage from comprising an active compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen. For instance, the “an effective amount, ” when used in connection with treatment of B cell-related diseases or conditions, refers to the fusion proteins as disclosed herein in an amount or concentration effective to treat the said diseases or conditions.

The term “prevent, ” “prevention” or “preventing, ” as used herein, with reference to a certain disease condition in a mammal, refers to preventing or delaying the onset of the disease, or preventing the manifestation of clinical or subclinical symptoms thereof.

The term “pharmaceutically acceptable, ” as used herein, means that the vehicle, diluent, excipient and/or salts thereof, are chemically and/or physically is compatible with other ingredients in the formulation, and the physiologically compatible with the recipient.

As used herein, the term “a pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient pharmacologically and/or physiologically compatible with a subject and an active agent, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) , and includes, but is not limited to pH adjuster, surfactant, adjuvant and ionic strength enhancer. For example, the pH adjuster includes, but is not limited to, phosphate buffer; the surfactant includes, but is not limited to, cationic, anionic, or non-ionic surfactant, e.g., Tween-80; the ionic strength enhancer includes, but is not limited to, sodium chloride.

As used herein, the term “adjuvant” refers to a non-specific immunopotentiator, which can enhance immune response to an antigen or change the type of immune response in an organism when it is delivered together with the antigen to the organism or is delivered to the organism in advance. There are a variety of adjuvants, including, but not limited to, aluminium adjuvants (for example, aluminum hydroxide) , Freund’s adjuvants (for example, Freund’s complete adjuvant and Freund’s incomplete adjuvant) , coryne bacterium parvum, lipopolysaccharide, cytokines, and the like. Freund's adjuvant is the most commonly used adjuvant in animal experiments now. Aluminum hydroxide adjuvant is more commonly used in clinical trials.

Albumin binding molecules

Serum albumin is synthesized in the liver and its high concentration in plasma is the result of a cellular recycling pathway in which albumin is bound by the neonatal Fc receptor (FcRn) at endosomal pH and released at neutral pH at the cell surface. Serum albumin-binding proteins offer several important advantages over Fc fusions. While fusion to Fc is a robust strategy that not only increases serum half-life and provides effector function, the resulting proteins are IgG-like in size and geometry. In some instances, maintaining small size (e.g., increased tumor penetration) , lack of effector function (e.g., reduced cytokine release) , and nonnatural molecular geometries (e.g., improved engagement of bispecifics and biparatopics) are highly desirable.

In some aspects, the disclosure provides albumin binding molecules that specifically bind to serum albumin, such as a mammalian serum albumin, including human, cyno, mouse, rat, feline and canine serum albumins. An albumin binding molecule, in a general sense, may include any molecule that specifically binds to albumin and may include albumin antagonist and anti-albumin antibody or albumin-binding portion thereof. “albumin antagonist” refers to any chemical compound or biological molecule that blocks albumin activities. “Anti-albumin antibody or albumin-binding portion thereof” includes, but not limited to, a chimeric antibody, a humanized antibody, a human antibody, a single-domain antibody, a scFv, Fab or Fab’ that binds to albumin. The albumin binding molecule is not limited to a polypeptide or a protein and may comprise other components such as nucleotides, hybrids, glucans and a combination thereof. As exemplified herein, the albumin-binding molecule may be an anti-albumin antibody or albumin binding protein. Albumin binding proteins may also be referred to as “albumin binding moieties” when they are fused with therapeutic proteins (e.g. antibodies) to provide half-life-extending functions.

In some embodiments, albumin binding proteins as disclosed herein comprise at least one (e.g. one, two, three, four) immunoglobulin single variable domain that specifically binds to albumin. In some preferable embodiments, the immunoglobulin single variable domain is a VHH. The VHH alone is able to specifically bind to serum albumin like a whole antibody.

The serum albumin-binding proteins as disclosed herein have at least one of the following properties:

(i) moderate-to high-affinity binding to the albumins of multiple species, enabling continuity between preclinical animal studies and human immunotherapy, at neutral pH and endosomal pH (pH 5.5-6) ;

(ii) binding to albumin without disrupting the interaction of albumin with FcRn; and

(iii) retention of albumin binding upon fusion to diverse biologics in which the albumin-binding moiety may be located at the N-terminal, C-terminal or in the interval of the fusion molecule.

As demonstrated in the Examples, the albumin binding antibodies as disclosed herein could bind to human serum albumin at pH 5.5 and pH 7.4 buffers with middle to high affinity and cross-reacts with cyno monkey albumin, mouse albumin, cat albumin and dog albumin; show good developability and fusion with the VHHs prolongs the T_1/2 of a therapeutic protein from several minutes to 30-40 hours in mice and preserved the in vivo efficacy and potency of therapeutic protein. In some embodiments, the albumin binding proteins cross-reacts to cyno and mouse albumin with similar affinities, facilitating preclinical animal studies.

The albumin binding proteins of the disclosure binds to human serum albumin with a middle to high affinity at both neutral pH and endosomal pH. The binding of the albumin binding proteins to albumin can be assessed using one or more techniques well established in the art, for instance, ELISA, flow cytometry, and Surface Plasmon Resonance (SPR) . For example, the binding of the albumin binding proteins, including the binding kinetics (e.g., KD value) can be tested in BIAcore binding assays. As determined by SPR, an albumin binding protein (and specifically an albumin binding VHH) of the disclosure binds to a human albumin with a KD of 1×10^-7 M or less, a KD of 5×10^-8 M or less, a KD of 1×10^-8 M or less, a KD of 5×10^-9 M or less, a KD of 4×10^-9 M or less or a KD of 3×10^-9 M or less.

Albumin-binding VHHs

VHHs have the particular advantage that they are highly soluble and very stable and do not have a tendency to aggregate. VHHs derived from Camelidae antibodies are among the smallest intact antigen-binding domains known (approximately 15 kDa, or 10 times smaller than a conventional IgG) and hence are well suited towards delivery to dense tissues, accessing limited space between macromolecules and fusion with therapeutic proteins.

In some embodiments, albumin binding molecules as disclosed herein comprise at least one (e.g. one, two, three, four) VHH that specifically binds to albumin. Further, the albumin binding molecule may be a single-domain antibody consisting of one VHH. Like a whole antibody, a single-domain antibody is able to bind selectively to a specific antigen. In some other embodiments, the albumin binding molecule comprises a VHH fused to a heterologous protein, for example, an antigen binding moiety that binds to a targeted antigen related to a disease or disorder. The antigen binding moiety may be selected from a whole antibody, a scFv, a Fab, a scFv, among others. The fusion of albumin binding VHH to the antigen binding moiety facilitates the extension of the half life of the therapeutic molecule in vivo.

In some embodiments, the albumin binding VHH as disclosed herein comprises one, two, or all three CDRs of the amino acid sequence as set forth in any of SEQ ID NOs: 7-12. The VHHs as disclosed herein are not limited to a specific biological source from which they have been obtained or to a specific method of preparation. In some embodiments, the albumin binding VHH as disclosed herein is camelid. In some embodiments, the albumin binding VHH as disclosed herein is humanized. In some embodiments, the albumin binding VHH as disclosed herein comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework. In some embodiments, the albumin binding VHH as disclosed herein is affinity-maturated.

In some embodiments, the albumin binding VHH as disclosed herein comprises:

a CDR1 comprising SEQ ID No: 1 or 4, or an amino acid sequence that differs from SEQ ID No: 1 or 4 by an amino acid addition, deletion or substitution of not more than 2 amino acids (e.g. 2 amino acids, 1 amino acid) ;

a CDR2 comprising SEQ ID No: 2, or an amino acid sequence that differs from SEQ ID No: 2 by an amino acid addition, deletion or substitution of not more than 2 amino acids (e.g. 2 amino acids, 1 amino acid) ; and

a CDR3 comprising SEQ ID No: 3, 5 or 6, or an amino acid sequence that differs from SEQ ID No: 3, 5 or 6 by an amino acid addition, deletion or substitution of not more than 2 amino acids (e.g. 2 amino acids, 1 amino acid) .

The substitution is preferably a conservative substitution. In some embodiments, the albumin binding VHH as disclosed herein comprises:

a CDR1 comprising the amino acid sequence of GRAX₁SSYA, wherein X₁ is F or P; a CDR2 comprising the amino acid sequence of VARIGDTT; and a CDR3 comprising the amino acid sequence of AGGQTIAVYX₂TPNMYTX₃, wherein X₂ is D or S, X₃ is Y or A.

The assignment of amino acids to each CDR may be in accordance with one of the numbering schemes provided by the Kabat definition, the Chothia definition, the IMGT numbering, the AbM definition, the EU definition, and/or the contact definition, all of which are well known in the art. See Kabat et al. (1991) Sequences of Proteins of Immunological Interest (5^th Ed. ) , US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242; Chothia et al., 1987, PMID: 3681981; Chothia et al., 1989, PMID: 2687698; MacCallum et al., 1996, PMID: 8876650; or Dubel, Ed. (2007) Handbook of Therapeutic Antibodies, 3^rd Ed., Wily-VCH Verlag GmbH and Co.; and http: //www. imgt. org/. In some embodiments, the CDRs are according to IMGT numbering.

As will be appreciated by those in the art, the exact numbering and placement of the CDRs can be different among different numbering systems. It should be understood that the disclosure of a variable heavy sequence, a variable light sequence and/or a VHH sequence in the application includes the disclosure of the associated (inherent) CDRs. Accordingly, the disclosure of each variable region is a disclosure of the CDRs (e.g., CDRl, CDR2 and CDR3) . Two antibodies having the same VH, VL or VHH CDRs means that their CDRs are identical when determined by the same approach (e.g., the Kabat, AbM, Chothia, Contact, and IMGT numbering approaches as known in the art) .

Variable regions and CDRs in an antibody sequence can be identified according to general rules that have been developed in the art (such as, the Kabat, Chothia and IMGT numbering system) or by aligning the sequences against a database of known variable regions. Methods for identifying these regions are described in Kontermann and Dubel, eds., Antibody Engineering, Springer, New York, NY, 2001 and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000. Exemplary databases of antibody sequences are described in, and can be accessed through, the “Abysis" website at www. bioinf. org. uk/abs (maintained by A.C. Martin in the Department of Biochemistry &Molecular Biology University College London, London, England) and the VBASE2 website at www. vbase2. org, as described in Retter et al., Nucl. Acids Res., 33 (Database issue) : D671 -D674 (2005) . For example, sequences are analyzed using the Abysis database, which integrates sequence data from Kabat, IMGT and the Protein Data Bank (PDB) with structural data from the PDB. See Dr. Andrew C.R. Martin's book chapter Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg, ISBN-13: 978-3540413547, also available on the website bioinforg. uk/abs) . The Abysis database website further includes general rules that have been developed for identifying CDRs which can be used in accordance with the teachings herein. Two VHHs having the same CDRs means that their CDRs are identical when determined by the same approach (e.g., the Kabat approach, the Chothia approach, or the IMGT numbering as known in the art) .

In some embodiments, the CDR1 is according to IMGT numbering, comprising the amino acid sequence as set forth in SEQ ID NO: 1; the CDR2 is according to IMGT numbering, comprising the amino acid sequence as set forth in SEQ ID NO: 2; and the CDR3 is according to IMGT numbering, comprising the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, the single variable domain is camelid. In some embodiments, the single variable domain is humanized. In some embodiments, the single variable domain comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework. In some embodiments, the single variable domain has been back mutated in the framework region and/or futher affinity-maturated.

In some embodiments, the CDR1 is according to IMGT numbering, comprising the amino acid sequence as set forth in SEQ ID NO: 1; the CDR2 is according to IMGT numbering, comprising the amino acid sequence as set forth in SEQ ID NO: 2; and the CDR3 is according to IMGT numbering, comprising the amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, the single variable domain is camelid. In some embodiments, the single variable domain is humanized. In some embodiments, the single variable domain comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework. In some embodiments, the single variable domain has been back mutated in the framework region and/or futher affinity-maturated.

In some embodiments, the CDR1 is according to IMGT numbering, comprising the amino acid sequence as set forth in SEQ ID NO: 4; the CDR2 is according to IMGT numbering, comprising the amino acid sequence as set forth in SEQ ID NO: 2; and the CDR3 is according to IMGT numbering, comprising the amino acid sequence as set forth in SEQ ID NO: 6. In some embodiments, the single variable domain is camelid. In some embodiments, the single variable domain is humanized. In some embodiments, the single variable domain comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework. In some embodiments, the single variable domain has been back mutated in the framework region and/or futher affinity-maturated.

In some embodiments, the albumin binding VHH comprises or consists of:

(A) the amino acid sequence as set forth in any one of SEQ ID NOs: 7-12;

(B) an amino acid sequence which is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%identical to any one of SEQ ID NOs: 7-12; or

(C) an amino acid sequence with addition, deletion and/or substitution of one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids in the framework regions compared with any one of SEQ ID NOs: 7-12.

The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988) ) which has been incorporated into the ALIGN program (version 2.0) , using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percentage of identity between two amino acid sequences can be determined by the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 444-453 (1970) ) which has been incorporated into the GAP program in the GCG software package (available at http: //www. gcg. com) , using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

Additionally or alternatively, the protein sequences of the present disclosure can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al.(1990) J. MoI. Biol. 215: 403-10. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the antibody molecules of the disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25 (17) : 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www. ncbi. nlm. nih. gov.

In some further embodiments, the anti-albumin VHHs may contain conservative substitution or modification of amino acids in the framework regions. It is understood in the art that certain conservative sequence modification can be made which do not remove antigen binding. See, e.g., Brummell et al. (1993) Biochem 32: 1180-8; de Wildt et al. (1997) Prot. Eng. 10: 835-41; Komissarov et al. (1997) J. Biol. Chem. 272: 26864-26870; Hall et al. (1992) J. Immunol. 149: 1605-12; Kelley and O’ Connell (1993) Biochem. 32: 6862-35; Adib-Conquy et al. (1998) Int. Immunol. 10: 341-6 and Beers et al. (2000) Clin. Can. Res. 6: 2835-43.

The term “conservative substitution” , as used herein, refers to amino acid substitutions which would not disadvantageously affect or change the essential properties of a protein/polypeptide comprising the amino acid sequence. For example, a conservative substitution may be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions wherein an amino acid residue is substituted with another amino acid residue having a similar side chain, for example, a residue physically or functionally similar (such as, having similar size, shape, charge, chemical property including the capability of forming covalent bond or hydrogen bond, etc. ) to the corresponding amino acid residue. The families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having alkaline side chains (for example, lysine, arginine and histidine) , amino acids having acidic side chains (for example, aspartic acid and glutamic acid) , amino acids having uncharged polar side chains (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan) , amino acids having nonpolar side chains (for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine) , amino acids having β-branched side chains (such as threonine, valine, isoleucine) and amino acids having aromatic side chains (for example, tyrosine, phenylalanine, tryptophan, histidine) . Therefore, a corresponding amino acid residue is preferably substituted with another amino acid residue from the same side-chain family. Methods for identifying amino acid conservative substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32: 1180-1187 (1993) ; Kobayashi et al., Protein Eng. 12 (10) : 879-884 (1999) ; and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997) , which are incorporated herein by reference) .

In some embodiments, the albumin-binding protein comprises one, two or all three CDRs of any one of SEQ ID NOs: 7-12. In some embodiments, the albumin-binding VHH comprises FRW1-CDR1-FRW2-CDR2-FRW3-CDR3-FRW4, and wherein CDR1 is consisted of an amino acid sequence as set forth in SEQ ID No: 1 or 4, CDR2 is consisted of an amino acid sequence as set forth in SEQ ID No: 2, and CDR3 is consisted of an amino acid sequence as set forth in SEQ ID No: 3, 5 or 6. In some embodiments, the FRW1 and FRW4 at the N and C terminal of the VHH comprised in the albumin-binding VHH may be truncated or extended.

In some specific embodiments, the albumin binding VHH consists of the amino acid sequence as set forth in any one of SEQ ID NOs: 7-12. Such VHHs are exemplified below and designated as T6016-P1R2-6D10, T6016-P1R2-6D10-z5, T6016-P1R2-6D10-z4-R1-34G9, T6016-P1R2-6D10-z5-R1-34G9, T6016-P1R2-6D10-z4-R1-34G9-m2 and T6016-P1R2-6D10-z5-R1-34G9-m2, respectively.

BCMA-binding molecules

In some aspects, the disclosure provides BCMA binding molecules. A BCMA binding molecule, in a general sense, may include any molecule that specifically binds to BCMA. In some circumstances, “BCMA binding molecule” may include BCMA antagonist and anti-BCMA antibody or BCMA-binding portion thereof. “BCMA antagonist” refers to any chemical compound or biological molecule that blocks BCMA activities. “Anti-BCMA antibody or BCMA-binding portion thereof” includes, but not limited to, a chimeric antibody, a humanized antibody, a human antibody or a single-domain antibody, a scFv, Fab or Fab’ that binds to BCMA. The BCMA binding molecule is not limited to a polypeptide or a protein and may comprise other components such as nucleotides, hybrids, glucans and a combination thereof. As exemplified herein, the BCMA-binding molecule may be an anti-BCMA antibody or BCMA binding protein. A BCMA binding protein may also be referred to as a “BCMA binding moiety” when it is fused with a heterologous protein, such as an albumin-binding VHH, forming a multi-specific fusion protein.

In some embodiments, BCMA binding molecules as disclosed herein comprise at least one (e.g. one, two, three, four) VHH that specifically binds to BCMA. Further, the BCMA binding molecule may be a single-domain antibody and consisted of one VHH. In some other embodiments, the BCMA binding molecule comprises a VHH fused to an immunoglobulin Fc region, for example, Fc-domain of IgG (e.g., IgG4 or IgG1) . In some specific embodiments, the Fc-domain is an Fc-domain of human IgG1. By fusing a VHH to a Fc domain, it may be more efficient to recruit effector functions. Also, the fusion of VHH to Fc domain may help the BCMA binding molecule to form a dimer and may also help the extension of the half life of BCMA binding molecule in vivo. 1. In some embodiments, the single variable domain is camelid. In some embodiments, the single variable domain is humanized. In some embodiments, the single variable domain comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework. In some embodiments, the single variable domain has been back mutated in the framework region and/or futher affinity-maturated.

In some embodiments, BCMA binding proteins as disclosed herein comprise at least one (e.g. one, two, three, four) immunoglobulin single variable domain that specifically binds to BCMA. In some preferable embodiments, the immunoglobulin single variable domain is a VHH. The VHH alone is able to specifically bind to BCMA like a whole antibody.

The BCMA-binding proteins as disclosed herein have one or more of the following properties:

(a) bind to human BCMA and cyno BCMA with EC50 at nM grade, as measured by FACS;

(b) bind to human and cyno BCMA with a KD at nM grade, as measured by SPR; and

(c) have a good thermal stability.

The binding of the BCMA binding proteins to BCMA can be assessed using one or more techniques well established in the art, for instance, ELISA, flow cytometry, and Surface Plasmon Resonance (SPR) . For example, the binding of the BCMA binding proteins, including the binding kinetics (e.g., KD value) can be tested in BIAcore binding assays. As determined by SPR, a BCMA binding protein (and specifically a BCMA binding VHH) of the disclosure binds to a human BCMA with a KD of 1×10^-9 M or less, a KD of 5×10^-10 M or less, a KD of 1×10^-10 M or less, or a KD of 5×10^- ¹¹ M or less.

In some embodiments, the BCMA-binding protein comprises a VHH, wherein the VHH comprises:

a CDR1 comprising SEQ ID No: 36, or an amino acid sequence that differs from SEQ ID No: 36 by an amino acid addition, deletion or substitution of not more than 2 amino acids (e.g. 2 amino acids, 1 amino acid) ;

a CDR2 comprising SEQ ID No: 37, or an amino acid sequence that differs from SEQ ID No: 37 by an amino acid addition, deletion or substitution of not more than 2 amino acids (e.g. 2 amino acids, 1 amino acid) ; and

a CDR3 comprising SEQ ID No: 38, or an amino acid sequence that differs from SEQ ID No: 38 by an amino acid addition, deletion or substitution of not more than 2 amino acids (e.g. 2 amino acids, 1 amino acid) .

Preferably, the substitution is a conservative substitution. In some embodiments, the BCMA-binding VHH comprises a CDR1, CDR2 and CDR3, wherein:

the CDR1 comprises the amino acid sequence of GSIX₄SINA, wherein is D or W;

the CDR2 comprises the amino acid sequence of ISSGGFP; and

the CDR3 comprises the amino acid sequence of NAEX₅VWGGX₆IYNY, wherein X₅ is R or E, X₆ is K or R.

IIn some embodiments, the BCMA-binding VHH comprises a CDR1, CDR2 and CDR3 comprising the amino acid sequence of SEQ ID Nos: 36, 37 and 38. In some embodiments, the BCMA-binding VHH comprises the amino acid sequence of SEQ ID Nos: 36, 37 and 40, respectively. In some embodiments, the BCMA-binding VHH comprises a CDR1, CDR2 and CDR3 comprising the amino acid sequence of SEQ ID Nos: 36, 37 and 41, respectively. In some embodiments, the BCMA-binding VHH comprises a CDR1, CDR2 and CDR3 comprising the amino acid sequence of SEQ ID Nos: 43, 37 and 40, respectively. In some embodiments, the BCMA-binding VHH comprises a CDR1, CDR2 and CDR3 comprising the amino acid sequence of SEQ ID Nos: 43, 37 and 41, respectively. In some embodiments, the BCMA-binding VHH comprises a CDR1, CDR2 and CDR3 comprising the amino acid sequence of SEQ ID Nos: 43, 37 and 38, respectively. In some embodiments, the CDR1, CDR2 and CDR3 of the BCMA binding VHH are according to IMGT numbering.

In some specific embodiments, the BCMA-binding VHH comprises:

(A) the amino acid sequence as set forth in any of SEQ ID NOs: 44-51;

(B) an amino acid sequence which is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%identical to any of SEQ ID NOs: 44-51; or

(C) an amino acid sequence with addition, deletion and/or substitution of one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids in the framework regions compared with any of SEQ ID NOs: 44-51.

In some embodiments, the BCMA-binding protein comprises one, two or all three CDRs of any one of SEQ ID NOs: 44-51. In some embodiments, the albumin-binding VHH comprises FRW1-CDR1-FRW2-CDR2-FRW3-CDR3-FRW4, and wherein CDR1 is consisted of an amino acid sequence as set forth in SEQ ID No: 36 or 43, CDR2 is consisted of an amino acid sequence as set forth in SEQ ID No: 37, and CDR3 is consisted of an amino acid sequence as set forth in SEQ ID No: 38, 40 or 41. In some embodiments, the FRW1 and FRW4 at the N and C terminal of the VHH comprised in the albumin-binding VHH may be truncated or extended.

In some specific embodiments, the BCMA binding VHH consists of the amino acid sequence as set forth in any one of SEQ ID NOs: 44-51. Such VHHs are exemplified in the Examples and designated as W3566-FP20R3-1D5-z2, W3566-FP20R3-1D5-z2-m17, W3566-FP20R3-1D5-z2-m36, W3566-FP20R3-1D5-z2-m42, W3566-FP20R3-1D5-z2-m49, W3566-FP20R3-1D5-z2-m50, W3566-FP20R3-1D5-z2-m51, W3566-FP20R3-1D5-z2-m52, respectively.

Methods for obtaining albumin binding VHHs

The VHHs as disclosed herein may be made by the skilled artisan according to methods known in the art or any future method. For example, VHHs may be obtained by immunizing a camel and obtaining hybridoma's therefrom, or by cloning a library of VHHs using molecular biology techniques known in the art and subsequently selecting by using phage display.

Specifically, a VHH can be obtained by immunization of llamas or alpacas with the desired antigen and subsequent isolation of the mRNA coding for heavy-chain antibodies. By reverse transcription and polymerase chain reaction, a gene library of single-domain antibodies containing several million clones is produced. Screening techniques like phage display and ribosome display help to identify the clones binding the antigen. In phage display, a library of (e.g., human) antibodies is synthesized on phages, the library is screened with the antigen of interest or an antibody-binding portion thereof, and the phage that binds the antigen is isolated, from which one may obtain the immunoreactive fragments. Methods for preparing and screening such libraries are well known in the art and kits for generating phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; and the Stratagene SurfZAP^TM phage display kit, catalog no. 240612) . There also are other methods that can be used in generating and screening antibody display libraries (see, e.g., Barbas et al., Proc. Natl. Acad. Sci. USA 88: 7978-7982 (1991) ) .

When the most potent clones have been identified, their DNA sequence is optimized, for example, by affinity maturation or humanization. Humanization may prevent immunological reactions of the human organism against the antibody.

Accordingly, the VHHs can be obtained (1) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) by “humanization” of a naturally occurring VHH domain or by expression of a nucleic acid encoding a such humanized VHH domain; (4) by “camelization” of a naturally occurring VH domain from any animal species, in particular a species of mammal, such as from a human being, or by expression of a nucleic acid encoding such a camelized VH domain; (5) by “camelisation” of a “domain antibody” or “dAb” , or by expression of a nucleic acid encoding such a camelized VH domain; (6) using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences; (7) by preparing a nucleic acid encoding a VHH using techniques for nucleic acid synthesis, followed by expression of the nucleic acid thus obtained; (8) subjecting heavy chain antibodies or VHHs to affinity maturation, to mutagenesis (e.g. random mutagenesis or site-directed mutagenesis) and/or any other technique (s) in order to increase the affinity and/or specificity of the VHH; and/or (8) by any combination of the foregoing. Suitable methods and techniques for performing the above-described steps are known in the art and will be clear to the skilled person. By way of example, methods of obtaining VHH domains binding to a specific antigen or epitope have been described in WO2006/040153 and WO2006/122786.

Single-domain antibodies are usually generated by PCR cloning of variable domain repertoire from blood, lymph node, or spleen cDNA obtained from immunized animals into a phage display vector. Antigen-specific single-domain antibodies are commonly selected by panning phase libraries on immobilized antigen, e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on Streptavidin beads, or membrane proteins expressed on the surface of cells. The affinity of dAbs can often been improved by mimicking this strategy in vitro, for instance, by site directed mutagenesis of the CDR regions and further rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH, and low antigen concentrations) (Wesolowski et al., Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. Med Microbiol Immunol (2009) 198: 157-174) .

Binding VHHs with improved properties in view of therapeutic application, e.g. enhanced affinity or decreased immunogenicity, may be obtained from individual binding molecules by techniques known in the art, such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences) , CDR grafting, humanizing, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing, also termed "sequence optimization" , as described herein. Reference is, for example, made to standard handbooks, as well as to the further description and Examples.

In some embodiments, a VHH may be truncated at the N-terminus or C-terminus such that it comprise only a partial FR1 and/or FR4, or lacks one or both of those framework regions, so long as the VHH substantially maintain antigen binding and specificity.

Fusion proteins comprising the albumin-binding proteins

Many small serum albumin-binding proteins (primarily antibody fragments) have been used to extend the serum half-lives of biologics, including streptococcal albumin-binding domains (ABDs) , DARPins, immunoglobulin variable heavy chain (VH) and variable light chain (VL) domains, camelid heavy chain-only variable (VHH) domains, shark new antigen receptor variable (VNAR) domains, a fragment variable (Fv) , and a fragment antigen binding (Fab) .

In some aspects, provided herein are fusion proteins comprising the albumin-binding proteins (preferably VHHs) as disclosed above fused to at least one heterogeneous protein, which is generally an antibody or antigen-binding portion thereof that binds to one or more other antigen (i.e. different from albumin) . The antibody or antigen-binding portion thereof may be referred to as “antigen-binding moiety” when they are fused with the albumin-binding protein to form the fusion protein.

In some embodiments, the fusion protein comprises an albumin-binding protein (preferably a VHH) as disclosed above fused to an antigen-binding moiety. The albumin-binding protein may be at the C-terminal or N-terminal of the antigen-binding moiety. In some embodiments, the fusion protein comprises one or more (e.g. 2, 3) copies of the antigen-binding moiety, e.g. the fuson protein comprises two copies of the antigen-binding moiety and the albumin-binding protein is located between the two copies of the antigen-binding moiety. In some embodiments, the fusion protein comprises one or more (e.g. 2, 3) copies of the albumin-binding protein.

In some other embodiments, the fusion protein comprises an albumin-binding protein (preferably a VHH) as disclosed above fused to two antigen-binding moieties, such as two different antigen-binding moieties (a first antigen-binding moiety and a second antigen-binding moiety) . The first and the second antigen-binding moieties may bind to two different antigens selecting from a group comprising tumor associated antigens, T cell associated antigens and B cell associated antigens, immune-checkpoint molecules, which are related to tumors, immune disorders and infections and other diseases. Alternatively, the first and the second antigen-binding moieties may bind to two different epitopes of a same antigen.

In some specific embodiments, the fusion protein may comprise, from N terminal to C terminal, (albumin-binding VHH) - (1^st antigen binding moiety) - (2^nd antigen binding moiety) .

In some specific embodiments, the fusion protein may comprise, from N terminal to C terminal, (albumin-binding VHH) - (2^nd antigen binding moiety) - (1^st antigen binding moiety) .

In some specific embodiments, the fusion protein may comprise, from N terminal to C terminal, (1^st antigen binding moiety) - (2^nd antigen binding moiety) - (albumin-binding VHH) .

In some specific embodiments, the fusion protein may comprise, from N terminal to C terminal, (2^nd antigen binding moiety) - (1^st antigen binding moiety) - (albumin-binding VHH) .

In some specific embodiments, the fusion protein may comprise, from N terminal to C terminal, (1^st antigen binding moiety) - (albumin-binding VHH) - (2^nd antigen binding moiety) .

In some specific embodiments, the fusion protein may comprise, from N terminal to C terminal, (2^nd antigen binding moiety) - (albumin-binding VHH) - (1^st antigen binding moiety) .

The “-” in the formats described above represents an operable linkage, i.e. direct linking or linking via a linker, as long as the functions of the two linked parts are not affected. In some embodiments, the linker may be a multimer of GGGGS (or G4S or Gly4Ser) . Those include (G4S) n linker with n=1-5 or derivatives thereof.

In some embodiments, the fusion protein comprises one or more (e.g. 2, 3) copies of the first or the second antigen-binding moiety, e.g. the fuson protein comprises two copies of the first antigen-binding moiety. Further, the two copies of the first antigen-binding moiety are located at one side of the albumin-binding protein while the second antigen-binding moiety is located at the other side of the albumin-binding protein, wherein the two copies of the first antigen-binding moiety are operably linked (directly or via a linker) to each other. In some alternative embodiments, the two copies of the first antigen-binding moiety are located at different sides of the albumin-binding protein. In some specific embodiments, the fusion protein may comprise, from N terminal to C terminal, (1^st antigen binding moiety) - (1^st antigen binding moiety) - (albumin-binding VHH) - (2^nd antigen binding moiety) , (1^st antigen binding moiety) - (albumin-binding VHH) - (1^st antigen binding moiety) - (2^nd antigen binding moiety) or (1^st antigen binding moiety) - (albumin-binding VHH) - (2^nd antigen binding moiety) - (1^st antigen binding moiety) , etc.

Preferably, the repetitive copy/coies of the antigen-binding moiety is in VHH format. In some embodiments, the fusion protein comprises one or more (e.g. 2, 3) copies of the albumin-binding protein.

The antigen-binding moiety may adopt a variety of formats, such as but not limited to, a Fab, a Fab', a F (ab') ₂, an Fv fragment, a single-chain antibody molecule (scFv) and a VHH. In some embodiments, the fusion protein as disclosed herein is a single polypeptide comprising operably linked components. A single polypeptide allows for facile production and manufacturing of the fusion proteins as they can be encoded by single cDNA molecule to be easily incorporated into a vector. Further, for monomeric single polypeptide, there are no chain pairing issues or a requirement for dimerization. As shown in the Examples, the fusion proteins herein have a low tendency to aggregate.

In some embodiments, the antigen binding moiety are derived from antibodies which are already known on the market, such as any of the following antibodies: Isatuximab, daratumumab, elotuzumab, trastuzumab, pertuzumab, sacituzumab, abciximab, adalimumab, alefacept, alemtuzumab, basiliximab, belimumab, bezlotoxumab, canakinumab, certolizumab pegol, cetuximab, daclizumab, denosumab, efalizumab, golimumab, inflectra, ipilimumab, ixekizumab, natalizumab, nivolumab, olaratumab, omalizumab, palivizumab, panitumumab, pembrolizumab, rituximab, tocilizumab, trastuzumab, secukinumab, and ustekinumab. In some other embodiments, the antigen binding moiety are derived from antibodies developed de novo. By “derived from” , it is generally meant herein that the antigen-binding moiety comprises the CDR sequences of the parent antibody, and preferably, comprises the variable regions of the parent antibody. In some embodiments, the antigen-binding moiety comprises the variants of the CDR sequences of the parent antibody which retain the antigen-binding specificity. Preferably, the variable regions (or at least the CDR regions) of the first and second antigen binding moieties are same as those of the antibodies which are already known or developed de novo.

Theoretically, the parent antibodies that can derive the antigen-binding moieties can include all monoclonal antibodies that have specificity for a certain antigen, such as antibodies against tumor related antigens or pathways, e.g. PD-1/PD-L1, TIM-3, LAG-3, VEGF, HER2, CTLA-4, BMPR1B, E16, STEAP1, MUC16, MPF, Napi2b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, HER2, NCA, MDP, IL20Ra, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD22, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, FcRH5, TENB2, PMEL17, TMEFF1, GDNF-Ra1, Ly6E, TMEM46, Ly6G6D, LGR5, RET, Ly6K, GPR19, GPR54, ASPHD1, Tyrosinase, TMEM118, GPR172A, CD33 and CLL-1; antibodies against leukocyte receptors, e.g., MHC, CD2, CD3, CD4, CD7, CD8, CD25, CD28, CD40, CD45, CD58, CD80, CD86 or their ligands; CD3 engager antibodies, NK engager antibodies; ADCC enabling anti-Tumor associated antigens; monoclonal antibodies to TNF, among others. The antibodies may include but not limited to, trastuzumab, pertuzumab, sacituzumab, abciximab, adalimumab, alefacept, alemtuzumab, basiliximab, belimumab, bezlotoxumab, canakinumab, certolizumab pegol, cetuximab, daclizumab, denosumab, efalizumab, golimumab, inflectra, ipilimumab, ixekizumab, natalizumab, nivolumab, olaratumab, omalizumab, palivizumab, panitumumab, pembrolizumab, rituximab, tocilizumab, trastuzumab, secukinumab, and ustekinumab.

In some embodiments, the fusion protein comprises an albumin binding VHH as disclosed herein fused to a scFv or VHH that specifically binds to a disease related antigen. In some embodiments, the fusion protein comprises an albumin binding VHH as disclosed herein fused to a scFv or VHH that specifically binds to a tumor associated antigen. In some embodiments, the fusion protein comprises an albumin binding VHH as disclosed herein fused to a scFv or VHH that specifically binds to a T cell associated antigen. In some embodiments, the fusion protein comprises an albumin binding VHH as disclosed herein fused to a scFv or VHH that specifically binds to a B cell associated antigen. In some embodiments, the fusion protein comprises three components: an albumin binding VHH as disclosed herein, a VHH or scFv that specifically binds to a first antigen and a VHH or scFv that specifically binds to a second antigen, wherein targeting both the first antigen and the second antigen have an additive or synergistic effect in preventing or treating a certain disease, such as a cancer, an immune disease (including an autoimmune disease) and an infection.

The albumin-binding proteins as disclosed herein may be fused to or conjugated to antigen-binding moieties that bind to a numerous variety of antigens, as desired. The term “disease associated antigen” refers to a target antigen that plays a role in modulating, controlling, alleviating, or aggravating a certain disease. For example, the term “tumor associated antigen (TAA) ” may include a target antigen expressed by tumor cells, however may be expressed by the cognate cell (or healthy cells) prior to transforming into a tumor; a target antigen presented only by tumor cells and not by normal, i.e. non-tumor cells; a target antigen found in both tumor cells and non-tumor cells, but is overexpressed on tumor cells when compared to non-tumor cells or are accessible for antibody binding in tumor cells due to the less compact structure of the tumor tissue compared to non-tumor tissue; a target antigen located on the vasculature of a tumor; and a target antigen that participates in modulating the proliferation of tumor via its interaction with other molecules in the pathways.

Illustrative examples of a disease associated antigen are BCMA, CD3, LAG-3, CD10, CD19, CD20, CD22, CD21, CD22, CD25, CD30, CD33, CD34, CD37, CD44v6, CD45, CD133, Fms-like tyrosine kinase 3 (FLT-3, CD135) , chondroitin sulfate proteoglycan 4 (CSPG4, melanoma-associated chondroitin sulfate proteoglycan) , Epidermal growth factor receptor (EGFR) , Her2neu, Her3, IGFR, IL3R, fibroblast activating protein (FAP) , CDCP1, Derlin1, Tenascin, frizzled 1-10, the vascular antigens VEGFR2 (KDR/FLK1) , VEGFR3 (FLT4, CD309) , PDGFR-alpha (CD140a) , PDGFR-beta (CD140b) Endoglin, CLEC14, Tem1-8, and Tie2. Further examples may include A33, CAMPATH-1 (CDw52) , Carcinoembryonic antigen (CEA) , Carboanhydrase IX (MN/CA IX) , de2-7 EGFR, EGFRvIII, EpCAM, Ep-CAM, Folate-binding protein, G250, Fms-like tyrosine kinase 3 (FLT-3, CD135) , c-Kit (CD117) , CSF1R (CD115) , HLA-DR, IGFR, IL-2 receptor, IL3R, MCSP (Melanoma-associated cell surface chondroitin sulphate proteoglycane) , Muc-1, Prostate-specific membrane antigen (PSMA) , Prostate stem cell antigen (PSCA) , Prostate specific antigen (PSA) , and TAG-72.

In some embodiments, the first antigen-binding moiety is a BCMA-binding moiety, and the second antigen-binding moiety is a CD3-binding moiety, or vice versa.

A fusion protein that specifically binds to BCMA, CD3 and albumin

In one aspect, provided herein is a fusion protein comprising three components: an albumin-binding protein (preferably a VHH) as disclosed herein, a BCMA-binding moiety as disclosed herein and a CD3-binding moiety. In some embodiments, the fusion protein as disclosed herein comprises a BCMA-binding moiety from a BCMA-binding VHH clone selected from W3566-FP20R3-1D5-z2-m17, W3566-FP20R3-1D5-z2, W3566-FP20R3-1D5-z2-m36, W3566-FP20R3-1D5-z2-m42, W3566-FP20R3-1D5-z2-m49, W3566-FP20R3-1D5-z2-m50, W3566-FP20R3-1D5-z2-m51, W3566-FP20R3-1D5-z2-m52 (also named as W3566-FP20R3-1D5-z2-4F10) . In some embodiments, the fusion protein as disclosed herein comprises a BCMA-binding moiety from a BCMA-binding VHH clone W3566-FP20R3-1D5-z2-4F10.

In some embodiments, the fusion protein as disclosed herein comprises a CD3-binding moiety from an anti-CD3 monospecific or multispecific antibody. In some embodiments, the CD3-binding moiety is a scFv comprising one, two, or all three heavy chain CDRs of the amino acid sequence as set forth in SEQ ID NO: 23. In some embodiments, the CD3-binding moiety is a scFv comprising one, two, or all three light chain CDRs of the amino acid sequence as set forth in SEQ ID NO: 24 or 34. In some embodiments, the CD3-binding moiety is a scFv comprising all six CDRs of the amino acid sequence as set forth in SEQ ID NO: 25 or 35.

In some embodiments, the CD3-binding moiety is a scFv and comprises:

a HCDR1 comprising SEQ ID No: 17, or an amino acid sequence that differs from SEQ ID No: 13 by an amino acid addition, deletion or substitution of not more than 2 amino acids (e.g. 2 amino acids, 1 amino acid) ;

a HCDR2 comprising SEQ ID No: 18, or an amino acid sequence that differs from SEQ ID No: 14 by an amino acid addition, deletion or substitution of not more than 2 amino acids (e.g. 2 amino acids, 1 amino acid) ; and

a HCDR3 comprising SEQ ID No: 19, or an amino acid sequence that differs from SEQ ID No: 15 by an amino acid addition, deletion or substitution of not more than 2 amino acids (e.g. 2 amino acids, 1 amino acid) .

a LCDR1 comprising SEQ ID No: 20, or an amino acid sequence that differs from SEQ ID No: 13 by an amino acid addition, deletion or substitution of not more than 2 amino acids (e.g. 2 amino acids, 1 amino acid) ;

a LCDR2 comprising SEQ ID No: 21, or an amino acid sequence that differs from SEQ ID No: 14 by an amino acid addition, deletion or substitution of not more than 2 amino acids (e.g. 2 amino acids, 1 amino acid) ; and

a LCDR3 comprising SEQ ID No: 22, or an amino acid sequence that differs from SEQ ID No: 15 by an amino acid addition, deletion or substitution of not more than 2 amino acids (e.g. 2 amino acids, 1 amino acid) .

Preferably, the substitution is a conservative substitution. In some specific embodiments, the CD3-binding moiety is a scFv and comprises: a HCDR1 comprising SEQ ID NO: 17, a HCDR2 comprising SEQ ID NO: 18, a HCDR3 comprising SEQ ID NO: 19; a LCDR1 comprising SEQ ID NO: 20, a LCDR2 comprising SEQ ID NO: 21, a LCDR3 comprising SEQ ID NO: 22. In some embodiments, the CD3-binding moiety is a scFv comprising a HCDR1 as set forth in SEQ ID No: 17, a HCDR2 as set forth in SEQ ID No: 18, a HCDR3 as set forth in SEQ ID No: 19, and/or a LCDR1 as set forth in SEQ ID No: 20, a LCDR2 as set forth in SEQ ID No: 21, a LCDR3 as set forth in SEQ ID No: 22. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CD3-binding moiety is a scFv comprising a linker joining the VH and VL regions. The linker may be a peptide linker such as (G4S) n or derivatives thereof such as GGSSRSSSSGGGGSGGGG. Many other linker sequences have been proposed, which are well-known in the art.

In some specific embodiments, the CD3-binding moiety comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH comprises:

(A) the amino acid sequence as set forth in SEQ ID NO: 23;

and/or the VL comprises:

(A) the amino acid sequence as set forth in SEQ ID NO: 24 or 34;

As described herein, the fusion protein of the present invention comprises at least one serum albumin binding component. In some embodiments, the fusion proteins have at least one BCMA-binding moiety and at least one serum albumin binding component, and optionally at least one CD3-binding moiety. The order of these three binding components could be any possible order, e.g. the BCMA-binding moiety, CD3-binding moiety or serum albumin binding component can be N-terminal or C-terminal or in the middle. In some embodiments, the albumin binding moiety is located between the BCMA-binding moiety and CD3-binding moiety. In some embodiments, the albumin binding moiety is operably linked to the BCMA-binding moiety and CD3-binding moiety at its N and C terminal respectively. In some embodiments, the albumin binding moiety is located between the BCMA-binding moiety and CD3-binding moiety. In some embodiments, the albumin binding moiety is operably linked to the BCMA-binding moiety and CD3-binding moiety at its C and N terminal respectively. The fusion proteins may comprise other antigen-binding moieties instead of BCMA or CD3 binding, depending on the needs for the treatment of diseases.

In some exemplified embodiments, the present invention provides fusion proteins that comprise at least one BCMA-binding moiety, at least one CD3-binding moiety and at least one serum albumin binding protein. Examples of the amino acid sequences of the fusion proteins are set forth in SEQ ID NOs: 31-33. “At least one” binding component (BCMA, CD3 or serum albumin) when used herein includes that a fusion protein of the present invention may contain one, two, three, four or five BCMA-, CD3-, and/or serum albumin binding components (i.e., entities/units) which are preferably represented by VHH or scFv as described herein.

The albumin-, BCMA-and/or CD3-binding components with improved properties in view of therapeutic application, e.g. enhanced affinity or decreased immunogenicity, may be obtained from individual antibodies by techniques known in the art, such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences) , CDR grafting, humanizing, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing.

Nucleic Acid Molecules Encoding Antibodies of the Disclosure

In some aspects, the disclosure is directed to an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding the albumin binding proteins (e.g. the albumin binding VHHs) as disclosed herein. In some embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence that encodes any one of SEQ ID NOs: 7-12. In some embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence that hybridizes under high stringency conditions to the complementary strand of the nucleic acid sequence that encodes any one of SEQ ID NOs: 7-12.

In some aspects, the disclosure is directed to an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding the fusion proteins comprising the albumin binding proteins (e.g. the albumin binding VHHs) as disclosed herein. In some embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence that encodes any one of SEQ ID NOs: 31-33.

In some aspects, the disclosure is directed to an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding the BCMA binding proteins (e.g. the BCMA binding VHHs) as disclosed herein. In some embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence that encodes any one of SEQ ID NOs: 44-51. In some embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence that hybridizes under high stringency conditions to the complementary strand of the nucleic acid sequence that encodes any one of SEQ ID NOs: 44-51.

In some aspects, the disclosure is directed to an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding the fusion proteins comprising the BCMA binding proteins (e.g. the BCMA binding VHHs) as disclosed herein. In some embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence that encodes any one of SEQ ID NOs: 31-33.

In some aspects, the disclosure is directed to a vector comprising the nucleic acid sequence as disclosed herein.

A vector in the context of the present disclosure may be any suitable vector, including chromosomal, non-chromosomal, and synthetic nucleic acid vectors (anucleic acid sequence comprising a suitable set of expression control elements) . Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In some embodiments, a BCMA binding fusion protein is comprised in a naked DNA or RNA vector, including, for example, a linear expression element (as described in for instance Sykes and Johnston, Nat Biotech 17, 355-59 (1997) ) , a compacted nucleic acid vector (as described in for instance US 6,077,835 and/or WO 00/70087) , a plasmid vector such as pBR322, pUC 19/18, or pUC 118/119, a “midge” minimally-sized nucleic acid vector (as described in for instance Schakowski et al., Mol Ther 3, 793-800 (2001) ) , or as a precipitated nucleic acid vector construct, such as a CaP04-precipitated construct (as described in for instance WO200046147, Benvenisty and Reshef, PNAS USA 83, 9551-55 (1986) , Wigler et al., Cell 14, 725 (1978) , and Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981) ) . Such nucleic acid vectors and the usage thereof are well known in the art (see for instance US 5,589,466 and US 5,973,972) .

In one embodiment, the vector is suitable for expression of the albumin binding proteins, the BCMA binding proteins or fusion proteins in a bacterial cell. Examples of such vectors include expression vectors such as BlueScript (Stratagene) , pIN vectors (Van Heeke &Schuster, J Biol Chem 264, 5503-5509 (1989) , pET vectors (Novagen, Madison WI) and the like) . A vector may also or alternatively be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be employed. Suitable vectors include, for example, vectors comprising constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH (reviewed in: F. Ausubel et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987) , and Grant et al., Methods in Enzymol 153, 516-544 (1987) ) .

A vector may also or alternatively be a vector suitable for expression in mammalian cells, e.g. a vector comprising glutamine synthetase as a selectable marker, such as the vectors described in Bebbington (1992) Biotechnology (NY) 10: 169-175.

A nucleic acid and/or vector may also comprise a nucleic acid sequence encoding a secretion/localization sequence, which can target a polypeptide, such as a nascent polypeptide chain, to the periplasmic space or into cell culture media. Such sequences are known in the art, and include secretion leader or signal peptides.

The vector may comprise or be associated with any suitable promoter, enhancer, and other expression-facilitating elements. Examples of such elements include strong expression promoters (e. g., human CMV IE promoter/enhancer as well as RSV, SV40, SL3-3, MMTV, and HIV LTR promoters) , effective poly (A) termination sequences, an origin of replication for plasmid product in E. coli, an antibiotic resistance gene as selectable marker, and/or a convenient cloning site (e.g., a polylinker) . Nucleic acids may also comprise an inducible promoter as opposed to a constitutive promoter such as CMV IE.

In an even further aspect, the disclosure relates to a host cell comprising the vector specified herein above.

Thus, the present disclosure also relates to a recombinant eukaryotic or prokaryotic host cell which produces a bispecific antibody of the present disclosure, such as a transfectoma. A bispecific antibody may be expressed in a recombinant eukaryotic or prokaryotic host cell, such as a transfectoma, which produces the bispecific antibody of the disclosure as defined herein.

Examples of host cells include yeast, bacterial, plant and mammalian cells, such as CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER. C6 or NSO cells or lymphocytic cells. For example, in one embodiment, the host cell may comprise a first and second nucleic acid construct stably integrated into the cellular genome. In another embodiment, the present disclosure provides a cell comprising a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, which comprises a first and second nucleic acid construct as specified above.

In an even further aspect, the disclosure relates to a transgenic non-human animal or plant comprising nucleic acids encoding the albumin binding proteins, the BCMA binding proteins or fusion proteins as disclosed herein. In one aspect, the disclosure relates to a nucleic acid construct encoding one or more amino acid sequences set out in the sequence listing.

In one aspect, the disclosure relates to a method for producing the albumin binding proteins, the BCMA binding proteins or fusion proteins according to any one of the embodiments as disclosed herein, comprising the steps of culturing a host cell as disclosed herein comprising an expression vector or more than one expression vectors expressing the albumin binding proteins, the BCMA binding proteins or fusion proteins, and purifying said proteins from the culture media. In one aspect, the disclosure relates to a host cell comprising an expression vector as defined above. In one embodiment, the host cell is a recombinant eukaryotic, recombinant prokaryotic, or recombinant microbial host cell.

Pharmaceutical Compositions

In some aspects, the disclosure is directed to a pharmaceutical composition comprising the albumin binding proteins, the BCMA binding proteins or the fusion proteins as disclosed herein and a pharmaceutically acceptable carrier. In some aspects, the present disclosure provides a pharmaceutical composition comprising a nucleic acid encoding the albumin binding proteins, the BCMA binding proteins or the fusion proteins as disclosed herein and a pharmaceutically acceptable carrier. In some aspects, the present disclosure provides a pharmaceutical composition comprising a cell expressing the albumin binding proteins, the BCMA binding proteins, or the fusion proteins as disclosed herein and a pharmaceutically acceptable carrier.

Components of the compositions

The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or a drug. The pharmaceutical compositions of the disclosure also can be administered in a combination therapy with, for example, another immune-stimulatory agent, anti-cancer agent, an antiviral agent, or a vaccine. A pharmaceutically acceptable carrier can include, for example, a pharmaceutically acceptable liquid, gel or solid carriers, an aqueous medium, a non-aqueous medium, an anti-microbial agent, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispersing agent, a chelating agent, a diluent, adjuvant, excipient or a nontoxic auxiliary substance, other known in the art various combinations of components or more.

Suitable components may include, for example, antioxidants, fillers, binders, disintegrating agents, buffers, preservatives, lubricants, flavorings, thickening agents, coloring agents, emulsifiers or stabilizers such as sugars and cyclodextrin. Suitable anti-oxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercapto glycerol, thioglycolic acid, Mercapto sorbitol, butyl methyl anisole, butylated hydroxy toluene and/or propylgalacte. For example, in a solvent containing an fusion protein of the present disclosure, compositions include one or more anti-oxidants such as methionine, reducing antibody or antigen binding fragment thereof may be oxidized. The oxidation reduction may prevent or reduce a decrease in binding affinity, thereby enhancing antibody stability and extended shelf life. Thus, in some embodiments, the present disclosure provides a composition comprising one or more fusion proteins and one or more anti-oxidants such as methionine. The present disclosure further provides a variety of methods, wherein fusion proteins are mixed with one or more anti-oxidants, such as methionine, so that the fusion proteins can be prevented from oxidation, to extend their shelf life and/or increased activity.

To further illustrate, pharmaceutical acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, nonaqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcelluose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifying agents such as Polysorbate 80 (TWEEN-80) , sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid) , ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents utilized as carriers may be added to pharmaceutical compositions in multiple-dose containers that include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

Administration, Formulation and Dosage

The pharmaceutical composition of the disclosure may be administered in vivo, to a subject in need thereof, by various routes, including, but not limited to, oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or otherwise by implantation or inhalation. The subject compositions may be formulated into preparations in solid, semi-solid, liquid, or gaseous forms; including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols. The appropriate formulation and route of administration may be selected according to the intended application and therapeutic regimen.

Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.

Formulations suitable for parenteral administration (e.g., by injection) , include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) , in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in a liposome or other microparticulate) . Such liquids may additional contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, suspending agents, thickening agents, and solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Similarly, the particular dosage regimen, including dose, timing and repetition, will depend on the particular individual and that individual's medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance rate, etc. ) .

Frequency of administration may be determined and adjusted over the course of therapy, and is based on reducing the number of proliferative or tumorigenic cells, maintaining the reduction of such neoplastic cells, reducing the proliferation of neoplastic cells, or delaying the development of metastasis. In some embodiments, the dosage administered may be adjusted or attenuated to manage potential side effects and/or toxicity. Alternatively, sustained continuous release formulations of a subject therapeutic composition may be appropriate.

It will be appreciated by one of skill in the art that appropriate dosages can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, the severity of the condition, and the species, sex, age, weight, condition, general health, and prior medical history of the patient. The amount of compound and route of administration will ultimately be at the discretion of the physician, veterinarian, or clinician, although generally the dosage will be selected to achieve local concentrations at the site of action that achieve the desired effect without causing substantial harmful or deleterious side-effects.

In general, the fusion proteins of the disclosure may be administered in various ranges. These include about 5 μg/kg body weight to about 100 mg/kg body weight per dose; about 50 μg/kg body weight to about 5 mg/kg body weight per dose; about 100 μg/kg body weight to about 10 mg/kg body weight per dose. Other ranges include about 100 μg/kg body weight to about 20 mg/kg body weight per dose and about 0.5 mg/kg body weight to about 20 mg/kg body weight per dose. In certain embodiments, the dosage is at least about 100 μg/kg body weight, at least about 250 μg/kg body weight, at least about 750 μg/kg body weight, at least about 3 mg/kg body weight, at least about 5 mg/kg body weight, at least about 10 mg/kg body weight per dose.

In any event, the fusion proteins of the disclosure is preferably administered as needed to a subject in need thereof. Determination of the frequency of administration may be made by persons skilled in the art, such as an attending physician based on considerations of the condition being treated, age of the subject being treated, severity of the condition being treated, general state of health of the subject being treated and the like.

In certain preferred embodiments, the course of treatment involving the fusion proteins of the disclosure will comprise multiple doses of the selected drug product over a period of weeks or months. More specifically, the fusion proteins of thedisclosure may be administered once every day, every two days, every four days, every week, every ten days, every two weeks, every three weeks, every month, every six weeks, every two months, every ten weeks or every three months. In this regard, it will be appreciated that the dosages may be altered or the interval may be adjusted based on patient response and clinical practices.

Dosages and regimens may also be determined empirically for the disclosed therapeutic compositions in individuals who have been given one or more administration (s) . For example, individuals may be given incremental dosages of a therapeutic composition produced as described herein. In selected embodiments, the dosage may be gradually increased or reduced or attenuated based respectively on empirically determined or observed side effects or toxicity. To assess efficacy of the selected composition, a marker of the specific disease, disorder or condition can be followed as described previously. For cancer, these include direct measurements of tumor size via palpation or visual observation, indirect measurement of tumor size by x-ray or other imaging techniques; an improvement as assessed by direct tumor biopsy and microscopic examination of the tumor sample; the measurement of an indirect tumor marker (e.g., PSA for prostate cancer) or a tumorigenic antigen identified according to the methods described herein, a decrease in pain or paralysis; improved speech, vision, breathing or other disability associated with the tumor; increased appetite; or an increase in quality of life as measured by accepted tests or prolongation of survival. It will be apparent to one of skill in the art that the dosage will vary depending on the individual, the type of neoplastic condition, the stage of neoplastic condition, whether the neoplastic condition has begun to metastasize to other location in the individual, and the past and concurrent treatments being used.

Compatible formulations for parenteral administration (e.g., intravenous injection) will comprise the fusion proteins as disclosed herein in concentrations of from about 10 μg/ml to about 100 mg/ml. In certain selected embodiments, the concentrations of the fusion proteins will comprise 20 μg/ml, 40 μg/ml, 60 μg/ml, 80 μg/ml, 100 μg/ml, 200 μg/ml, 300, μg/ml, 400 μg/ml, 500 μg/ml, 600 μg/ml, 700 μg/ml, 800 μg/ml, 900 μg/ml or 1 mg/ml. In other preferred embodiments, concentrations of the fusion proteins will comprise 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 8 mg/ml, 10 mg/ml, 12 mg/ml, 14 mg/ml, 16 mg/ml, 18 mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, 35 mg/ml, 40 mg/ml, 45 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml or 100 mg/ml.

Applications of the Disclosure

In some aspects, the present disclosure provides a method of extending the half-life or stability of therapeutic drugs in vivo. The method comprises incorporating, conjugating or fusing an albumin binding VHH into/with the therapeutic drug (generally proteins, e.g. antibodies) . The resultant fusion proteins would have a much longer clinical effect in the bodies of the subjects (e.g. half-life extended by at least a few days) , reducing administration frequency and without interfering the therapeutic effect of the drug per se.

In some aspects, the present disclosure provides a BCMA binding molecule. Further, the present disclosure provides a fusion protein comprising a BCMA binding moiety, optionally a CD3 binding moiety as well as the albumin binding VHHs. Thus, the present disclosure also provides a method of preventing or treating a BCMA-related disorder in a subject, which comprises administering to the subject (for example, a human) in need of treatment a therapeutically effective amount of the BCMA binding molecule or the fusion proteins as disclosed herein. For example, the B cell-related disorder is selected from cancers such as lymphoma and autoimmune diseases such as systemic lupus erythematosus (SLE) .

The disclosure provides methods of recognizing BCMA and binding to various type of cells, especially B cells. The methods comprise administering the fusion proteins of the disclosure, as described above. Types of cells that may be depleted by the methods of the disclosure include, without limitation, plasma cells, B cells, memory B cells (including switched, unswitched, and double negative) , lymphoma cells derived from B cells, and cells that express BCMA, a protein similar thereto, the extracellular domain thereof, or a polypeptide similar to the extracellular domain thereof.

In a still further aspect, the disclosure provides methods of treating or preventing or delaying a B-cell mediated disorder. The method includes administering to a subject in which such treatment or prevention or delay is desired, a fusion protein of the disclosure in an amount sufficient to treat, prevent, or delay a tumorigenic or immunoregulatory condition in the subject. In some embodiments, the subject is a human. In other embodiments, the subject is a non-human mammal. In some embodiments, administration of the fusion proteins of the disclosure blocks BCMA-mediated signalling in the subject, which may result in one or more of cell death, inhibition, reduction, or cessation of cell proliferation.

In some embodiments, the fusion proteins of the disclosure use BCMA to "target" B cell lymphomas. In essence, such targeting can be generalized as follows: fusion proteins of the disclosure specific to the BCMA surface antigen of B cells are, e.g., injected into a subject and specifically bind to the BCMA cell surface antigen of (ostensibly) both normal and malignant B cells; this binding leads to the destruction and/or depletion of neoplastic B cells. Additionally, chemical agents or radioactive labels having the potential to destroy cancer cells and/or tumors can be conjugated to the fusion proteins of the disclosure such that the agent is specifically “delivered” to the targeted B cells, such as, e.g., neoplastic B cells. In some embodiments, the methods of the disclosure comprise administering a fusion protein that is not conjugated to a chemical agent or radioactive label. In some embodiments, the methods of the disclosure comprise administering a fusion protein that is not conjugated to a cytotoxic agent.

B cell-related disorders include, without limitation, autoimmune diseases involving inappropriate B cell activity and B cell lymphomas. B cell lymphomas include, without limitation, multiple myeloma, plasmacytoma, Hodgkins' lymphoma, follicular lymphomas, small non-cleaved cell lymphomas, endemic Burkitt's lymphoma, sporadic Burkitt's lymphoma, marginal zone lymphoma, extranodal mucosa-associated lymphoid tissue lymphoma, nodal monocytoid B cell lymphoma, splenic lymphoma, mantle cell lymphoma, large cell lymphoma, diffuse mixed cell lymphoma, immunoblastic lymphoma, primary mediastinal B cell lymphoma, pulmonary B cell angiocentric lymphoma, and small lymphocytic lymphoma. The fusion proteins of the disclosure may also be used to treat cancers in which the cancer cells express BCMA. The B cell-related disorders additionally include B cell proliferations of uncertain malignant potential, such as, for example, lymphomatoid granulomatosis and post-transplant lymphoproliferative disorder.

The conditions diagnosed, treated, prevented or delayed using the fusion proteins of the disclosure can additionally be an immunoregulatory disorder. These disorders include those that are autoimmune in nature such as, for example, systemic lupus erythematosus, rheumatoid arthritis, myasthenia gravis, autoimmune hemolytic anemia, idiopathic thrombocytopenia purpura, anti-phospholipid syndrome, Chagas' disease, Grave's disease, Wegener's granulomatosis, poly-arteritis nodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, anti-phospholipid syndrome, ANCA associated vasculitis, Goodpasture's disease, Kawasaki disease, and rapidly progressive glomerulonephritis. The fusion proteins of the disclosure may also have application in plasma cell disorders such as heavy-chain disease, primary or immunocyte-associated amyloidosis, and monoclonal gammopathy of undetermined significance (MGUS) .

Compositions and methods of treatment using the fusion proteins of the disclosure can be used with any condition associated with undesired BCMA-expressing cell proliferation.

A variety of cancers where BCMA is implicated, whether malignant or benign and whether primary or secondary, may be treated or prevented with a method provided by the disclosure. The cancers may be solid cancers or hematologic malignancies. In some embodiments, examples of cancer include but not limited to B-cell cancers, including B-cell lymphoma (including low grade/follicular non-Hodgkin’s lymphoma (NHL) ; small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom’s Macroglobulinemia; chronic lymphocytic leukemia (CLL) ; acute lymphoblastic leukemia (ALL) ; Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliierative disorder (PTLD) , as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors) , B-cell proliferative disorders, and Meigs’ syndrome. More specific examples include, but are not limited to, relapsed or refractory NHL, front line low grade NHL, Stage III/IV NHL, chemotherapy resistant NHL, precursor B lymphoblastic leukemia and/or lymphoma, small lymphocytic lymphoma, B-cell chronic lymphocytic leukemia and/or prolymphocytic leukemia and/or small lymphocytic lymphoma, B-cell prolymphocytic lymphoma, immunocytoma and/or lymphoplasmacytic lymphoma, lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone-MALT lymphoma, nodal marginal zone lymphoma, hairy cell leukemia, plasmacytoma and/or plasma cell myeloma, low grade/follicular lymphoma, intermediate grade/follicular NHL, mantle cell lymphoma, follicle center lymphoma (follicular) , intermediate grade diffuse NHL, diffuse large B-cell lymphoma, aggressive NHL (including aggressive front-line NHL and aggressive relapsed NHL) , NHL relapsing after or refractory to autologous stem cell transplantation, primary mediastinal large B-cell lymphoma, primary effusion lymphoma, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL, Burkitt’s lymphoma, precursor (peripheral) large granular lymphocytic leukemia, mycosis fungoides and/or Sezary syndrome, skin (cutaneous) lymphomas, anaplastic large cell lymphoma, angiocentric lymphoma.

In some embodiments, examples of cancer further include, but are not limited to, B-cell proliferative disorders, which further include, but are not limited to, lymphomas (e.g., B-Cell Non-Hodgkin’s lymphomas (NHL) ) and lymphocytic leukemias. Such lymphomas and lymphocytic leukemias include e.g. a) follicular lymphomas, b) Small Non-Cleaved Cell Lymphomas/Burkitt’s lymphoma (including endemic Burkitt’s lymphoma, sporadic Burkitt’s lymphoma and Non-Burkitt’s lymphoma) , c) marginal zone lymphomas (including extranodal marginal zone B-cell lymphoma (Mucosa-associated lymphatic tissue lymphomas, MALT) , nodal marginal zone B-cell lymphoma and splenic marginal zone lymphoma) , d) Mantle cell lymphoma (MCL) , e) Large Cell Lymphoma (including B-cell diffuse large cell lymphoma (DLCL) , Diffuse Mixed Cell Lymphoma, Immunoblastic Lymphoma, Primary Mediastinal B-Cell Lymphoma, Angiocentric Lymphoma-Pulmonary B-Cell Lymphoma) , f) hairy cell leukemia, g ) lymphocytic lymphoma, Waldenstrom’s macroglobulinemia, h) acute lymphocytic leukemia (ALL) , chronic lymphocytic leukemia (CLL) /small lymphocytic lymphoma (SLL) , B cell prolymphocytic leukemia, i) plasma cell neoplasms, plasma cell myeloma, multiple myeloma, plasmacytoma, and/or j) Hodgkin’s disease.

In some other embodiments, the disorder is an autoimmune disease. Examples of autoimmune diseases that may be treated with the fusion proteins include autoimmune encephalomyelitis, lupus erythematosus, and rheumatoid arthritis. The fusion proteins may also be used to treat or prevent infectious disease, inflammatory disease (such as allergic asthma) and chronic graft-versus-host disease.

A skilled person would readily appreciate that, the diseases are not limited to those listed above. The albumin binding proteins disclosed herein may be fused with antibodies or antigen-binding portions thereof targeting a numerous variety of antigens (thus may be used for the treatment of other diseases) , and the formed fusion proteins that have an extended half-life may have an improved therapeutic effect in the treatment of other diseases.

Combined use

The fusion proteins may be used in combination with a different anti-cancer agent, a cytotoxic agent, a chemotherapeutic agent, or a cell immunotherapy. For example, the fusion proteins of the disclosure may be administered in conjunction with antibody C2B8 of US Patent 5,736,137, also known as RITUXAN^TM. The fusion proteins of the disclosure may be administered in conjunction with CART therapy.

Cellular immunotherapies

In some embodiments, the fusion proteins as disclosed herein are used in combination with a cellular immunotherapy, also known as adoptive cell therapy. As is generally known, cellular immunotherapy is a form of treatment that uses the cells of human body’s immune system to eliminate cancer. Some of these approaches involve directly isolating our own immune cells and simply expanding their numbers (e.g. performed by activating and expanding the immune cells of patient outside of the body and infused into the patient) , whereas others involve genetically engineering immune cells (via gene therapy) to enhance their cancer-fighting capabilities. Cellular immunotherapies can be deployed in different ways, including but not limited to Tumor-Infiltrating Lymphocyte (TIL) therapy, Engineered T Cell Receptor (TCR-T) therapy, Chimeric Antigen Receptor (CAR) T Cell therapy, CAR NK Cell therapy, Natural Killer (NK) Cell therapy.

In some embodiments, the fusion proteins may be used in combination with an additional anti-tumor therapy, such astumor-infiltrating lymphocyte (TIL) therapy, T cell receptor T cell (TCR-T) therapy, chimeric antigen receptor (CAR) T cell therapy, and NK cell therapy, as well as targeted therapy and chemotherapy. The administration of the additional anti-tumor therapy may be performed before, after or simultaneously with the administration of the fusion proteins or the pharmaceutical composition as disclosed herein.

Chemotherapies

The term “anti-cancer agent” or “anti-proliferative agent” means any agent that can be used to treat a cell proliferative disorder such as cancer, and includes, but is not limited to, cytotoxic agents, cytostatic agents, anti-angiogenic agents, debulking agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapies, radiation therapy and anti-metastatic agents and immunotherapeutic agents. It will be appreciated that, in selected embodiments as discussed above, such anti-cancer agents may comprise conjugates and may be associated with the disclosed fusion proteins prior to administration. More specifically, in certain embodiments selected anti-cancer agents will be linked to the unpaired cysteines of the engineered fusion proteins to provide engineered conjugates as set forth herein. Accordingly, such engineered conjugates are expressly contemplated as being within the scope of the instant disclosure. In other embodiments, the disclosed anti-cancer agents will be given in combination with site-specific conjugates comprising a different therapeutic agent as set forth above.

As used herein the term “cytotoxic agent” means a substance that is toxic to the cells and decreases or inhibits the function of cells and/or causes destruction of cells. In certain embodiments, the substance is a naturally occurring molecule derived from a living organism. Examples of cytotoxic agents include, but are not limited to, small molecule toxins or enzymatically active toxins of bacteria (e.g., Diptheria toxin, Pseudomonas endotoxin and exotoxin, Staphylococcal enterotoxin A) , fungal (e.g., α-sarcin, restrictocin) , plants (e.g., abrin, ricin, modeccin, viscumin, pokeweed anti-viral protein, saporin, gelonin, momoridin, trichosanthin, barley toxin, Aleuritesfordii proteins, dianthin proteins, Phytolaccamericana proteins (PAPI, PAPII, and PAP-S) , Momordica charantia inhibitor, curcin, crotin, chlorambu officinalis inhibitor, gelonin, mitegellin, restrictocin, phenomycin, neomycin, and the tricothecenes) or animals, (e.g., cytotoxic Rnases, such as extracellular pancreatic Rnases; Dnase I, including fragments and/or variants thereof) .

For the purposes of the instant disclosure a “chemotherapeutic agent” comprises a chemical compound that non-specifically decreases or inhibits the growth, proliferation, and/or survival of cancer cells (e.g., cytotoxic or cytostatic agents) . Such chemical agents are often directed to intracellular processes necessary for cell growth or division, and are thus particularly effective against cancerous cells, which generally grow and divide rapidly. For example, vincristine depolymerizes microtubules, and thus inhibits cells from entering mitosis. In general, chemotherapeutic agents can include any chemical agent that inhibits, or is designed to inhibit, a cancerous cell or a cell likely to become cancerous or generate tumorigenic progeny (e.g., TIC) . Such agents are often administered, and are often most effective, in combination, e.g., in regimens such as CHOP or FOLFIRI.

Examples of anti-cancer agents that may be used in combination with the site-specific constructs of the present disclosure (either as a component of a site specific conjugate or in an unconjugated state) include, but are not limited to, alkylating agents, alkyl sulfonates, aziridines, ethylenimines and methylamelamines, acetogenins, a camptothecin, bryostatin, callystatin, CC-1065, cryptophycins, dolastatin, duocarmycin, eleutherobin, pancratistatin, a sarcodictyin, spongistatin, nitrogen mustards, antibiotics, enediyne antibiotics, dynemicin, bisphosphonates, esperamicin, chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folic acid analogues, purine analogs, androgens, anti-adrenals, folic acid replenisher such as frolinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, an epothilone, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansinoids, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide, procarbazine, polysaccharide complex (JHS Natural Products, Eugene, OR) , razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2’, 2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine) ; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ( “Ara-C” ) ; cyclophosphamide; thiotepa; taxoids, chloranbucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, vinblastine; platinum; etoposide (VP-16) ; ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) , topoisomerase inhibitor RFS 2000; difluorometlhylornithine; retinoids; capecitabine; combretastatin; leucovorin; oxaliplatin; inhibitors of PKC-alpha, Raf, H-Ras, EGFR and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators, aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, and anti-androgens; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog) ; antisense oligonucleotides, ribozymes such as a VEGF expression inhibitor and a HER2 expression inhibitor; vaccines, rIL-2; topoisomerase 1 inhibitor; Vinorelbine and Esperamicins and pharmaceutically acceptable salts, acids or derivatives of any of the above.

Combined use with radiotherapies

The present disclosure also provides for the combination of the fusion proteins with radiotherapy (i.e., any mechanism for inducing DNA damage locally within tumor cells such as gamma-irradiation, X-rays, UV-irradiation, microwaves, electronic emissions and the like) . Combination therapy using the directed delivery of radioisotopes to tumor cells is also contemplated, and the disclosed conjugates may be used in connection with a targeted anti-cancer agent or other targeting means. Typically, radiation therapy is administered in pulses over a period of time from about 1 to about 2 weeks. The radiation therapy may be administered to subjects having head and neck cancer for about 6 to 7 weeks. Optionally, the radiation therapy may be administered as a single dose or as multiple, sequential doses.

Pharmaceutical packs and kits

Pharmaceutical packs and kits comprising one or more containers, comprising one or more doses of the fusion proteins are also provided. In certain embodiments, a unit dosage is provided wherein the unit dosage contains a predetermined amount of a composition comprising, for example, the fusion proteins, with or without one or more additional agents. For other embodiments, such a unit dosage is supplied in single-use prefilled syringe for injection. In still other embodiments, the composition contained in the unit dosage may comprise saline, sucrose, or the like; a buffer, such as phosphate, or the like; and/or be formulated within a stable and effective pH range. Alternatively, in certain embodiments, the composition may be provided as a lyophilized powder that may be reconstituted upon addition of an appropriate liquid, for example, sterile water or saline solution. In certain preferred embodiments, the composition comprises one or more substances that inhibit protein aggregation, including, but not limited to, sucrose and arginine. Any label on, or associated with, the container (s) indicates that the enclosed conjugate composition is used for treating the neoplastic disease condition of choice.

The present disclosure also provides kits for producing single-dose or multi-dose administration units of the fusion proteins and, optionally, one or more anti-cancer agents. The kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic and contain a pharmaceutically effective amount of the disclosed fusion proteins. In other preferred embodiments, the container (s) comprise a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) . Such kits will generally contain in a suitable container a pharmaceutically acceptable formulation of the fusion proteins and, optionally, one or more anti-cancer agents in the same or different containers. The kits may also contain other pharmaceutically acceptable formulations, either for diagnosis or combined therapy. For example, in addition to the fusion proteins of the present disclosure such kits may contain any one or more of a range of anti-cancer agents such as chemotherapeutic or radiotherapeutic drugs; anti-angiogenic agents; anti-metastatic agents; targeted anti-cancer agents; cytotoxic agents; and/or other anti-cancer agents.

More specifically the kits may have a single container that contains the disclosed fusion proteins, with or without additional components, or they may have distinct containers for each desired agent. Alternatively, the fusion proteins and any optional anti-cancer agent of the kit may be maintained separately within distinct containers prior to administration to a patient. The kits may also comprise a second/third container means for containing a sterile, pharmaceutically acceptable buffer or other diluents such as bacteriostatic water for injection (BWFI) , phosphate-buffered saline (PBS) , Ringer's solution and dextrose solution.

When the components of the kit are provided in one or more liquid solutions, the liquid solution is preferably an aqueous solution, with a sterile aqueous or saline solution being particularly preferred. However, the components of the kit may be provided as dried powder (s) . When reagents or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container.

As indicated briefly above the kits may also contain a means by which to administer the fusion proteins and any optional components to a patient, e.g., one or more needles, I. V. bags or syringes, or even an eye dropper, pipette, or other such like apparatus, from which the formulation may be injected or introduced into the animal or applied to a diseased area of the body. The kits of the present disclosure will also typically include a means for containing the vials, or such like, and other component in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vials and other apparatus are placed and retained.

Sequence Listing

Appended to the instant application is a sequence listing comprising a number of amino acid sequences. The following Tables A-G provide a summary of the included sequences. The illustrative albumin binding VHHs as disclosed herein are collectively referred to as TAD6016 VHHs. The illustrative BCMA binding VHHs as disclosed herein are collectively referred to as W3566 VHHs.

Table A

CDR Sequences of anti-albumin VHHs

Table B

Amino acid sequences of anti-albumin VHHs

Table C

CDR sequences of anti-BCMA VHHs

Table D

Amino acid sequences of anti-BCMA VHHs

Table E

Amino acid sequence of the constant region of VHH-Fc antibody

Table F

Sequences of anti-CD3 scFvs

Table G

Sequences of constructed antibodies

EXAMPLES

The present disclosure, thus generally described, will be understood more readily by reference to the following Examples, which are provided by way of illustration and are not intended to be limiting of the present disclosure. The Examples are not intended to represent that the experiments below are all or the only experiments performed.

Example 1: Preparation of Materials

1.1 Antigen preparation

Human serum Albumin (HSA) was purchased from Sigma (A7736) . Cynomolgus Monkey (cyno) Albumin protein was purchased from Abcam (ab184894) . Mouse Albumin protein was purchased from Abcam (ab183228) or in-house produced based on mouse serum Albumin sequence (Genbank Accession BC024643) . Human FcRn protein was purchased from R&D (8639-FC) .

1.2 Generation of Benchmark Antibody (BMK)

Two anti-human Albumin VHH BMKs were synthesized according to the sequences of ALB-11 in patent US8703131B2 and 10G in patent WO 2017/201488 Al, respectively, and named as T6016-BMK4 and T6016-BMK6 herein. The gene encoding the BMK was constructed into pET-BAC vector and transformed into BL21 (DE3) E. coli (ThermoFisher) and cultured in ZYM Medium (ThermoFisher) at 25 ℃ for 2 days. The culture supernatants were harvested for protein purification by Ni-NTA column (GE Healthcare, 175248) .

Example 2: Generation of anti-Albumin VHHs

2.1 Generation of parental VHH

Anti-Albumin VHHs were generated by phage display technology from an in-house constructed native VHH phage library. Briefly, recombinant human Albumin protein immobilized in 5 ml immune tube was used for bio-panning. The positive VHH clones binding to human Albumin were selected and tested for cross-binding to cyno Albumin or mouse Albumin by ELISA. The ELISA were done in PBST buffer (50 mM Na₂HPO₄/NaH₂PO₄, 150 mM NaCl, 0.05%Tween20) of pH 5.5 and pH 7.4, respectively. The clones showing good binding in two pH buffers were sent to Biosune (Shanghai, China) for nucleotide sequencing of VHH genes. One VHH clone designated as T6016-P1R2-6D10 was selected and its sequence was shown in Table 1.

2.2 Humanization

“Best Fit” approach was used to humanize T6016-P1R2-6D10. Amino acid sequences of VHH framework regions were blasted against human germline V-gene database, and humanized VHH sequences were generated by replacing human CDR sequences in the top hit with VHH CDR sequences using Kabat CDR definition. Several residues in the framework region were back mutated to VHH to maintain the affinity.

The designed VHH variants and parental VHH proteins were produced in BL21 (DE3) E. coli. The expression of his-and c-Myc-tag fused VHH protein in BL21 (DE3) supernatant was confirmed by SDS-PAGE, and then purified using Ni-NTA column. The purified variants were tested for koff kinetics to human, cyno and mouse Albumin by SPR technology and the variants with proper affinity were selected as humanized antibody leads.

2.3 Affinity maturation

To further increase the target binding affinity of the humanized leads, affinity maturation was performed. Each amino acid of three complementary-determining regions (CDR1, CDR2, and CDR3) of the parental clone was individually mutated to other 20 amino acids using a site-directed mutagenesis method. DNA primers containing an NNS codon encoding twenty amino acids were used to introduce mutation to each targeted CDR position. The individual degenerate primers of phosphorylated were used in site-directed mutagenesis reactions. 200 ng of the reaction products was electroporated into BL21 (DE3) and expressed.

The mutagenesis clones were screened by ELISA assay. Affinity improved single clones were expressed and purified. The purified variants were tested for K_off rate using SPR technology. The point mutations in VHH determined to be beneficial for binding to antigen were further combined to gain enhanced affinity synergy. The combinatorial mutants were synthesized in GENEWIZ and expressed in BL21 (DE3) E. Coli. Supernatants of the mutants were tested by SPR K_off rate for choosing affinity improved variants.

T6016-P1R2-6D10-Z4-R1-34G9, T6016-P1R2-6D10-Z4-R1-34G9-m2 and T6016-P1R2-6D10-Z5-R1-34G9-m2 were then chosen and their affinity to human, cyno and mouse Albumin were confirmed by full kinetic SPR. The sequences of T6016-P1R2-6D10, its humanized variants (including affinity matured variants) T6016-P1R2-6D10-z5, T6016-P1R2-6D10-Z4-R1-34G9, T6016-P1R2-6D10-Z5-R1-34G9, T6016-P1R2-6D10-Z4-R1-34G9-m2 and T6016-P1R2-6D10-Z5-R1-34G9-m2 are shown in Table 1.

Table 1. The sequences of anti-albumin VHH and its variants

Example 3: In vitro characterization of anti-albumin VHHs

3.1 SPR affinity to human, cyno, mouse, cat or dog albumin at pH 5.5 and pH 7.4

Full kinetic affinity of testing VHH antibodies to the antigens were detected by SPR assay using Biacore 8K. Biotin conjugated human, cyno and mouse albumin were captured on a streptavidin immobilized CM5 sensor chip (GE) , respectively. The running buffers used for the pH dependent binding of VHHs are PBST buffer (50 mM Na₂HPO₄/NaH₂PO₄, 150 mM NaCl, 0.05%Tween20) of pH5.5 and pH 7.4, respectively. Each antibody at a series of concentrations were diluted in running buffers of pH 7.4 and pH 5.5, then injected over the sensor chip at a flow rate of 30 uL/min for an association phase of 120 s, followed by 300 s dissociation. The chip was regenerated by 10 mM glycine (pH 1.5) after each binding cycle. The sensorgrams of blank surface and buffer channel were subtracted from the test sensorgrams. The experimental data was fitted by 1: 1 model using Langmiur analysis.

The SPR affinities of VHHs to human, cyno, mouse, cat or dog albumin at pH 5.5 and pH 7.4 are shown in Table 2A-E.

Table 2A. Full kinetic affinity to human Albumin by SPR.

Table 2B. Full kinetic affinity to cyno Albumin by SPR.

Table 2C. Full kinetic affinity to mouse Albumin by SPR.

Table 2D. Full kinetic affinity to Cat Albumin by SPR.

Table 2E. Full kinetic affinity to Dog Albumin by SPR.

The affinities of T6016-Z4-m2 and T6016-Z5-m2 to human, cyno and mouse Albumin in buffer of pH 7.4 and pH 5.5 were increased by affinity maturation. Their affinities are as similar as that of T6016-BMK4 and T6016-BMK6, as shown in Table 2A-C. The affinities of T6016-Z5-m2 to cat and dog Albumin were also high, but BMK4 and BMK6 show no or weak binding with cat and dog Albumin, as shown in Table 2D-E.

3.2 SPR pairwise binning test

Antibody epitope binning to biotin conjugated human Albumin was detected by SPR assay using Biacore 8K. VHH samples were immobilized on a CM5 sensor chip (GE) . 100 nM biotin conjugated human Albumin was injected over the sensor chip to detect the binding activity to VHH samples. 5000 nM VHH samples were injected over the sensor chip to detect the non-specific binding. Afterwards, 100 nM biotin conjugated human Albumin was incubated with 5000 nM VHH samples at 25℃ for 1 h. Then, 300 nM biotin conjugated human Albumin and incubated mixtures were injected over the chip at a flow rate of 30 ul/min for an association phase of 180 s, followed by 300 s dissociation. The chip was regenerated by 10 mM glycine (pH 1.5) after each binding cycle. The sensorgrams of blank surface and buffer channel were subtracted from the test sensorgrams.

As shown in Figures 1A-1C, using T6016-Z4-34G9 as a representative of T6016-P1R2-6D10 derived variants, the SPR pairwise binning results show that the T6016-P1R2-6D10 derived variants have different epitope bin with T6016-BMK4 and T6016-BMK6, while T6016-BMK4 and T6016-BMK6 have same/similar epitope bin.

3.3 Thermal stability by differential scanning fluorimetry (DSF) assay

DSF assay was performed on7 Flex Real-Time PCR system (Applied Biosystems, Thermo Fisher Scientific) . Briefly, 19 μL of antibody solution was mixed with 1 μL of 80x SYPRO Orange solution (Invitrogen-S6651) and transferred to the MicroAmp^TM Optical 96-Well Reaction Plate (Applied Biosystems-N8010560) . The plate was sealed with the MicroAmp^TM Optical Adhesive Film (Applied Biosystems-4311971) and centrifugated at 3,000 rpm for 5 min to remove any air bubbles. The plate was heated from 26 ℃ to 95 ℃ at a rate of 0.9 ℃/min, and the resulting fluorescence data was collected. The negative derivatives of the fluorescence changes with respect to different temperatures were calculated, and the maximal absolute value was defined as melting temperature T_m. Sometimes protein could have multiple unfolding transitions, and the reported T_m value in Figure 2 and Table 3 referred to the melting temperature of the first unfolding transition event. Data collection and T_m calculation were conducted automatically by theReal Time PCR software (v1.3) .

Table 3 illustrates the temperature of hydrophobic exposure (Th ℃) for several anti-albumin VHH variants

Table 3. DSF results of BMKs and T6016-P1R2-6D10 and its variants.

The DSF results in table 3 show that Tm values of T6016-P1R2-6D10 were higher than BMK Abs, indicating improved thermal stability. Meanwhile, the humanized and affinity-matured VHHs of T6016-P1R2-6D10 also showed comparable or higher values than BMK Abs. The improved and excellent thermal stability of T6016-P1R2-6D10 and its derived variants will facilitate anti-Albumin VHH based druggability.

3.4 Radius (nm) measurement by Dynamic Light Scattering (DLS)

Radius measurement was investigated using DynaPro Plate Reader III (Wyatt DynaproTM) . 5 acquisitions were collected for each protein sample while each acquisition time was 5 s. Each well contained 7.5 μL of solution in 1536 plate (Aurora microplate) . For each measurement, the diffusion coefficient was determined. Radius were calculated automatically by the operation software (DYNAMICS 7.8.1.3) .

Table 4 below illustrates the radius of antibodies investigated using DynaPro Plate Reader III.

Table 4. Radius (nm) measurement by DLS

As shown in Table 4, the radiuses of antibodies were all around 2nm, suggesting no obvious oligomeric forms of protein was detected.

3.5 Diffusion interaction parameter (kD) measurement by DLS

kD measurement was investigated using DynaPro Plate Reader III (Wyatt DynaproTM) . 5 acquisitions were collected for each protein sample while each acquisition time was 5s. Each well contained 7.5 μL of solution at protein concentrations of 1.25, 2.5, 5, 10 mg/mL and original dose (10-20 mg/mL) in 1536 plate (Aurora microplate) . For each measurement, the diffusion coefficient was determined and plotted against protein concentration. kD values were calculated automatically by the operation software (DYNAMICS 7.8.1.3) .

Table 5 illustrates the DLS-kD values of antibodies investigated using DynaPro Plate Reader III.

Table 5. Diffusion interaction parameter (kD) measurement by DLS

As shown in Table 5, TAD6016-P1R2-6D10 and its variants had higher DLS-kD values than BMKs, indicating a lower aggregation propensity.

3.6 Hydrophobicity interaction chromatography HPLC (HIC-HPLC)

Hydrophobicity property of antibody was detected by HPLC 1260 Infinity II system (Agilent Technologics^TM) with TSKgel butyl-NPR column (Tosoh-0042168) . Each sample was diluted to 0.5 mg/ml with PBS buffer and 20 μL diluted sample was injected into the column, and separated with a flow rate of 0.5 ml/min for 61 min. The running buffer is 25 mM sodium phosphate, pH7.0 (Buffer A) and 25 mM sodium phosphate, 1.5 M (NH₄) ₂SO₄, pH7.0 (Buffer D) . The running gradient was 100%to 0%Buffer D from 3 to 53 min. The peak retention was detected with UV light of the wavelength at 280 nm and 230 nm. The retention time was analyzed with HIC-HPLC analysis method to integrate all peak areas from 8 min to 20 min. The operation and analysis software is the OpenLab CDS Workstation (v2.6.0.691) .

Table 6 illustrates the hydrophobicity property of antibody detected by HPLC 1260 Infinity II system with TSKgel butyl-NPR column.

Table 6. Hydrophobicity interaction chromatography HPLC (HIC-HPLC)

As shown in Table 6, the retention times of T6016-P1R2-6D10 and its variants were lower than that of BMKs, indicating lower hydrophobicity and higher solubility, which are useful in preparing high concentration antibody formulations.

Example 4: Generation of anti-BCMA VHHs and VHH-Fc fusion proteins (WBP3566 antibodies)

4.1 Antigen Generation

DNA sequences encoding the extracellular domain sequence of human BCMA (Uniprot No. Q02223-1) was synthesized in Sangon Biotech (Shanghai, China) , and then subcloned into modified pcDNA3.3 expression vectors with MBP tag and AVI-His tag or human Fc tag and AVI-His tag in C-terminal. Expi293 cells (Invitrogen-A14527) were transfected with the purified expression vectors. Cells were cultured for 5 days and supernatant was collected for protein purification using Ni-NTA column (GE Healthcare, Cat. 175248) or Protein A column (GE Healthcare, Cat. 175438) . The obtained human BCMA ECD protein was analyzed by SDS-PAGE and SEC, and then stored at -80℃. Cynomolgus BCMA (Uniprot No. G7Q0I4-1) was purchased from AcroBiosystems (ACRO BCA-C52H7, Cat. BCA-C52H7) .

4.2 Production of Benchmark Antibodies (BMKs)

DNA sequences encoding the variable regions of a benchmark anti-BCMA antibody, W356-BMK7 (monoclonal antibody of EngMab, Mab42 as disclosed in patent application WO2018083204A1) was synthesized in Sangon Biotech (Shanghai, China) , and then subcloned into modified pcDNA3.3 expression vectors with constant region of human IgG1 or IgG4.

The plasmids containing VH and VL genes were co-transfected into Expi293 cells. Cells were cultured for 5 days and supernatant was collected for protein purification using Protein A column (GE Healthcare, 175438) . The obtained antibodies were analyzed by SDS-PAGE and SEC, and then stored at -80℃.

4.3 Establishment of Stable Cell Lines/Cell Pool

Using Lipofectamine 2000, 293F cells were transfected with the expression vector containing gene encoding cynomolgus BCMA (XP_001106892.1) . Cells were cultured in medium containing proper selection marker Cynomolgus BCMA high expression stable cell line were selected after limited dilution.

4.4 Generation of anti-BCMA VHHs

Anti-BCMA VHHs were generated by immunization of Camelidae animals and phage display technology. Briefly, Alpacas (Vicugna pacos) were subcutaneously immunized with hFc tagged human BCMA ECD protein and hFc tagged mouse BCMA ECD protein. After immunization, peripheral blood was collected for construction of phage library displaying VHH fragments. After bio-panning with corresponding target ECD proteins or target cell line, the positive VHH clones binding to BCMA were selected.

4.5 VHH sequencing

The positive E. coli clones selected by target specific binding ELISA and FACS with E. coli supernatants were sent to Biosune (Shanghai, China) for nucleotide sequencing of VHH gene. The sequencing results were analyzed using CLC Main Workbench (Qiagen, Hilden, Germany) .

4.6 Antibody Humanization

One VHH with high affinity and specificity to BCMA was selected for humanization, to reduce the risk of immunogenicity when used in clinical trials. “Best Fit” approach was used to humanize VHH chains. Amino acid sequences of VHH framework regions were blasted against human germline V-gene database, and humanized VHH sequences were generated by replacing human CDR sequences in the top hit with VHH CDR sequences using Kabat CDR definition. Several residues in the framework region were back mutated to VHH to maintain the affinity.

Humanized VHH genes were synthesized in GENEWIZ and expressed in BL21. After testing on BCMA binding using SPR, the variant referred to as W3566-FP20R3-1D5-z2 with proper affinity was selected as humanized antibody lead. The sequence is shown in Table C.

4.7 Affinity maturation of VHH Antibodies

To increase the affinity of W3566-FP20R3-1D5-z2 to human BCMA, each amino acid of three complementary-determining regions (CDR1, CDR2, and CDR3) of parental clone was individually mutated to other 20 amino acids using a site-directed mutagenesis method. DNA primers containing a NNS codon encoding twenty amino acids were used to introduce mutation to each targeted CDR position. The individual degenerate primers of phosphorylated were used in site-directed mutagenesis reactions. 200 ng of the reaction products was electroporated into BL21 and expressed. The point mutations in VHH determined to be beneficial for binding to antigen were further combined to gain enhanced affinity synergy. The combinatorial mutants were synthesized in GENEWIZ and expressed in BL21.

Supernatant of the mutants were detected by SPR. After affinity maturation, affinity matured VHHs showed enhanced SPR affinity compared with the parental VHH Ab and their sequences are listed in Table D.

4.8 Generation of Humanized VHH-Fc (human IgG1) fusion antibody

The clones of interest were converted to VHH-Fc (human IgG1) fusion antibodies. The VHH antibodies and VHH-Fc fusion antibodies are collectively referred to as WBP3566 antibodies herein. Briefly, the VHH genes were PCR amplified from the pET-bac vectors using VHH-specific cloning primers containing appropriate restriction sites then cloned by fusion into a modified human hIgG1 expression pcDNA3.3 vector to create corresponding clones of VHH-Fc (human IgG1) fusion antibody. 293F or Expi293 cells were transiently transfected with the vector for antibody expression. The cell culture supernatants containing antibodies were harvested and purified using Protein A chromatography.

4.9 Full kinetic binding affinity of VHH antibodies to human and monkey BCMA

WBP3566 VHH binding affinity to cyno BCMA and human BCMA was detected by SPR assay using Biacore 8K. hFc tag labeled cyno BCMA and human BCMA were captured on an anti-human IgG Fc antibody immobilized CM5 sensor chip (GE) , respectively. WBP3566 VHH samples at different concentrations were injected over the sensor chip at a flow rate of 30 uL/min for an association phase of 120 s, followed by 300-3000 s dissociation. The chip was regenerated by 10 mM glycine (pH 1.5) after each binding cycle.

The sensorgrams of blank surface and buffer channel were subtracted from the test sensorgrams. The experimental data was fitted by 1: 1 model using Langmiur analysis. Molecular weight of 15 kDa was used to calculate the molar concentration of WBP3566 VHH samples. The binding affinities of WBP3566 VHH antibodies to human and monkey BCMA are shown in Table 7.

Table 7A. Full kinetic binding affinity of anti-BCMA VHH to cyno and human BCMA.

Table 7B. Full kinetic binding affinity of benchmark antibody to cyno and human BCMA.

4.10 Binding of W3566 VHH-Fc (human IgG1) fusion antibodies to cell surface BCMA

Serial dilutions of testing antibodies and isotype control antibodies were incubated with human NCI-H929 cells or cyno BCMA transfected cells, respectively, then the binding of antibodies to the cell surface BCMA was detected by Goat Anti-Human IgG Alexa Fluor647 (Jackson, cat: 109-605-098) .

Figures 3A and 3B show that W3566 VHH-Fc (human IgG1) antibodies bind to cell surface human BCMA and cyno BCMA. The binding EC50 of W3566 antibodies to NCI-H929 cells are 2.8-5.9 nM and to cyno BCMA transfected cells are 1.2-1.4 nM.

4.11 Thermal stability by DSF assay

A DSF assay was performed using 7500 Fast Real-Time PCR system (Applied Biosystems) . Briefly, 19 μL of antibody solution in PBS was mixed with 1 μL of 62.5 X SYPRO Orange solution (Invitrogen) and added to a 96 well plate (Biosystems) . The plate was heated from 26 ℃ to 95 ℃ at a rate of 2 ℃/min, and the resulting fluorescence data were collected. The negative derivatives of the fluorescence changes with respect to different temperatures were calculated, and the maximal value was defined as melting temperature Th. If a protein has multiple unfolding transitions, the first two Th were reported, named as Th1 and Th2. Th1 is melting temperature Tm for comparisons between different proteins. Data collection and Th calculation were conducted automatically by its operation software. Once the plot of negative derivatives of different temperatures was reported by the software, the point in the plot where the curve starts to decrease from a pre-transition baseline could be roughly estimated as the onset temperature Ton.

The result indicates that W3566 Abs have normal DSF profiles and the Th1 (Tm) are 62.5～68.0 (Table 8) .

Table 8. Thermal stability (Tm) by DSF assay.

Example 5: Generation of multi-specific antibodies and in vivo Characterization

5.1 Generation of WT107-BMK3

An anti-CD3 × HSA × BCMA antibody (trispecific antibody of Harpoon, Seq ID No. 520 of patent WO2019075359A1) was used as a benchmark antibody and designated as WT107-BMK3 herein. WT107-BMK3 comprises an anti-CD3 scFv, an anti-HSA (human serum albumin) VHH and an anti-BCMA VHH operably linked in a single polypeptide chain. DNA sequence encoding full length of the anti-CD3 × HSA × BCMA antibody was synthesized in GENEWIZ (SuZhou, China) , and then subcloned into a modified pcDNA3.3 expression vector.

The plasmids were transfected into Expi293 cells. Cells were cultured for 5 days and supernatant was collected for protein purification using Ni-NTA column (GE Healthcare, 175438) and/or SEC column (GE Healthcare, 28990944) . The obtained antibody was analyzed by SDS-PAGE and SEC, and then stored at -80 ℃.

5.2 Generation of WT1070-cAb1

DNA sequences encoding anti-CD3 scFv and anti-BCMA VHH of WT107-BMK3 were amplified by PCR, and then the anti-CD3 scFv and anti-BCMA VHH were fused with a C-terminal HisX6 tag and cloned into a modified pcDNA3.3 expression vector.

5.3 Generation of WT1070-cAb2

WT1070-cAb2 was generated by replacing the anti-HSA VHH of WT107-BMK3 with T6016-Z5-m2. The DNA sequence encoding the anti-HSA VHH of WT107-BMK3 was replaced by the DNA sequence encoding T6016-Z5-m2, and then the DNA encoding the anti-CD3 × HSA × BCMA antibody with a C-terminal HisX6 tag was subcloned into a modified pcDNA3.3 expression vector.

5.4 Generation of WT1070-T3W3U32. D26-3. His

WT1070-T3W3U32. D26-3. His was constructed as follows: Anti-CD3 scFv (VH and VL from an anti-CD3 monoclonal antibody) , anti-HSA VHH (T6016-z5-m2) and anti-BCMA VHH (W3566-FP20R3-1D5-z2-m17) were fused by linkers, with a C-terminal HisX6 tag. And then DNA sequence encoding the antibody was cloned into modified pcDNA3.3 expression vector.

The plasmids of the trispecific antibody were transfected into Expi293 cells. Cells were cultured for 5 days and supernatant was collected for protein purification using Ni-NTA column (GE Healthcare, 175248) and/or SEC column (GE Healthcare, 28990944) . The obtained antibody was analyzed by SDS-PAGE and HPLC-SEC, and then stored at -80 ℃.

5.5 Mouse pharmacokinetics study

To determine if conjugation with TAD6016 VHHs would prolong the half-life of a protein of small size, a mouse PK study was investigated.

PK study method

96 healthy female C57BL/6 mice were used in the study. Animals were allowed to acclimate for at least 7 days in the specific-pathogen-free animal care unit before use. The animal facility is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care and the animal experiment/procedure was approved by the Institutional Animal Care and Use Committee.

In this study, 4 antibodies were evaluated. The antibodies for administration were prepared at specific concentration of dose administered in PBS. The antibodies were injected into the tail vein using a 25-G needle attached syringe at the following concentrations: WT107-BMK3 at 5.3 and 0.53 mg/kg, WT1070-cAb1 at 4.1 and 0.41 mg/kg, WT1070-cAb2 at 5.4 and 0.54 mg/kg, WT1070-T3W3U32. D26-3. His at 5.5 and 0.55 mg/kg with a dose volume of 10 mL/kg. At the predetermined time points before and after the intravenous dosing (15 and 30 mins, 1, 2, 6, 24, 48, 72, 120, 168, and 240h) , the mice were briefly anesthetized with the inhalation anesthetic Isoflurane. About 30 microliter of blood was collected via the dorsal or lateral side of the mouse orbital sinus using a micro-capillary glass tube. The blood was then transferred in a tube coated with EDTA-K2, which was centrifuged within 10 min after its collection at 8 000 rpm for 5 min. The top layer of plasma was transferred into a new set of tubes and stored at -30 ℃ until analysis. The results from 6 mice were collected for each data point.

The concentrations of antibody in plasma were determined by using a bioanalytical ELISA method. Briefly, 96-well ELISA plates were coated overnight at 4℃ with Recombinant Human CD3e (sino-10977-H08H) in carbonate-bicarbonate buffer (Thermo-28382) as capturing antibody. After washing and blocking, serial diluted plasma samples were added and then Biotin labeled human BCMA ECD protein with human Fc tag (hBCMA. hFc. AVI. Biotin, CD3+BCMA method) were used as detection antibody. HRP-streptavidin (Thermo-21127) and TMB (life technologies-002023) substrate were used for color development. The reaction was stopped after approximate 5～10 minutes through the addition of 2M HCl. The absorbance was read at 450 nm and 540 nm using a microplate spectrophotometer (M5e) . The OD value of the samples were substituted into the standard curve to obtain the plasma antibody concentration.

The plasma concentration of WT1070 antibodies in mouse was subjected to a non-compartmental pharmacokinetic data analysis by using the Phoenix WinNonlin software (version 8.1, Pharsight, Mountain View, CA) .

Table 9. PK parameters of antibodies in mice.

T_1/2: half life; Cl_obs (ml/day/kg) : average serum clearance; Vss: volume of distribution

The result shows that T6016-z5-m2 VHH conjugation in WT1070-T3W3U32. D26-3. His prolongs the T1/2 of the therapeutic protein to 30-40 hours in mice, similar to the natural half-life of mouse albumin. Similar T1/2 prolongation is observed for conjugation with T6016-z5-m2 VHH in the result of WT1070-cAb2.

5.6 In vivo antitumor efficacy study

To determine the efficacy of multispecific antibody containing TAD6016 VHHs, an NCI-H929/hPBMC mouse efficacy study was conducted.

A new trispecific antibody WT1070-U31W2T3. D5-2. His was generated. WT1070-U31W2T3. D5-2. His comprises an anti-CD3 scFv (derived from an anti-CD3 monoclonal antibody) , anti-albumin VHH T6016-P1R2-6D10-z5-34G9 and anti-BCMA VHH (W3566-FP20R3-1D5-z2-m52, also named as W3566-FP20R3-1D5-z2-4F10) operably linked together in a single polypeptide chain. The amino acid sequence of WT1070-U31W2T3. D5-2. His can be found in Table G.

Construction of WT1070-U31W2T3. D5-2. His was conducted using molecular biology protocol. Anti-CD3 scFv, anti-HSA VHH and anti-BCMA VHH were fused by linkers, with a C-terminal HisX6 tag. And then DNA coding region was cloned into modified pcDNA3.3 expression vector.

Method of the efficacy study

The efficacy study was tested in NCI-H929/hPBMC model in M-NSG female mice. Female M-NSG mice (Shanghai Model Organisms Center, Inc. ) of 12 week-old were used in the study. NCI-H929 cells were cultured in RPMI-1640 medium supplemented with 10%fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin and 0.05 mM 2-mercaptoethanol at 37 ℃ in an atmosphere of 5%CO₂. Tumor cells were harvested and counted followed by tumor inoculation.

Each mouse was inoculated subcutaneously at the right flank with NCI-H929 tumor cells (5.0×10⁶ cells in 100μL RPMI-1640 medium without FBS) , and was injected intraperitoneally with hPBMC cells (5.0×10⁶ cells in 200μL RPMI-1640 medium without FBS) at the same day. When the average tumor volume reached approximately to 170 mm³, animals were randomly grouped into 4 groups and each group contained 6 mice. The 4 groups of mice received the following intravenous injections every day for a total of 10 injections: G1 vehicle-PBS; G2 0.18 mg/kg of WT1070-U31W2T3. D5-2. His; G3 1.2 mg/kg of WT1070-U31W2T3. D5-2. His; G4 7.2 mg/kg of WT1070-U31W2T3. D5-2. His.

The first injection day was defined as day 0. Mice were weighed and tumor growth was measured twice a week using calipers. All the procedures related to animal handling, care and the treatment in the study were performed according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Model Organisms Center, Inc. Tumor volume was calculated with the formula (1/2 (length × width²) . The results were represented by mean and the standard error (Mean ± SEM) . Data were analyzed using Two way RM ANOVA test with GraphPad Prism 6.0 and p<0.05 was considered to be statistically significant. TGI (tumor growth inhalation) was calculated for each group using the formula: TGI (%) = [1- (Ti-T0) / (Vi-V0) ] ×100. Ti is the average tumor volume of a treatment group on a given day. T0 is the average tumor volume of the treatment group on the first day of treatment. Vi is the average tumor volume of the vehicle control group on the same day with Ti and V0 is the average tumor volume of the vehicle group on the first day of treatment.

One mouse in G2 WT1070-U31W2T3. D5-2. His (0.18mg/kg) group had exhibited some physical symptoms of GVHD and died during the experiment.

As shown in Figure 4, the mean tumor volume of G1 vehicle group was 2509.80 mm³ at day 17, which indicated NCI-H929/hPBMC model was well established. WT1070-U31W2T3. D5-2. His showed tumor growth inhibition in a dose response manner. Compared with vehicle group, G3 and G4 groups showed significant tumor growth inhibition (p value: p<0.001 by 2 way ANOVA of G1 vs G3, P< 0.0001 by 2 way ANOVA of G1 vs G4) , while G2 did not show significant tumor growth inhibition (p value: ns by 2 way ANOVA of G1 vs G2) . The TGI at day 17 of each group was 2.77%for G2, 31.03%for G3, 99.13%for G4.

Those skilled in the art will further appreciate that the present disclosure may be embodied in other specific forms without departing from the spirit or central attributes thereof. In that the foregoing description of the present disclosure discloses only exemplary embodiments thereof, it is to be understood that other variations are contemplated as being within the scope of the present disclosure. Accordingly, the present disclosure is not limited to the particular embodiments that have been described in detail herein. Rather, reference should be made to the appended claims as indicative of the scope and content of the disclosure.

Claims

A albumin-binding molecule comprising an immunoglobulin single variable domain, wherein the single variable domain comprises a CDR1, a CDR2 and a CDR3 same as those from a VHH as set forth in any of SEQ ID NOs: 7-12.
The albumin-binding molecule of claim 1, wherein the single variable domain comprises:

a CDR1 comprising the amino acid sequence of GRAX₁SSYA, wherein X₁ is F or P;

a CDR2 comprising the amino acid sequence of VARIGDTT; and

a CDR3 comprising the amino acid sequence of AGGQTIAVYX₂TPNMYTX₃, wherein X₂ is D or S, X₃ is Y or A.
The albumin-binding molecule of claim 1, wherein the single variable domain comprises:

(A) a CDR1 comprising SEQ ID NO: 1, a CDR2 comprising SEQ ID NO: 2, a CDR3 comprising SEQ ID NO: 3;

(B) a CDR1 comprising SEQ ID NO: 1, a CDR2 comprising SEQ ID NO: 2, a CDR3 comprising SEQ ID NO: 5; or

(C) a CDR1 comprising SEQ ID NO: 4, a CDR2 comprising SEQ ID NO: 2, a CDR3 comprising SEQ ID NO: 6.
The albumin-binding molecule of claim 1 or 2, wherein the single variable domain comprises:

(A) the amino acid sequence as set forth in any one of SEQ ID NOs: 7-12; or

(B) an amino acid sequence at least 85%, 90%, or 95%identical to the amino acid sequence as set forth in any one of SEQ ID NOs: 7-12.
The albumin-binding molecule of any of the preceding claims, wherein the single variable domain is a VHH, such as a humanized VHH domain, an affinity maturated VHH domain, and a human VHH domain.
The albumin-binding molecule of any of the preceding claims, wherein the single variable domain specifically binds to any of human, mouse, cynomolgus, feline and canine serum albumins.
The albumin-binding molecule of any of the preceding claims, comprising the single variable domain fused to a heterogenous protein.
The albumin-binding molecule of any of the preceding claims, wherein the heterogenous protein is an antigen-binding moiety in the format of an antibody or an antigen-binding fragment thereof, such as a Fab, a VHH, or a scFv.
An isolated nucleic acid molecule, comprising a nucleic acid sequence encoding the albumin-binding molecule of any of claims 1-8.
A vector comprising the isolated nucleic acid molecule of claim 9.
A host cell comprising the nucleic acid molecule of claim 9 or the vector of claim 10.
A pharmaceutical composition comprising the albumin-binding molecule of any of claims 1-8 and a pharmaceutically acceptable carrier.
A method for producing the albumin-binding molecule of any of claims 1-8, comprising:

expressing the albumin-binding molecule in a host cell comprising a nucleic acid sequence encoding the fusion protein; and

isolating the albumin-binding molecule from the host cell.
A BCMA-binding molecule comprising an immunoglobulin single variable domain, wherein the single variable domain comprises a CDR1, a CDR2 and a CDR3 same as those from a VHH as set forth in any of SEQ ID NOs: 44-51.
The BCMA-binding molecule of claim 14, wherein the single variable domain comprises:

a CDR1 comprising the amino acid sequence of GSIX₄SINA, wherein is D or W;

a CDR2 comprising the amino acid sequence of ISSGGFP; and

a CDR3 comprising the amino acid sequence of NAEX₅VWGGX₆IYNY, wherein X₅ is R or E, X₆ is K or R.
The BCMA-binding molecule of claim 15, wherein the single variable domain comprises:

(A) a CDR1, CDR2 and CDR3 comprising the amino acid sequence of SEQ ID Nos: 36, 37 and 38, respectively;

(B) a CDR1, CDR2 and CDR3 comprising the amino acid sequence of SEQ ID Nos: 36, 37 and 40, respectively;

(C) a CDR1, CDR2 and CDR3 comprising the amino acid sequence of SEQ ID Nos: 36, 37 and 41, respectively;

(D) a CDR1, CDR2 and CDR3 comprising the amino acid sequence of SEQ ID Nos: 43, 37 and 40, respectively;

(E) a CDR1, CDR2 and CDR3 comprising the amino acid sequence of SEQ ID Nos: 43, 37 and 41, respectively; or

(F) a CDR1, CDR2 and CDR3 comprising the amino acid sequence of SEQ ID Nos: 43, 37 and 38, respectively.
The BCMA-binding molecule of any of claims 14-16, wherein the single variable domain comprises:

(A) the amino acid sequence as set forth in any one of SEQ ID NOs: 44-51; or

(B) an amino acid sequence at least 85%, 90%, or 95%identical to the amino acid sequence as set forth in any one of SEQ ID NOs: 44-51.
The BCMA-binding molecule of any of claims 14-17, wherein the single variable domain is a VHH, such as a humanized VHH domain, an affinity maturated VHH domain, and a human VHH domain.
The BCMA-binding molecule of any of claims 14-18, wherein the single variable domain specifically binds to any of human, mouse and cynomolgus BCMAs.
The BCMA-binding molecule of any of claims 14-19, comprising the single variable domain fused to a heterogenous protein, such as an immunoglobulin IgG constant region, including IgG1, IgG2, IgG3 or IgG4 Fc region.
The BCMA-binding molecule of claim 20, wherein the heterogenous protein is an antigen-binding moiety in the format of an antibody or an antigen-binding fragment thereof, such as a Fab, a VHH, or a scFv.
An isolated nucleic acid molecule, comprising a nucleic acid sequence encoding the BCMA binding molecule of any of claims 14-21.
A vector comprising the isolated nucleic acid molecule of claim 22.
A host cell comprising the nucleic acid molecule of claim 22 or the vector of claim 23.
A pharmaceutical composition comprising the BCMA binding molecule of any of claims 14-21 and a pharmaceutically acceptable carrier.
A method for producing the BCMA binding molecule of any of claims 14-21, comprising:

expressing the BCMA binding molecule in a host cell comprising a nucleic acid sequence encoding the fusion protein; and

isolating the BCMA binding molecule from the host cell.
A method for modulating a BCMA related immune response in a subject, comprising administering to the subject the BCMA binding molecule of any of claims 14-21 or the pharmaceutical composition of claim 25 to the subject.
A method for depleting B cells (such asB cells, memory B cells, and plasma cells) in a subject, comprising administering to the subject the BCMA-binding molecule as defined in any of claims 14-21 to the subject.
A method for preventing or treating B cell-related disorders in a subject, comprising administering an effective amount of the BCMA-binding molecule as defined in any of claims 14-21 to the subject.
The method of claim 29, wherein the B cell-related disorder is selected from cancers and immune disorders.
The method of claim 30, wherein the cancer is lymphoma selected from multiple myeloma, plasmacytoma, Hodgkins' lymphoma, follicular lymphomas, small non-cleaved cell lymphomas, endemic Burkitt's lymphoma, sporadic Burkitt's lymphoma, marginal zone lymphoma, extranodal mucosa-associated lymphoid tissue lymphoma, nodal monocytoid B cell lymphoma, splenic lymphoma, mantle cell lymphoma, large cell lymphoma, diffuse mixed cell lymphoma, immunoblastic lymphoma, primary mediastinal B cell lymphoma, pulmonary B cell angiocentric lymphoma, and small lymphocytic lymphoma.
The BCMA-binding molecule of any of claims 14-21 for use in treating or preventing B cell-related disorders in a subject.
A kit, comprising a container comprising the BCMA-binding molecule of any of claims 14-21.