CN117157322A - Binding agents targeting tumor and/or immune cells - Google Patents

Binding agents targeting tumor and/or immune cells Download PDF

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Publication number
CN117157322A
CN117157322A CN202280025997.9A CN202280025997A CN117157322A CN 117157322 A CN117157322 A CN 117157322A CN 202280025997 A CN202280025997 A CN 202280025997A CN 117157322 A CN117157322 A CN 117157322A
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amino acid
acid sequence
seq
binding agent
domain
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Inventor
姚书刚
R·沃加查克
侯文洋
L·达克鲁兹
D·S·杨
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Canadian Shang Mu Zeng Road Biotechnology Co ltd
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Canadian Shang Mu Zeng Road Biotechnology Co ltd
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Priority claimed from PCT/CA2022/050442 external-priority patent/WO2022204793A1/en
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Abstract

The present invention relates generally to binding agents capable of targeting tumor cells and/or immune cells. The binding agents of the invention comprise one or more antigen binding domains of a heavy chain antibody (HcAb) capable of binding to DR2, to PD-1 and/or to CD 47. The binding agents of the invention may be in the form of monomers or multimers, which may be monospecific or multispecific.

Description

Binding agents targeting tumor and/or immune cells
Technical Field
The present invention relates generally to binding agents capable of targeting tumor cells and/or immune cells. The binding agents of the invention comprise one or more antigen binding domains of a VHH antibody capable of binding to DR2, to PD-1 and/or to CD 47. The binding agents of the invention may be in the form of monomers or multimers, and may be monospecific or multispecific.
Background
Camels and cartilaginous fish naturally produce antibodies composed of only functional homodimeric heavy chain antibodies (HCAb) (Hamers-Casterman et al, 1993; muydermans and Smider, 2016). The heavy chain of HCAb lacks the first constant domain (CH 1) and differs from classical antibodies only in some of the amino acid substitutions normally involved in light chain pairing (Muyldermans et al, 1994; vu et al, 1997). These substitutions (Val 37Phe/Tyr, gly44Glu, leu45Arg and Trp47 Gly) are present in framework region 2 (FR 2). Antigen binding fragments of HCAb are referred to as VHH or VHH has a molecular weight of about 15kDa, which makes it suitable for applications requiring enhanced tissue penetration or rapid clearance, such as radioisotope-based imaging. However, for therapeutic applications, VHH half-life is often required to be extended in order to minimize renal clearance and optimize therapeutic efficacy (De Vlieger et al, antibodies 8 (1), 1-22,2019). Although methods have been utilized to extend VHH half-life, such as pegylation, N-glycosylation, HSA or other carrier protein infusion, such constructs may introduce immunogenicity or have limited success.
VHH have been utilized as building blocks for the production of bispecific and multispecific antibodies. In some studies, bivalent constructs showed a comparison to monovalent formsIncreased affinity or avidity (establishment et al, 2001; copbieters et al, 2006; hmila et al, 2008; simmons et al, 2006 and Hultberg et al, 2011,and (2010), fridy et al, 2014).
Some VHH-based therapeutics are currently in the late stage of the study or have been approved by the FDA. These therapeutic agents include the bivalent monospecific antibody capecitabine (cappucizumab) against the antigen vWF approved for thrombocytopenic purpura (Duggan, 2018). Trivalent nanobody complex ALX-0171 against RSV is in the late stage of development for respiratory syncytial virus infection (Detallea et al 2015). ALX-0061 is monovalent against the antigen IL-6R but linked to HSA nanobodies to extend half-life, which is in the clinical development stage, for RA and SLE indications (Van Roy et al 2015). The research drug ALX-0761 contains three nanobodies against the antigens IL-17A, IL-17F and HAS and is being developed for psoriasis (Svecova et al, 2019). anti-RANKL ALX-0141 is bivalent, against the antigen RANKL, which is linked to HSA to extend half-life (Schoen et al, 2013). Oxolablab (Ozoralizumab) is a bivalent nanobody, directed against the antigen tnfα, which is linked to HSA to extend half-life (Fleischmann et al 2012).
Applicants have developed novel binding agents containing one or more antigen binding domains that target tumor cells and/or immune cells.
Disclosure of Invention
The present invention relates generally to binding agents capable of targeting tumor cells and/or immune cells and the like.
In some embodiments, the binding agents of the invention are capable of binding to DR2, binding to PD-1, and/or binding to CD 47. In other embodiments of the invention, the binding agent may bind to DR 2-expressing cells, to PD-1-expressing cells, and/or to CD 47-expressing cells.
In some embodiments, the DR 2-expressing cell comprises a tumor cell.
In some embodiments, the PD-1 expressing cell comprises an immune cell.
In some embodiments, the CD 47-expressing cells comprise immune cells or tumor cells.
Thus, in some embodiments, the binding agent may comprise an antigen binding domain capable of binding to DR2 or to a cell expressing DR2, an antigen binding domain capable of binding to PD-1 or to a cell expressing PD-1, and/or an antigen binding domain capable of binding to CD47 or to a cell expressing CD 47. According to the invention, the binding agent may also comprise an additional antigen binding domain.
In some embodiments, the antigen binding domain capable of binding to DR2 or to a cell expressing DR2 is antigen binding domain 1 (ABD 1) as described herein. In some embodiments, the antigen binding domain capable of binding to DR2 or to a cell expressing DR2 is not limited to antigen binding domain 1 (ABD 1).
Thus, in some embodiments, the binding agent may comprise at least one antigen binding domain 1 (ABD 1).
In some embodiments, the binding agent may comprise at least one antigen binding domain 1 (ABD 1) and at least one other antigen binding domain.
In some cases, the binding agent may comprise at least one antigen binding domain 1 (ABD 1), and may also bind to immune cells. Thus, in some embodiments, the binding agent comprises at least one antigen binding domain 1 (ABD 1), and at least one antigen binding domain that binds to an immune cell.
In some embodiments, the antigen binding domain capable of binding to PD-1 or to a cell expressing PD-1 is antigen binding domain 2 (ABD 2) as described herein. In some embodiments, an antigen binding domain capable of binding to PD-1 or to a cell expressing PD-1 is not limited to antigen binding domain 2 (ABD 2).
Thus, in some embodiments, the binding agent may comprise at least one antigen binding domain 2 (ABD 2).
In some embodiments, the binding agent may comprise at least one antigen binding domain 2 (ABD 2) and at least one other antigen binding domain.
In some cases, the binding agent may comprise at least one antigen binding domain 2 (ABD 2), and may also bind to tumor cells. Thus, in some embodiments, the binding agent comprises at least one antigen binding domain 2 (ABD 2), and at least one antigen binding domain that binds to a tumor cell.
In some embodiments, the antigen binding domain capable of binding to CD47 or to a cell expressing CD47 is antigen binding domain 3 (ABD 3) as described herein. In some embodiments, the antigen binding domain capable of binding to CD47 or to a cell expressing CD47 is not limited to ABD3.
Thus, in some embodiments, the binding agent may comprise at least one antigen binding domain 3 (ABD 3).
In some embodiments, the binding agent may comprise at least one antigen binding domain 3 (ABD 3) and at least one other antigen binding domain.
In some cases, the binding agent may comprise at least one antigen binding domain 3 (ABD 3), and may also bind to tumor cells. Thus, in some embodiments, the binding agent comprises at least one antigen binding domain 3 (ABD 3), and at least one antigen binding domain that binds to a tumor cell. In other examples, the binding agent may comprise at least one antigen binding domain 3 (ABD 3), and may also bind to immune cells. In some embodiments, the binding agent comprises at least one antigen binding domain 3 (ABD 3), and at least one antigen binding domain that binds to an immune cell.
In certain instances, the binding agent is capable of binding to DR2 and to PD-1. Thus, in some embodiments, the binding agent comprises at least one antigen binding domain that binds DR2 and at least one antigen binding domain that binds PD-1.
In certain instances, the binding agent is capable of binding DR2 and CD 47. Thus, in some embodiments, the binding agent comprises at least one antigen binding domain that binds DR2, and at least one antigen binding domain that binds CD 47.
In certain instances, the binding agent is capable of binding to DR2, PD-1, and to CD 47. Thus, in some embodiments, the binding agent comprises at least one antigen binding domain that binds DR2, at least one antigen binding domain that binds PD-1, and at least one antigen binding domain that binds CD 47.
In certain instances, the binding agent is capable of binding to PD-1 and to CD 47. Thus, in some embodiments, the binding agent comprises at least one antigen binding domain that binds to PD-1 and at least one antigen binding domain that binds to CD 47.
In certain instances, the binding agent is capable of binding to PD-1, to CD47, and to tumor cells. Thus, in some embodiments, the binding agent comprises at least one antigen binding domain that binds to PD-1, at least one antigen binding domain that binds to CD47, and at least one antigen binding domain that binds to tumor cells.
In certain instances, the binding agent is capable of binding to PD-1 and to tumor cells. Thus, in some embodiments, the binding agent comprises at least one antigen binding domain that binds to PD-1 and at least one antigen binding domain that binds to a tumor cell.
In certain instances, the binding agent is capable of binding to CD47 and to tumor cells. Thus, in some embodiments, the binding agent comprises at least one antigen binding domain that binds CD47 and at least one antigen binding domain that binds tumor cells.
In certain instances, the binding agent is capable of binding to CD47 and to immune cells. Thus, in some embodiments, the binding agent comprises at least one antigen binding domain that binds to CD47, and at least one antigen binding domain that binds to immune cells.
In some embodiments, the binding agent comprises one or more antigen binding domains, wherein at least one of the antigen binding domains is antigen binding domain 1 (ABD 1).
In some embodiments, the binding agent comprises one or more antigen binding domains, wherein at least one of the antigen binding domains is antigen binding domain 2 (ABD 2).
In some embodiments, the binding agent comprises one or more antigen binding domains, wherein at least one of the antigen binding domains is antigen binding domain 3 (ABD 3).
In some embodiments, the binding agent comprises one or more antigen binding domains, wherein at least one of the antigen binding domains is selected from the group consisting of:
antigen binding domain 1 (ABD 1),
antigen binding domain 2 (ABD 2) or
Antigen binding domain 3 (ABD 3).
In some embodiments, the binding agent may comprise an antigen binding domain that targets an epitope other than the epitope covered by ABD1, ABD2, and ABD 3. Thus, the binding agent may comprise an antigen binding domain having the same or different specificity as ABD1, ABD2, and/or ABD 3.
In some embodiments, the binding agent may comprise more than one antigen binding domain.
For example, in some embodiments, the binding agent may comprise two or more antigen binding domains, three or more antigen binding domains, four or more antigen binding domains, five or more antigen binding domains, six or more antigen binding domains, seven or more antigen binding domains, eight or more antigen binding domains, nine or more antigen binding domains, ten or more antigen binding domains.
In some embodiments, the binding agent may comprise between one and twelve antigen binding domains.
In some embodiments, the binding agents of the invention may be monospecific.
In some embodiments, the binding agents of the invention may be multispecific.
In some embodiments, the binding agents of the invention may be monovalent.
In some embodiments, the binding agents of the invention may be multivalent.
In some embodiments, the binding agents of the present invention may be in the form of monomers.
In some embodiments, the binding agents of the invention may be in dimeric or higher order forms, such as trimers, tetramers, pentamers, and the like (e.g., multimers).
In some cases, the antigen binding domain of the binding agent is derived from a heavy chain antibody. In some cases, the heavy chain antibodies may be obtained by immunization of camelidae or transgenic animals.
In some embodiments, the binding agent comprises at least one antigen binding domain that binds DR2 and comprises a complementarity determining region as described herein.
In some embodiments, the binding agent comprises at least one antigen binding domain that binds to PD-1 and comprises a complementarity determining region as described herein.
In some embodiments, the binding agent comprises at least one antigen binding domain that binds CD47 and comprises a complementarity determining region as described herein.
In some embodiments, the antigen binding domain is on one or more polypeptide chains.
In some embodiments, the antigen binding domain antigens are on the same polypeptide chain.
In some embodiments, the binding agent comprises a single polypeptide chain.
In some embodiments, the binding agent comprises two polypeptide chains. Thus, the binding agent may be in the form of a dimer.
In some embodiments, the binding agent comprises more than two polypeptide chains, such as three or more polypeptide chains, four or more polypeptide chains, five or more polypeptide chains, six or more polypeptide chains, seven or more polypeptide chains, eight or more polypeptide chains, nine or more polypeptide chains, ten or more polypeptide chains. Thus, the binding agent may be in the form of a multimer. For example, a binding agent comprising three polypeptide chains is referred to herein as a trimer, a binding agent comprising four polypeptide chains is referred to herein as a tetramer, and the like.
In some embodiments, the binding agent comprises at least two polypeptide chains capable of assembling to form a dimer, and wherein each polypeptide chain comprises one or more antigen binding domains.
In some embodiments, the two polypeptide chains are capable of assembling to form a dimer, and each polypeptide chain comprises a different antigen binding domain.
In some embodiments, the two polypeptide chains are capable of assembling to form a dimer, and each polypeptide chain comprises the same antigen binding domain.
In some embodiments, each polypeptide chain comprises the same antigen binding domain 1 (ABD 1).
In some embodiments, each polypeptide chain comprises the same antigen binding domain 2 (ABD 2).
In some embodiments, each polypeptide chain comprises the same antigen binding domain 3 (ABD 3).
In some exemplary embodiments, the binding agent comprises at least two polypeptide chains that assemble to form a dimer, and each polypeptide chain comprises the same antigen binding domain 1 (ABD 1) and the same antigen binding domain 2 (ABD 2).
In some other exemplary embodiments, the binding agent comprises at least two polypeptide chains that assemble to form a dimer, and each polypeptide chain comprises the same antigen binding domain 1 (ABD 1) and the same antigen binding domain 3 (ABD 3).
In further exemplary embodiments, the binding agent comprises at least two polypeptide chains that assemble to form a dimer, and each polypeptide chain comprises the same antigen binding domain 2 (ABD 2) and the same antigen binding domain 3 (ABD 3).
In yet further exemplary embodiments, the binding agent comprises at least two polypeptide chains that assemble to form a dimer, and each polypeptide chain comprises the same antigen binding domain 1 (ABD 1), the same antigen binding domain 2 (ABD 2), and the same antigen binding domain 2 (ABD 3).
In some cases, the antigen binding domain 1 (ABD 1), antigen binding domain 2 (ABD 2), and antigen binding domain 2 (ABD 3) may occupy the same position in each of the two polypeptide chains.
In some cases, the antigen binding domain 1 (ABD 1), antigen binding domain 2 (ABD 2), and antigen binding domain 2 (ABD 3) may occupy different positions in each of the two polypeptide chains.
In some embodiments, each polypeptide chain of the binding agent is the same.
In some embodiments, each polypeptide chain of the binding agent is different.
In some embodiments, the two polypeptide chains are capable of assembling to form a homodimer.
In some embodiments, the two polypeptide chains are capable of assembling to form a heterodimer.
In some embodiments, the antigen binding domains are separated by an amino acid sequence.
In some embodiments, the amino acid sequence is a linker.
The binding agents of the invention encompass, for example, an antigen binding domain as disclosed herein, a single polypeptide chain as disclosed herein, a dimer of a polypeptide chain as disclosed herein, or a multimer of a polypeptide chain as disclosed herein.
Thus, the binding agent of the invention may have the following pattern: antibodies and antigen binding fragments thereof, antibody-like molecules (Fc-, CH 3-fusions, etc.), fusions with protein scaffolds, immune cell modulators, etc.
Advantageously, the binding agents of the present invention may have the formula Ia, formula Ib, formula Ic, formula II, formula III, formula IV, formula V, formula VI, formula VII or formula VIII and similar versions thereof or the versions of formula I, formula II, formula III, formula IIIa and formula IIIb, formula IV, formula V, formula VI, formula VII or formula VIII disclosed herein as disclosed in PCT/CA2020/051753 filed on 18 th month of 2020, the entire contents of which are incorporated herein by reference.
In some embodiments, the binding agent consists of a polypeptide chain comprising one or more antigen binding domains and a dimerization domain that allows at least two polypeptide chains to form a dimer.
In some embodiments, the binding agents of the invention may comprise one or more polypeptide chains, each independently comprising in N-terminal to C-terminal fashion an amino acid sequence of formula I:
X-[(Ab a )-(L b )] m -(DD)-[(L c )-(Ab d )] n -Y
wherein m is 0, 1, 2 or an integer greater than 2;
wherein n is 0, 1, 2 or an integer greater than 2;
wherein m and n are not both 0;
wherein Ab a 、Ab d Each represents an antigen binding domain, wherein at least one of the antigen binding domains is antigen binding domain 1 (ABD 1), antigen binding domain 2 (ABD 2), or antigen binding domain 3 (ABD 3);
wherein X or Y is independently present or absent and comprises an amino acid sequence;
wherein L is b 、L c Each independently comprising one or more linkers; and is also provided with
Wherein DD represents the dimerization domain.
In some embodiments, the polypeptide chain comprises two or more antigen binding domains, three or more antigen binding domains, four or more antigen binding domains, five or more antigen binding domains, six or more antigen binding domains, and the like.
In some embodiments, the polypeptide chain comprises 1-12 antigen binding domains.
In some embodiments, the binding agent comprises one polypeptide chain.
In some embodiments, the binding agent comprises two polypeptide chains, three polypeptide chains, four polypeptide chains, five polypeptide chains, six polypeptide chains, seven polypeptide chains, eight polypeptide chains, nine polypeptide chains, ten polypeptide chains, or more than ten polypeptide chains.
In some embodiments, the polypeptide chains may be covalently linked.
In some embodiments, the polypeptide chains may be non-covalently linked.
In some embodiments, the polypeptide chains may associate via electrostatic interactions.
In some embodiments wherein m is 2 or an integer greater than 2, [ (Ab) a )-(L b )]The units are identical or different.
In some embodiments wherein n is 2 or an integer greater than 2, [ (L) c )-(Ab d )]The units are identical or different.
In some embodiments, when m is 2 or an integer greater than 2, each Ab a Are the same or different.
In some embodiments, when n is 2 or an integer greater than 2, each Ab d Are the same or different.
In some embodiments, ab a Representing ABD1, ABD2, or ABD3.
In some embodiments, ab d Representing ABD1, ABD2, or ABD3.
In some embodiments, ab d Representing ABD1, ABD2, or ABD3.
In some embodiments, the one or more polypeptide chains further comprise a hinge region of an antibody or antigen binding fragment thereof. For example, in some embodiments, L b Is the hinge region of an antibody or antigen binding fragment thereof.
In some embodiments, the hinge region is a native hinge region from IgG1, igG2, igG3, or IgG 4.
In some embodiments, the hinge region is a mutant hinge region having at least 70% identity to a native hinge region from IgG1, igG2, igG3, or IgG 4.
In some embodiments, each of the one or more linkers independently has a length of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid residues.
In some embodiments, each of the one or more connectors is independently a flexible connector, a screw connector, or a rigid connector.
In some embodiments, the flexible linker is a GS linker. In some embodiments, the flexible linker comprises one or more GGGGS units as described herein.
In some embodiments, the rigid linker comprises a plurality of PA repeat sequences as described herein.
In some embodiments, the helical linker comprises one or more EAAAK units as described herein.
In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains comprises formula II: x- (Ab) a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 ) Y (formula II).
In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains comprises formula III: x- (Ab) a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 ) Y (formula III).
In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains comprises formula IV: x- (Ab) a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 ) Y (formula IV).
In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains comprises formula V: x- (Ab) a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 ) Y (formula V).
In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains comprises formula VI: x- (Ab) a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-(L c3 )-(Ab d3 ) Y (formula VI).
In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains comprises formula VII: x- (Ab) a1 )-(L b3 )-(Ab a2 )-(L b2 )-(Ab a3 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 ) Y (formula VII).
In some embodiments, the binding agent comprises one or more ofA plurality of polypeptide chains, wherein at least one of the polypeptide chains comprises formula VIII: x- (Ab) a1 )-(L b3 )-(Ab a2 )-(L b2 )-(Ab a3 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-(L c3 )-(Ab d3 ) Y (formula VIII).
In some embodiments, ab a1 、Ab a2 、Ab a3 、Ab d1 、Ab d2 Or Ab d3 Each independently comprising an antigen binding domain.
In some embodiments, ab a1 、Ab a2 、Ab a3 、Ab d1 、Ab d2 Or Ab d3 Each independently represents an antigen binding domain.
In some embodiments, ab a1 、Ab a2 、Ab a3 、Ab d1 、Ab d2 Or Ab d3 Is an antigen binding domain 1 (ABD 1), an antigen binding domain 2 (ABD 2), or an antigen binding domain 3 (ABD 3).
In some embodiments, ab a1 、Ab a2 、Ab a3 、Ab d1 、Ab d2 Or Ab d3 Is not antigen binding domain 1 (ABD 1), antigen binding domain 2 (ABD 2), or antigen binding domain 3 (ABD 3).
In some embodiments, L b1 A hinge region comprising a linker or a plurality of linkers and/or an antibody or antigen binding fragment thereof. In some embodiments, the hinge region is a natural hinge region as disclosed herein. In other embodiments, the hinge region is a mutant hinge region as disclosed herein.
In some embodiments, L b2 、L b3 、L c1 、L c2 L and L c3 Each independently comprising a linker or a plurality of linkers as disclosed herein.
In some embodiments, the polypeptide chain comprises an antigen binding domain ABD1 as disclosed herein and an antigen binding domain that binds to an immune cell.
In some embodiments, the polypeptide chain comprises an antigen binding domain ABD2 as disclosed herein and an antigen binding domain that binds to a tumor cell.
In some embodiments, the polypeptide chain comprises an antigen binding domain ABD3 as disclosed herein and an antigen binding domain that binds to a tumor cell.
In some embodiments, the polypeptide chain comprises an antigen binding domain ABD3 as disclosed herein and an antigen binding domain that binds to an immune cell.
In some embodiments, the polypeptide chain comprises at least one antigen binding domain selected from ABD1, ABD2, and ABD3 as disclosed herein.
In some embodiments, the polypeptide chain comprises at least two antigen binding domains selected from ABD1, ABD2, and ABD3 as disclosed herein.
In some embodiments, the polypeptide chain comprises at least three antigen binding domains selected from ABD1, ABD2, and ABD3 as disclosed herein.
In some embodiments, the dimerization domain comprises an immunoglobulin dimerization domain. Other dimerization domains known to those of skill in the art are contemplated herein, including leucine zippers and the like.
In some embodiments, the dimerization domain comprises IgG, igM, igA, igD or IgE dimerization domain (from human or animal IgGss, igM, igAs, igDs or IgE).
In some embodiments, the dimerization domain comprises a CH3 domain of an antibody. The dimerization domain may also comprise a CH2 domain of an antibody.
In some exemplary embodiments, the dimerization domain comprises a native CH3 domain.
In some exemplary embodiments, the dimerization domain comprises a mutant CH3 domain.
In some embodiments, the dimerization domain comprises native CH2 and native CH3 domains.
In some embodiments, the dimerization domain comprises a native CH2 and a mutant CH3 domain.
In some embodiments, the dimerization domain comprises mutant CH2 and mutant CH3 domains.
In some embodiments, the dimerization domain comprises mutant CH2 and native CH3 domains.
In some embodiments, the dimerization domain does not comprise a CH1 domain.
In some embodiments, the dimerization domain does not comprise a CH4 domain.
In some embodiments, the dimerization domain comprises the Fc region of an antibody or a portion thereof.
In some embodiments, the polypeptide chains comprise at least two or at least three antigen binding domains and a dimerization domain, which allows two polypeptide chains to be assembled to form a multivalent and/or multispecific binding agent.
In some embodiments, the antigen binding domain comprises or consists of an antigen binding domain of a single domain antibody (sdAb).
In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH or VHH).
In certain embodiments, the VHH is derived from a human, mouse, rat, or the like.
In some embodiments, the VHH is from a transgenic mouse or rat capable of expressing a camelized mouse or rat VHH, a VHH from other species (e.g., human, etc.), or a camelized VHH from other species (e.g., a camelized human VHH, etc.).
In some embodiments, the binding agent does not comprise a light chain variable region (VL or VLL).
In some embodiments, the binding agent comprises a light chain variable region (VL or VLL).
In some embodiments, the antigen binding domain is a single chain variable fragment (ScFv).
In some embodiments, the antigen binding domain is from V NAR Fragments.
In other embodiments, the antigen binding domain of the polypeptide chain comprises a combination of any of the following antigen binding domains: single structureDomain antibodies (sdabs), heavy chain variable regions (VH or VHH), light chain variable regions (VL or VLL), single chain variable fragments (ScFv), and/or V NAR Fragments.
In some embodiments, the sdAb or VHH is from a camelidae antibody.
In some embodiments, the camelid antibody is from dromedarion, camel, alpaca, and the like.
In other embodiments, the sdAb or VHH is from a cartilaginous fish antibody.
In some embodiments, the cartilage fish antibody is a shark antibody.
In some embodiments, each antigen binding domain specifically binds to a different epitope.
In other embodiments, each antigen binding domain specifically binds to a different antigen.
In other embodiments, each antigen binding domain specifically binds to a different protein.
In some embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen binding domain that binds to a protein expressed by a tumor. In other embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen binding domain that binds to and modulates the activity of a protein expressed by a tumor.
In some embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen binding domain that binds to an immune checkpoint protein. In other embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen binding domain that binds to and modulates the activity of an immune checkpoint protein.
In some embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen binding domain that binds to an immune cell protein. In other embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen binding domain that binds to and modulates the activity of an immune cell protein. However, in other embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen binding domain that binds to or engages and recruits or redirects immune cells.
In some embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen binding domain that binds to Peripheral Blood Mononuclear Cells (PBMCs). In other embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen binding domain that binds to and modulates the activity of PBMCs. In still other embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen binding domain that binds to PBMCs and recruits or redirects PBMCs.
In some embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen binding domain that binds to a T cell protein. In other embodiments, the binding agent may bind to a T cell protein and may modulate its activity. In yet other embodiments, the binding agent may bind to a T cell protein and may recruit or redirect T cells. In some embodiments, the binding agent may bind to and/or modulate the activity of activated T cells.
In some embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen binding domain that binds to a protein expressed by a tumor and at least one antigen binding domain that binds to an immune cell and recruits or redirects the immune cell.
In some embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen binding domain that binds to a protein expressed by a tumor, at least one antigen binding domain that binds to an immune checkpoint protein, and at least one antigen binding domain that binds to an immune cell and recruits or redirects the immune cell.
In some embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen binding domain that modulates an immune checkpoint inhibitor.
In some embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen binding domain that binds a tumor antigen, at least one antigen binding domain that binds an immune checkpoint protein, and at least one antigen binding domain that binds a T cell.
In some embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen binding domain that binds a tumor antigen, at least one antigen binding domain that binds an immune checkpoint protein, and at least one antigen binding domain that binds CD 47.
In some embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen binding domain that modulates CD47 function.
In some embodiments, the binding agent comprises a polypeptide chain that comprises at least one antigen binding domain that binds CD47 and enhances macrophage function by blocking sirpa/CD 47 interaction.
In some embodiments, the binding agent comprises a polypeptide chain comprising an antigen binding domain that specifically binds to a receptor.
In some embodiments, the binding agent comprises a polypeptide chain comprising an antigen binding domain that specifically binds to a tumor antigen and an antigen binding domain that specifically binds to an immunomodulatory agent.
In some embodiments, the antigen binding domain that specifically binds to a tumor antigen is located N-terminal to the dimerization domain and the antigen binding domain that specifically binds to an immunomodulatory agent is located C-terminal to the dimerization domain.
In some embodiments, the immunomodulator is an immune checkpoint protein, cytokine, chemokine or immune receptor or immune co-receptor.
It will be appreciated herein that a given antigen binding domain may bind to epitopes present in different proteins. Thus, in some embodiments, the antigen binding domain or binding agent comprising the same may bind to more than one protein. In some embodiments, the antigen binding domain or binding agent may have affinity for more than one protein.
In some embodiments, the binding agent comprises at least two polypeptide chains capable of forming a dimer.
In some embodiments, the two polypeptide chains are identical.
In some embodiments, the two polypeptide chains are different.
In some embodiments, the polypeptide chains are identical and the binding agent is a homodimer.
In some embodiments, the polypeptide chains are different and the binding agent is a heterodimer.
In some embodiments, the binding agent is multispecific.
In some embodiments, the binding agent is bispecific, trispecific, or tetraspecific.
In some embodiments, the binding agent comprises one or more polypeptide chains that are multispecific.
In some embodiments, the binding agent comprises one or more polypeptide chains that are bispecific, trispecific, or tetraspecific.
In some embodiments, the one or more polypeptide chains have the same valency and specificity.
In some embodiments, the one or more polypeptide chains have different valencies and specificities.
In some embodiments, the one or more polypeptide chains are each an antibody heavy chain.
The binding agent of any one of the preceding claims, wherein the binding agent is an antibody or antigen binding fragment thereof.
In some embodiments, the binding agent is a bispecific antibody.
In some embodiments, the bispecific antibody further comprises a first antibody light chain and a second antibody light chain.
In some embodiments, one or more of the antigen binding domains are humanized.
In some embodiments, one or more of the antigen binding domains are partially humanized.
In some embodiments, the antigen binding domain comprises one or more human frameworks.
In some embodiments, X or Y is independently selected from a linker, cytokine, chemokine, tag, masking domain, phage capsid protein (pIII, pVI, pV, pVII or pIX), antigen binding domain, or a combination thereof.
In some embodiments, the polypeptide chain is conjugated to a therapeutic moiety.
In some embodiments, the polypeptide chain is conjugated to a detectable moiety.
In some embodiments, the polypeptide chain is conjugated to a protein that allows for extended half-life.
In some embodiments, the polypeptide chain is linked to a nanoparticle.
Other aspects and embodiments of the invention relate to a composition comprising at least one binding agent disclosed herein.
In some embodiments, the compositions comprise monomers, dimers, and mixtures thereof.
In some embodiments, greater than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the first polypeptide chain and the second polypeptide chain are present in the composition as dimers.
In some embodiments, greater than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the first polypeptide chain and the second polypeptide chain are present in the composition as homodimers.
In some embodiments, greater than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the first polypeptide chain and the second polypeptide chain are present in the composition as heterodimers.
Other aspects and embodiments of the invention relate to a pharmaceutical composition comprising a binding agent as disclosed herein and a pharmaceutically acceptable carrier.
Still other aspects and embodiments of the invention relate to a nucleic acid or set of nucleic acids encoding a polypeptide chain and/or binding agent disclosed herein.
As disclosed herein, the nucleic acid may be in the form of a DNA segment.
Further aspects and embodiments of the invention relate to a vector comprising a nucleic acid disclosed herein or a set of vectors comprising a nucleic acid disclosed herein.
Further aspects and embodiments of the invention relate to a cell expressing a polypeptide chain or binding agent disclosed herein.
Further aspects and embodiments of the invention relate to a cell comprising a nucleic acid or vector disclosed herein.
Further aspects and embodiments of the invention relate to a kit comprising a binding agent disclosed herein.
Still further aspects and embodiments of the invention relate to a kit comprising a nucleic acid as disclosed herein.
Still further aspects and embodiments of the invention relate to a kit comprising a vector as disclosed herein.
Still further aspects and embodiments of the invention relate to a kit comprising the cells disclosed herein.
In another aspect and embodiment, the invention relates to a method of treating a disorder or disease comprising administering a binding agent disclosed herein.
In a further aspect and embodiment, the present invention relates to a method of treating a disorder or disease comprising administering a composition disclosed herein.
In a further aspect and embodiment, the present invention relates to a method of treating a disorder or disease comprising administering a pharmaceutical composition disclosed herein.
In certain embodiments, the disorder or disease is cancer.
In some embodiments, the disorder or disease is a solid tumor.
In some embodiments, the disorder or disease is advanced metastatic solid cancer.
In certain embodiments, the disorder or disease is hematological cancer.
In certain embodiments, the condition or disease is lung cancer.
In some embodiments, the lung cancer is metastatic.
In some embodiments, the disorder or disease is small cell lung cancer.
In some embodiments, the disorder or disease is non-small cell lung cancer.
In some embodiments, the disorder or disease is myeloma.
In some embodiments, the disorder or disease is prostate cancer.
In some embodiments, the condition or disease is breast cancer.
In some embodiments, the disorder or disease is rectal cancer.
In some embodiments, the disorder or disease is pancreatic cancer.
In some embodiments, the disorder or disease is glioblastoma.
In some embodiments, the condition or disease is an infection.
In some embodiments, the condition or disease is an immune disorder.
In other aspects and embodiments, the invention relates to a method of making a binding agent disclosed herein, the method comprising transforming a cell with one or more vectors comprising a nucleic acid disclosed herein.
In some embodiments, the method may further comprise isolating and/or purifying the binding agent from the impurities.
In other embodiments, the method may further comprise separating and/or purifying the heterodimer from the monomer and/or homodimer.
In other embodiments, the method may further comprise separating and/or purifying the homodimer from the monomer and/or heterodimer.
In some embodiments, the method further comprises conjugating the binding agent to a therapeutic moiety, a detectable moiety, or a protein that allows for an extended half-life, or to a nanoparticle.
Further scope, applicability, and advantages of the present invention will become apparent from the non-limiting detailed description given hereinafter. It should be understood, however, that the detailed description, while indicating some exemplary embodiments of the invention, is given by way of illustration only and with reference to the accompanying drawings.
Brief description of the drawings
Fig. 1: a graph showing the binding profile of D2-specific binding agents (VHH-hinge-CH 2-CH 3) on D2 protein liposomes. The mock proteoliposomes were included as negative controls.
Fig. 2: the table listing EC50 s of D2 specific binders (VHH-hinge-CH 2-CH 3) in GPCR BRET assays transfected with various G protein receptors is presented. Dopamine was used as a positive control to show the reaction of GPCRs with G proteins including gαz, gαi2, gαoa and barre 2. The negative control was a 4HEM specific binding agent (VHH-hinge-CH 2-CH 3) (KC 018) comprising the antigen binding domain shown in SEQ ID NO: 114.
Fig. 3: a heat map of the change in gene expression in NCI-H69 cells after treatment with a binding agent comprising a D2-specific antigen binding domain (VHH-hinge-CH 2-CH 3) (KC 013; SEQ ID NO: 73) is shown. The negative control was a 4HEM specific binding agent (VHH-hinge-CH 2-CH 3) (KC 018) comprising the antigen binding domain shown in SEQ ID NO: 114.
Fig. 4A-4B: a pattern of down-regulated signaling pathways and protein classes representing panher analysis from the RNAseq results of fig. 3.
Fig. 5A-5C: a diagram showing the down-regulated genes verified by qRT-PCR NCI-H69 (FIG. 5A), NCI-H82 (FIG. 5B) and HEK-D2 (FIG. 5C) after treatment with a binding agent (KC 013; SEQ ID NO: 73) comprising a D2 specific antigen binding domain (VHH-hinge-CH 2-CH 3).
Fig. 6A-6B: a diagram representing the pharmacokinetics of single dose injection of trastuzumab in SCID mice (fig. 6a: iv injection) (fig. 6b: ip injection) of a binding agent comprising a D2-specific antigen-binding domain (VHH-hinge-CH 2-CH 3) (KC 013; SEQ ID NO: 73).
Fig. 7A-7C: a graph showing tumor kinetics and in vivo efficacy of binding agents (KC 013; SEQ ID NO: 73) comprising a D2-specific antigen binding domain (VHH-hinge-CH 2-CH3, solid line) in NCG mice with tumors of the Small Cell Lung Cancer (SCLC) xenograft model compared to isotype control (dashed line) in NCI-H727 tumor prevention model (fig. 7A), NCI-H69 tumor prevention model (fig. 7B) or NCI-H510A tumor prevention model (fig. 7C). The negative control was a 4HEM specific binding agent (VHH-hinge-CH 2-CH 3) (KC 018) comprising the antigen binding domain shown in SEQ ID NO: 114.
Fig. 8: tables are presented for the percent tumor growth inhibition in various tumor-preventing SCLC tumor models in SCID mice after treatment with a D2 specific binding agent (VHH-hinge-CH 2-CH 3) comprising the amino acid sequence shown in SEQ ID NO:71 (KC 011), SEQ ID NO:72 (KC 012), SEQ ID NO:73 (KC 013), or SEQ ID NO:74 (KC 014).
Fig. 9A: the pattern of the binding profile of PD-1 specific binding agents (KC 036; SEQ ID NO: 94) for human, cynomolgus monkey and rat recombinant PD1 protein or control protein (BSA) is shown. The negative control was a binding agent comprising a HEWL-specific antigen binding domain (KC 035: SEQ ID NO: 93).
Fig. 9B: the pattern of the binding curve of PD-1 specific binding agent (KC 036; SEQ ID NO: 94) versus the anti-PD-1 antibody palbockizumab (Pembrolizumab) (positive control) for human recombinant PD1 protein is shown. The negative control was a binding agent comprising a HEWL-specific antigen binding domain (KC 035: SEQ ID NO: 93).
Fig. 9C: the pattern of binding curves of the CD47 specific binding agent (KC 015; SEQ ID NO: 75) to human recombinant CD47 protein is shown. A negative control for KC015 is a binding agent comprising a HEWL-specific antigen binding domain (KC 016: SEQ ID NO: 76). The positive control was monoclonal antibody clone B6H12. Isotype control of clone B6H12 was used as a mouse IgG isotype control antibody.
Fig. 9D: a graph showing the binding profile of CD47 specific binding agent KC015 to cynomolgus monkey CD47 protein. The negative control KC016 is a binding agent comprising a HEWL-specific antigen binding domain. The positive control was monoclonal antibody clone B6H12. Isotype control of clone B6H12 was used as a mouse IgG isotype control antibody.
Fig. 9E: a graph showing the binding profile of CD47 specific binding agent KC015 for various blood cell subtypes including T cells, monocytes, granulocytes, red Blood Cells (RBCs) and platelets.
Fig. 10: a pattern showing cell binding activity of a PD-1 specific binding agent (KC 036; SEQ ID NO: 94) obtained by flow cytometry on CHO-PD1 cells (FIG. 10B) and CHO parent cells (FIG. 10A). Commercial PD-1 antibodies were used as positive controls. The negative control was a binding agent comprising a HEWL-specific antigen binding domain (KC 035: SEQ ID NO: 93).
Fig. 11: a graph representing cytotoxicity data for PBMC-mediated death of THP-1 induced by a PD-1 specific binding agent (KC 036; SEQ ID NO: 94) in the presence of CD33 BiTE or in the presence of control BiTE 48 hours after incubation for activation of T cells via CD3 subunit. The negative control was a binding agent comprising a HEWL-specific antigen binding domain (KC 031: SEQ ID NO: 89). The positive control included palbociclib and nivolumab (nivolumab).
Fig. 12: the pattern of checkpoint inhibitory activity of PD-1 specific binding agents (KC 036; SEQ ID NO: 94) of a biological assay system using PD-1/PD-L1 blocking is shown. This system is an engineered Jurkat cell line expressing a luciferase reporter gene under the NFAT promoter. The negative control was a binding agent comprising a HEWL-specific antigen binding domain (KC 035: SEQ ID NO: 93). The anti-PD-1 antibody palbociclib was used as a positive control.
Fig. 13: a diagram representing a trispecific binding agent comprising anti-D2, anti-PD-1 and anti-CD 47 antigen binding domains with HEK293T cells and HEK293T-CD47 KO cells (CD 47 knockout cells) (KC 020; SEQ ID NO: 77) and a monospecific binding agent comprising the same anti-CD 47 antigen binding domain (KC 015: 75 VHH-hinge-CH 2-CH 3). Negative controls included corresponding forms comprising anti-HEWL antigen binding domains (KC 016 (SEQ ID NO: 76) and KC039 (SEQ ID NO: 97)). A commercially available anti-human CD47 antibody (clone CC2C 6) was used as a positive control.
Fig. 14: a graph representing binding of a binding agent comprising anti-D2 and anti-CD 47 antigen binding domains to human T cells (KC 040: SEQ ID NO: 84) compared to a control binding agent comprising an anti-HEWL antigen binding domain (KC 035: SEQ ID NO: 93) or an anti-CD 3 and anti-HEWL antigen binding domain (KC 034; SEQ ID NO: 92).
Fig. 15A-15B: a pattern of T cell activation (fig. 15A) and T cell dependent cytotoxicity (fig. 15B) induced by a binding agent comprising anti-D2 and anti-CD 47 antigen binding domains on human T cells (KC 040: SEQ ID NO: 84) at a 5:1E to T ratio is shown. Controls included medium only (negative control), binders comprising anti-HEWL antigen binding domain and the same anti-D2 antigen binding domain (negative control KC032; SEQ ID NO: 90) and OKT3 (positive control).
Fig. 16: illustrating the pattern of T cell activation following treatment with a binding agent comprising the D2 and CD47 specific antigen binding domain (KC 040: SEQ ID NO: 84) in the presence or absence of concanavalin A. Controls included binders comprising HEWL-binding domain specific antigen (KC 035: SEQ ID NO: 93) or anti-CD 3 and anti-HEWL antigen binding domains (positive control KC034; SEQ ID NO: 92) or OKT3 (positive control).
Fig. 17: the pattern of checkpoint inhibitory activity of a CD47 specific binding agent (KC 015; SEQ ID NO: 75) of a biological assay system is shown using Promega CD 47/SIRPalpha blocking. This system is an engineered THP-1 cell line expressing a luciferase reporter gene induced by an Fc gamma R protein conjugated to an Fc gamma R. The negative control was a binding agent comprising a HEWL-specific antigen binding domain (KC 016: SEQ ID NO: 76). The positive control was monoclonal antibody clone B6H12. Isotype control for clone B6H12 was a mouse IgG isotype control antibody.
Fig. 18: a graph representing tumor volume after treatment with a trispecific binding agent comprising anti-D2, anti-PD-1 and anti-CD 47 antigen binding domains (KC 021: SEQ ID NO: 78) in an established NCI-H82 xenograft model in NCG mice. The negative control included a binding agent comprising a HEWL-specific antigen binding domain (KC 031: SEQ ID NO: 89).
Fig. 19A-19B: the pattern of T-cell infiltration into NCI-H69 clusters (FIG. 19A) or activation of T-cells with the same agent after treatment with binders comprising anti-D2 and anti-PD-1 antigen binding domains (KC 037; SEQ ID NO: 95), anti-PD-1 and anti-CD 47 antigen binding domains (KC 038; SEQ ID NO: 96) or trispecific binders comprising the same anti-D2, anti-PD 1 and anti-CD 47 antigen binding domains (KC 020; SEQ ID NO:77 and KC021: SEQ ID NO: 78) is shown. Controls included a binding agent comprising a HEWL-specific antigen binding domain (KC 035: SEQ ID NO: 93).
Fig. 20A-20B: a graph representing tumor volume (FIG. 20A) and a table representing tumor growth inhibition after treatment with a trispecific binding agent (KC 020; SEQ ID NO:77,KC022;SEQ ID NO:79,KC023;SEQ ID NO:80,KC024;SEQ ID NO:81,KC025;SEQ ID NO:82,KC026;SEQ ID NO:83) comprising different anti-D2 and the same anti-PD-1 and anti-CD 47 antigen binding domains (FIG. 20B).
Fig. 21A: a pattern of tumor volumes following treatment with CD47 specific binding agent (KC 015; SEQ ID NO: 75) in a NCG mice PBMC pre-implanted NCI-H82 xenograft model is shown. 1000 ten thousand PBMC were implanted into NCG mice 3 days prior to tumor cell implantation. Treatment was started 10 days after PBMC implantation twice weekly for 8 doses. The negative control used in the study was PBS.
Fig. 21B: tables showing the in vivo efficacy of KC020 (SEQ ID NO: 77) using various tumor cells including NCI-H69, NCI-H82, SCLC-21H and RKO in different models.
Fig. 21C: a graph showing tumor volume after treatment with anti-CD 47 specific binding agent KC015 in an established MDA-MB-231 xenograft model in SCID mice. The negative control used in the study was PBS. 500 ten thousand MDA-MB-231 cells were injected subcutaneously (s.c.) into the right abdomen of SCID mice. Mice received binder (8 mg/kg) treatment by intraperitoneal (i.p.) once a week for a total of eight doses.
Fig. 22: a pattern representing tumor volume after treatment with IgG1 or IgG4 isotype (KC 028; SEQ ID NO:86;KC029;SEQ ID NO:87,KC027:SEQ ID NO:85) of binding agents comprising anti-D2, anti-PD-1 and anti-CD 47 antigen binding domains. The control included a binding agent comprising an anti-HEWL antigen binding domain (KC 031: SEQ ID NO: 89).
Fig. 23A-23B: a graphical representation of biodistribution experiments (FIG. 23A) and a graphical representation showing accumulation of trispecific binders comprising anti-D2, anti-PD-1 and anti-CD 47 antigen binding domains (KC 021; SEQ ID NO: 78) in NCG mice (upper panel) and NCI-H69 (lower panel) xenograft models (FIG. 23B).
Fig. 24A-24B: a pattern of liver metastasis in hcd34+ humanized NCG mice with established NCI-H69 tumors treated with PBS or KC020 (SEQ ID NO: 77) (fig. 24A) and an H & E staining photograph showing tumors with disseminated tumor cells from PBS group instead of KC020 (SEQ ID NO: 77) group as shown in black boxes (fig. 24B).
Fig. 24C: a graph showing tumor kinetics and in vivo efficacy of KC020 in NCI-H82/PBMC co-implantation model at a 1:5E: T ratio in NCG mice.
Fig. 25A-25C: a graph showing the binding of KC020 (SEQ ID NO: 77) to D2 protein liposome (fig. 25A), human recombinant PD-1 protein (fig. 25B) and human recombinant CD47 protein (fig. 25C) subjected to stress conditions as measured by ELISA analysis. Pressure conditions included stirring for 3 days, thawing five times, and storage at 40 ℃ for two weeks.
Fig. 26A-26C: a pattern showing the binding of KC020 (SEQ ID NO: 77) to D2 protein liposome (fig. 26A), human recombinant PD-1 protein (fig. 26B) and human recombinant CD47 protein (fig. 26C) subjected to low pH pressure as measured by ELISA analysis. Pressure conditions included 4 hours and 48 hours of treatment at pH 3.5.
Fig. 27: a graph showing survival of KC 020-treated animals implanted with NCI-H82 SCLC tail vein transfer model. NCG mice were inoculated with 50,000 NCI-H82 cells and KC treated the next day. KC020 was treated every two weeks. The negative control used in the study was PBS.
Detailed Description
Definition of the definition
Unless otherwise indicated, the amino acid numbering indicated for the dimerization domains is according to the EU numbering system.
The use of the terms "a" and "an" and "the" and similar referents in the context of describing the embodiments (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.
The term "or" as used herein is to be understood as inclusive and to encompass "or" and "unless explicitly stated or apparent from the context.
As used herein, "and/or" should be taken to specifically disclose each specific feature or component (in the presence or absence of another feature or component).
Unless otherwise indicated, the terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to"). The term "consisting of" is considered to be closed.
The term "treatment" for the purposes of the present invention refers to therapeutic treatment and prophylactic or preventative measures of a tumor. The subject in need of treatment includes those already with the disorder and those prone to the disorder; and those to be prevented.
The term "about" or "approximately" with respect to an established value is meant to encompass a variation in the value. In some embodiments, the term "about" or "approximately" will generally mean within +/-10%, within +/-5%, within +/-4%, within +/-3%, within +/-2% or within +/-1% of the established value or range.
The term "functionally active" with reference to an antigen binding domain means that the antigen binding domain is capable of binding to its target, and optionally the antigen binding domain possesses one or more biological activities.
As used herein, the term "flexible linker" refers to a peptide comprising at least a portion consisting of flexible amino acid residues that allow adjacent modules to move relative to each other.
As used herein, the term "rigid linker" refers to a peptide comprising at least a portion of amino acids that exhibit a rigid structure and maintain a distance between the two modules.
As used herein, the term "helical linker" refers to a linker composed of amino acid residues in an alpha-helical configuration.
As used herein, the term "cleavable linker" refers to a peptide comprising an enzymatic cleavage site that is sensitive to a protease selected from the group consisting of ADAMS, ADAMTS, aspartic proteases, apoptotic proteases, cysteine cathepsins, cysteine proteases, metalloproteases, serine proteases, coagulation factor proteases, type II transmembrane serine proteases (TTSP), and combinations thereof.
As used herein, the term "monospecific" with respect to a polypeptide chain or binding agent refers to a polypeptide chain or binding agent that binds to a single antigen or epitope. Thus, a monospecific polypeptide chain or binding agent may have one antigen binding domain or more than one binding domain (which may be the same or different) which has the same specificity for a given antigen or epitope.
As used herein, the term "multispecific" with respect to a polypeptide chain or binding agent refers to a polypeptide chain or binding agent that binds to more than one antigen or epitope.
The term "multispecific" encompasses "bispecific", "trispecific", "tetraspecific", "penta", "hexa", and the like.
As used in the context of binding agents herein, the term "bispecific" refers to binding agents that bind to two different antigens or proteins or bind to two epitopes of the same antigen or protein.
As used herein, the expression "at least two polypeptide chains" and the like, such as in the expression "binding agent comprises at least two polypeptide chains" and the like, refers to the number of polypeptide chain species and not to absolute values.
It is understood herein that recitation of ranges of values in a format such as "a-B" includes each individual value and each sub-range of constituent and including such range. For example, the expression "1 to 10" includes subranges such as and not limited to "2 to 10", "2 to 9", "3 to 6", "5 to 7", and any individual value included therein, and includes 1 and 10, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
It is understood herein that the term "at least" with respect to a given value is intended to include that value and higher. For example, the term "at least 80%" includes "at least 81%", "at least 82%", "at least 83%", "at least 84%", "at least 85%", "at least 86%", "at least 87%", "at least 88%", "at least 89%", "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%", "at least 99.1%", "at least 99.2%", "at least 99.3%", "at least 99.4%", "at least 99.5%", "at least 99.6%", "at least 99.7%", "at least 99.8%" "at least 99.9%", and 100%.
Antigen binding domain (AB)
As disclosed herein, the binding agent may comprise one or more antigen binding domains.
The antigen binding domains of the invention may be selected for their ability to bind to a particular target. The antigen binding domains of the invention may also be selected for their functional properties or biological effects in vivo and/or in vitro, including, for example, their ability to modulate cellular processes such as gene expression, signal transduction, cell growth, cell viability, and the like.
For example, the binding agents of the invention comprise one or more antigen binding domains that each independently comprise one or more Complementarity Determining Regions (CDRs) of an antibody.
Thus, the specificity of the binding agents of the invention may be conferred by their antigen binding domains.
The binding agents of the invention may comprise an antigen binding domain capable of binding to DR2 or to a cell expressing DR2, an antigen binding domain capable of binding to PD-1 or to a cell expressing PD-1, and/or an antigen binding domain capable of binding to CD47 or to a cell expressing CD 47.
In some embodiments, the antigen binding domain is an antigen binding domain capable of binding to DR2 or to a cell expressing DR2, and includes, for example, antigen binding domain 1 (ABD 1).
In some embodiments, the antigen binding domain is an antigen binding domain capable of binding to PD-1 or to a cell expressing PD-1, and includes, for example, antigen binding domain 2 (ABD 2).
In some embodiments, the antigen binding domain is an antigen binding domain capable of binding to CD47 or to a cell expressing CD47, and includes, for example, antigen binding domain 3 (ABD 3).
In some embodiments, the antigen binding domain targets the same epitope or antigen as ABD1, ABD2, and/or ABD 3. In some embodiments, the antigen binding domain competes with ABD1, ABD2, and/or ABD3 for binding to its respective epitope or antigen.
In some embodiments, the antigen binding domain binds to at least one antigen that is not selected among DR2, PD-1 or CD 47.
In some embodiments, the binding agent may comprise more than one antigen binding domain.
For example, in some embodiments, the binding agent may comprise two or more antigen binding domains.
In some embodiments, the binding agent may comprise three or more antigen binding domains.
In some embodiments, the binding agent may comprise four or more antigen binding domains.
In some embodiments, the binding agent may comprise five or more antigen binding domains.
In some embodiments, the binding agent may comprise six or more antigen binding domains.
In some embodiments, the binding agent may comprise an antigen binding domain between one and twelve, such as between one and two, between one and three, between one and four, between one and five, between one and six, between one and seven, between one and eight, between one and nine, between one and ten, between one and eleven, between one and twelve, between two and three, between two and four, between two and five, between two and six, between two and seven, between two and eight, between two and ten, between two and eleven, between two and twelve, between three and seven, between three and nine, between three and ten, between three and eleven, between three and twelve, between four and five, between four and six, between four and seven, between four and eight, between four and nine, between four and ten, between four and eleven, between four and twelve, between five and six, between five and seven, between five and eight, between five and nine, between five and twelve, between seven and nine, between seven and ten, between seven and eleven, between seven and twelve, between eight and nine, between eight and ten, between eight and eleven, between eight and twelve, between nine and ten, between nine and eleven, between nine and twelve, between ten and eleven, between ten and twelve, or between eleven and twelve.
In some embodiments, a binding agent of the invention may comprise one or more antigen binding domains, and at least one of the antigen binding domains is capable of binding to a tumor cell.
In some embodiments, the binding agents of the invention may comprise one or more antigen binding domains and at least one of the antigen binding domains is capable of binding to an immunomodulatory agent. In some embodiments, at least one of the antigen binding domains is capable of binding to an immune checkpoint protein. In some embodiments, the immune checkpoint protein is PD-1.
In some embodiments, the binding agents of the invention may comprise one or more antigen binding domains, and at least one of the antigen binding domains is capable of binding to an immune cell. In some embodiments, at least one of the antigen binding domains is capable of binding to a protein expressed at the surface of an immune cell (e.g., T cell, NK cell, monocyte, macrophage, etc.).
In some embodiments, the binding agents of the invention may be multivalent and may comprise at least one antigen binding domain that binds tumor cells and at least one antigen binding domain that binds immune cells.
In some embodiments, the protein expressed at the surface of the immune cell is selected from CD47 or CD3.
In some embodiments, the protein expressed at the surface of the immune cell is CD47.
In some embodiments, the protein expressed at the surface of the immune cell is CD3.
The antigen binding domain may be derived from a natural antibody (of human or animal origin) or from a synthetic antibody.
In some embodiments, the antigen binding domain of the native antibody is engineered to form a single chain.
In some embodiments, the antigen binding domain may be obtained from an IgG such as IgG1, igG2, igG3, or IgG 4. In particular embodiments, the antigen binding domain is derived from a human IgG heavy chain.
In some embodiments, the antigen binding domain is obtainable as a heavy chain antibody (HCAb).
Exemplary embodiments of antigen binding domains include, for example and without limitation, single domain antibodies (sdabs), heavy chain variable regions (VH or VHH), light chain variable regions (VL or VLL), single chain variable fragments (scFv), V NAR Fragments and combinations thereof.
In some embodiments, the antigen binding domain of the binding agents disclosed herein is a VHH.
In some embodiments, the antigen binding domain of a binding agent disclosed herein is a VHH comprising a complementarity determining region of a VHH disclosed herein.
In some embodiments, the Complementarity Determining Regions (CDRs) of a VHH disclosed herein correspond to Kabat CDRs.
In some embodiments, the Complementarity Determining Regions (CDRs) of the VHHs disclosed herein correspond to IMGT CDRs.
In a specific embodiment, the binding agent of the invention may comprise an antigen binding domain VHH derived from a human or mouse or rat or derived from a transgenic mouse or rat, wherein the mouse or rat VHH has been camelized; a human VHH; a camelized human VHH; VHH of IgG1, igG2a, igG2b, igG2c or IgG3, or a combination thereof. Antibodies can be obtained by immunizing a mouse or rat or a transgenic mouse or rat lacking a functional CH1 domain in any of its heavy chains, igG1, igG2a, igG2b, igG2c, or IgG3, or a combination thereof, or a combination of VHH as described above, with an antigen of interest.
In a specific embodiment, the polypeptide chain of the invention may comprise an antigen binding domain of a camelidae antibody, such as a VHH of IgG2 or IgG 3. Camelid antibodies may be obtained by immunizing dromedaries, camels, horses or alpacas with an antigen of interest.
In some embodiments, the camelid antibody may be derived from a so-called old world camelid, such as dromedary camel (Camelus bactrianus), dromedary camel (Camelus dromaderus), or from a new world camelid, such as alpaca (Lama pacos), camel horse (Lama glama) and leptin camel (Lama vicugna).
In another embodiment, the polypeptide chain of the invention may comprise an antigen binding domain of a cartilaginous fish, such as the V of IgNAR NAR Fragments. V (V) NAR Fragments may be derived from shark antibodies.
If desired, the antigen binding domain of a non-human antibody may be humanized. For example, the framework regions of a non-human VH, VHH or HCAb may be modified to make it more human-like. Humanization of camelidae antibodies is discussed, for example, in Vincke C. Et al (J.Biol chem.) (2009, 284 (5): 3273-3284), the entire contents of which are incorporated herein by reference. Humanized camelidae antibodies can be prepared, for example, by grafting CDRs onto a universal humanized nanobody scaffold (e.g., h-NbBcII10 disclosed in Vincke c. Et al FGLA ) Obtained above. V (V) NAR Antibodies can be humanized by converting non-CDR residues to residues of the human reproductive V.kappa.1 sequence DPK9, as described by Kovalenko OV et al (J. Biol.Chem.2013, 288:17408-17419), the entire contents of which are incorporated herein by referenceIn the manner described herein. Thus, the polypeptide chains of the invention encompass humanized antigen binding domains.
In yet another specific embodiment, the antigen-binding domain may comprise a human VH (modified or unmodified). Human VH can be obtained, for example, from a synthetic human VH library. Modified human VH includes those in which some amino acid residues have been modified to make them more camelid-like (i.e. by camelisation).
Those skilled in the art will appreciate that the antigen binding domain may incorporate antibodies, antigen binding fragments, or antibody-like molecules, including, but not limited to, single domain antibodies (sdAb), VHHs, conventional antibodies or antigen binding fragments thereof, bispecific antibodies, single chain Fv-CH3 (scFv-CH 3) fusions, tandem-scFv-CH 3 (TaFv-CH 3) fusions, diabody-CH 3 (Db-CH 3) fusions, tandem Db-CH3 (TaDb-CH 3) fusions, single chain Db-CH3 fusions (scDb-CH 3), fab-CH3 fusions, single chain Fab-CH3 fusions, fab-scFv-CH3 fusions, dual Affinity Retargeting (DART) -CH3 fusions, fab-DART-CH3 fusions, single chain Fv-Fc (scFv-Fc) fusions, tandem-scFv-Fc (TaFv-Fc) fusions, diabody-Fc (Db-Fc) fusions, dab-Fc (Db-Fc), single chain Fab-Fc-fusions, single chain Fc-fusions, and the like. These antibody forms may have native CH3, mutated CH3 domains, native CH2-CH3 domains, or mutated CH2-CH3 domains as disclosed herein.
Illustrative embodiment of ABD1
Antigen binding domain 1 (ABD 1) may be selected, for example, from among antigen binding domains that bind DR2, some non-limiting exemplary embodiments of which are provided herein.
In some exemplary embodiments, a binding agent of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises heavy chain complementarity determining region 1 (CRDH 1) having the amino acid sequence set forth in SEQ ID No. 1, heavy chain complementarity determining region 2 (CDRH 2) having the amino acid sequence set forth in SEQ ID No. 2, and heavy chain complementarity determining region 3 (CDRH 3) having the amino acid sequence set forth in SEQ ID No. 3.
In some exemplary embodiments, a binding agent of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises CRDH1 having the amino acid sequence set forth in SEQ ID No. 4, CDRH2 having the amino acid sequence set forth in SEQ ID No. 5, and CDRH3 having the amino acid sequence set forth in SEQ ID No. 6.
In some exemplary embodiments, a binding agent of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises CRDH1 having the amino acid sequence set forth in SEQ ID No. 8, CDRH2 having the amino acid sequence set forth in SEQ ID No. 9, and CDRH3 having the amino acid sequence set forth in SEQ ID No. 10.
In some exemplary embodiments, a binding agent of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises CRDH1 having the amino acid sequence set forth in SEQ ID No. 11, CDRH2 having the amino acid sequence set forth in SEQ ID No. 12, and CDRH3 having the amino acid sequence set forth in SEQ ID No. 13.
In some exemplary embodiments, a binding agent of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises CRDH1 having the amino acid sequence set forth in SEQ ID No. 15, CDRH2 having the amino acid sequence set forth in SEQ ID No. 16, and CDRH3 having the amino acid sequence set forth in SEQ ID No. 17.
In some exemplary embodiments, a binding agent of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises CRDH1 having the amino acid sequence set forth in SEQ ID No. 18, CDRH2 having the amino acid sequence set forth in SEQ ID No. 19, and CDRH3 having the amino acid sequence set forth in SEQ ID No. 20.
In some exemplary embodiments, a binding agent of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises CRDH1 having the amino acid sequence set forth in SEQ ID No. 22, CDRH2 having the amino acid sequence set forth in SEQ ID No. 23, and CDRH3 having the amino acid sequence set forth in SEQ ID No. 24.
In some exemplary embodiments, a binding agent of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises CRDH1 having the amino acid sequence set forth in SEQ ID No. 25, CDRH2 having the amino acid sequence set forth in SEQ ID No. 26, and CDRH3 having the amino acid sequence set forth in SEQ ID No. 27.
In some exemplary embodiments, a binding agent of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises CRDH1 having the amino acid sequence set forth in SEQ ID No. 29, CDRH2 having the amino acid sequence set forth in SEQ ID No. 30, and CDRH3 having the amino acid sequence set forth in SEQ ID No. 31.
In some exemplary embodiments, a binding agent of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises CRDH1 having the amino acid sequence set forth in SEQ ID No. 32, CDRH2 having the amino acid sequence set forth in SEQ ID No. 33, and CDRH3 having the amino acid sequence set forth in SEQ ID No. 34.
In some exemplary embodiments, a binding agent of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises CRDH1 having the amino acid sequence set forth in SEQ ID No. 36, CDRH2 having the amino acid sequence set forth in SEQ ID No. 37, and CDRH3 having the amino acid sequence set forth in SEQ ID No. 38.
In some exemplary embodiments, a binding agent of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises CRDH1 having the amino acid sequence set forth in SEQ ID No. 39, CDRH2 having the amino acid sequence set forth in SEQ ID No. 40, and CDRH3 having the amino acid sequence set forth in SEQ ID No. 41.
In some exemplary embodiments, a binding agent of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises CRDH1 having the amino acid sequence set forth in SEQ ID No. 43, CDRH2 having the amino acid sequence set forth in SEQ ID No. 44, and CDRH3 having the amino acid sequence set forth in SEQ ID No. 45.
In some exemplary embodiments, a binding agent of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises CRDH1 having the amino acid sequence set forth in SEQ ID No. 46, CDRH2 having the amino acid sequence set forth in SEQ ID No. 47, and CDRH3 having the amino acid sequence set forth in SEQ ID No. 48.
In some exemplary embodiments, a binding agent of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises CRDH1 having the amino acid sequence set forth in SEQ ID No. 50, CDRH2 having the amino acid sequence set forth in SEQ ID No. 51, and CDRH3 having the amino acid sequence set forth in SEQ ID No. 52.
In some exemplary embodiments, a binding agent of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises CRDH1 having the amino acid sequence set forth in SEQ ID No. 53, CDRH2 having the amino acid sequence set forth in SEQ ID No. 54, and CDRH3 having the amino acid sequence set forth in SEQ ID No. 55.
In some exemplary embodiments, the binding agents of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises a heavy chain having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID No. 7. In some embodiments, the amino acid change may be located in one or more framework regions of SEQ ID NO. 7.
In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 7 and a CDR that is identical to the Kabat CDR of SEQ ID NO. 7.
In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 7 and a CDR that is identical to the IMGT CDR of SEQ ID NO. 7.
In some exemplary embodiments, the binding agents of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises a heavy chain having an amino acid sequence as shown in SEQ ID No. 7.
In some exemplary embodiments, the binding agents of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises a heavy chain having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID No. 14. In some embodiments, the amino acid change may be located in one or more framework regions of SEQ ID NO. 14.
In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 14 and a CDR that is identical to the Kabat CDR of SEQ ID NO. 14.
In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 14 and a CDR that is identical to the IMGT CDR of SEQ ID NO. 14.
In some exemplary embodiments, the binding agents of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises a heavy chain having an amino acid sequence as shown in SEQ ID No. 14.
In some exemplary embodiments, the binding agents of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises a heavy chain having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID No. 21. In some embodiments, the amino acid change may be located in one or more framework regions of SEQ ID NO. 21.
In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 21 and a CDR that is identical to the Kabat CDR of SEQ ID NO. 21.
In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 21 and a CDR that is identical to the IMGT CDR of SEQ ID NO. 21.
In some exemplary embodiments, the binding agents of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises a heavy chain having the amino acid sequence as shown in SEQ ID No. 21.
In some exemplary embodiments, the binding agents of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises a heavy chain having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID No. 28. In some embodiments, the amino acid change may be located in one or more of the framework regions of SEQ ID NO. 28.
In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 28 and a CDR that is identical to the Kabat CDR of SEQ ID NO. 28.
In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 28 and a CDR that is identical to the IMGT CDR of SEQ ID NO. 28.
In some exemplary embodiments, the binding agents of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises a heavy chain having the amino acid sequence as shown in SEQ ID No. 28.
In some exemplary embodiments, a binding agent of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises a heavy chain having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID No. 35. In some embodiments, the amino acid change may be located in one or more framework regions of SEQ ID NO. 35.
In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 35 and a CDR that is identical to the Kabat CDR of SEQ ID NO. 35.
In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 35 and a CDR that is identical to the IMGT CDR of SEQ ID NO. 35.
In some exemplary embodiments, the binding agents of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises a heavy chain having the amino acid sequence as shown in SEQ ID No. 35.
In some exemplary embodiments, a binding agent of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises a heavy chain having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID No. 42. In some embodiments, the amino acid change may be located in one or more framework regions of SEQ ID NO. 42.
In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 42 and a CDR that is identical to the Kabat CDR of SEQ ID NO. 42.
In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 42 and a CDR that is identical to the IMGT CDR of SEQ ID NO. 42.
In some exemplary embodiments, the binding agents of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 42.
In some exemplary embodiments, a binding agent of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises a heavy chain having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID No. 49. In some embodiments, the amino acid change may be located in one or more of the framework regions of SEQ ID NO. 49.
In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 49 and a CDR that is identical to the Kabat CDR of SEQ ID NO. 49.
In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 49 and a CDR that is identical to the IMGT CDR of SEQ ID NO. 49.
In some exemplary embodiments, the binding agents of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises a heavy chain having the amino acid sequence as shown in SEQ ID No. 49.
In some exemplary embodiments, the binding agents of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises a heavy chain having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID No. 56. In some embodiments, the amino acid change may be located in one or more of the framework regions of SEQ ID NO. 56.
In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 56 and a CDR that is identical to the Kabat CDR of SEQ ID NO. 56.
In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:56 and a CDR that is identical to the IMGT CDR of SEQ ID NO: 56.
In some exemplary embodiments, the binding agents of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises a heavy chain having the amino acid sequence as shown in SEQ ID No. 56.
Exemplary embodiment of ABD2
Antigen binding domain 2 (ABD 2) may be selected, for example, from among antigen binding domains that bind PD-1, some non-limiting exemplary embodiments of which are provided herein.
In some exemplary embodiments, a binding agent of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 2 (ABD 2) and comprises heavy chain complementarity determining region 1 (CRDH 1) having an amino acid sequence as set forth in SEQ ID No. 57, heavy chain complementarity determining region 2 (CRDH 2) having an amino acid sequence as set forth in SEQ ID No. 58, and heavy chain complementarity determining region 3 (CRDH 3) having an amino acid sequence as set forth in SEQ ID No. 59.
In some exemplary embodiments, a binding agent of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 2 (ABD 2) and comprises CRDH1 having the amino acid sequence set forth in SEQ ID No. 60, CDRH2 having the amino acid sequence set forth in SEQ ID No. 61, and CDRH3 having the amino acid sequence set forth in SEQ ID No. 62.
In some exemplary embodiments, the binding agents of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 2 (ABD 2) and comprises a heavy chain having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID No. 63. In some embodiments, the amino acid change may be located in one or more of the framework regions of SEQ ID NO. 63.
In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 63 and a CDR that is identical to the Kabat CDR of SEQ ID NO. 63.
In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 63 and a CDR that is identical to the IMGT CDR of SEQ ID NO. 63.
In some exemplary embodiments, the binding agents of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 2 (ABD 2) and comprises a heavy chain having the amino acid sequence as shown in SEQ ID No. 63.
Exemplary embodiment of ABD3
Antigen binding domain 3 (ABD 3) may be selected, for example, from the antigen binding domain that binds CD47, some non-limiting exemplary embodiments of which are provided herein.
In some exemplary embodiments, a binding agent of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 3 (ABD 3) and comprises heavy chain complementarity determining region 1 (CRDH 1) having the amino acid sequence set forth in SEQ ID No. 64, heavy chain complementarity determining region 2 (CDRH 2) having the amino acid sequence set forth in SEQ ID No. 65, and heavy chain complementarity determining region 3 (CDRH 3) having the amino acid sequence set forth in SEQ ID No. 66.
In some exemplary embodiments, a binding agent of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 3 (ABD 3) and comprises CRDH1 having the amino acid sequence set forth in SEQ ID No. 67, CDRH2 having the amino acid sequence set forth in SEQ ID No. 68, and CDRH3 having the amino acid sequence set forth in SEQ ID No. 69.
In some exemplary embodiments, the binding agents of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 3 (ABD 3) and comprises a heavy chain having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID No. 70. In some embodiments, the amino acid change may be located in one or more framework regions of SEQ ID NO. 70.
In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 70 and a CDR that is identical to the Kabat CDR of SEQ ID NO. 70.
In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 70 and a CDR that is identical to the IMGT CDR of SEQ ID NO. 70.
In some exemplary embodiments, the binding agents of the invention may comprise one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 3 (ABD 3) and comprises a heavy chain having the amino acid sequence as shown in SEQ ID No. 70.
Exemplary embodiments of other antigen binding domains
In some embodiments, the binding agent comprises one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1), antigen binding domain 2 (ABD 2), or antigen binding domain 3 (ABD 3), and at least one antigen binding domain binds to a different antigen.
In some embodiments, the binding agent may thus comprise an antigen binding domain that binds to CD 3. Such antigen binding domains include those known to those of skill in the art. Exemplary embodiments of antigen binding domains that bind CD3 are provided in SEQ ID NO. 115.
Thus, in some embodiments, the binding agent may comprise an antigen binding domain that binds DR2, PD-1 and/or CD 3. In other embodiments, the binding agent may comprise an antigen binding domain that binds DR2 and/or CD 3. In other embodiments, the binding agent may comprise an antigen binding domain that binds to PD-1 and/or CD 3.
In some embodiments, the binding agent comprises one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1), antigen binding domain 2 (ABD 2), or antigen binding domain 3 (ABD 3), and wherein at least one antigen binding domain is selected from, for example and without limitationSelected from the antigen binding domains that bind specifically to: CD3, CD36, DRD1, DRD2, DRD3, DRD4, DRD5, PD-L1, TROP2, CD147, MCT1, IL1RAP, AMIGO2, PTK7, MCT2, MCT4, NHE1, H+/K+ -ATPase, LAP, HLA-I A2, CD73, CD98, CEACAM5/6, ICAM-1, MCSP, fibronectin, beta 1 integrin, tetranectin (tetraspan) 8, CD164, CD59, CD63, CD44, CD166, cWF, TNF, IL-17A, IL-F, IL-6R, BCMA, TNF, RANKL, ADAMTS, VEGF, ang2, CX 3 CR1, CXCR4, tfR1 (CD 71), CXCR2, CD3, PD1, PDL-1, CTLA-4, CD8, LAG-3, OX40, CD27, CD122/IL2RB, TLR8/CD288, TIM-3, ICOS/CD278, NKG2A, A2AR, B7-H3, B7-H4, GITR/TNFRSF18, 4-IBB/CD137, KIR2DL1, KIR3DL2, SIRPalpha, CD47, VISTA, CD40, CD112, CD96, TOGOT, BTLA, TIGIT, CD4, VEGFR2, CD19, IGFR1, epCAM, EGFR, DLL3, CGRP, CD79B, CD28, CCR5, erbB3, erbB2, TGF beta 1, TGF beta 2, TGF beta 3, TGF beta R1, TGF beta R2, TLR 1, IDO2, TGF-4, TLR 7, TLR8, TLR 9, NOX2 or SIEC 7.
One or more polypeptide chains
The binding agents of the invention comprise one or more polypeptide chains.
Segments of DNA encoding the desired polypeptide chain sequences can be synthesized in vitro. The different DNA modules are assembled into a single piece in an organized and directed manner, and then cloned into an expression vector. The resulting polypeptide chain is thus composed of different modules forming a single chain.
Polypeptide chains of the invention include, for example and without limitation, the antigen binding domain, a linker, and a dimerization domain that facilitates assembly of at least two polypeptide chains.
In some embodiments, the polypeptide chains of the invention may be monospecific.
In some embodiments, the polypeptide chains of the invention may be multispecific.
In some embodiments, the polypeptide chains of the invention may be monovalent.
In some embodiments, the polypeptide chains of the invention may be multivalent.
In some cases, the polypeptide chain does not comprise a dimerization domain.
In some cases, the polypeptide chain comprises a dimerization domain.
In some aspects of the invention, the polypeptide chain may be monospecific.
Exemplary embodiments of monospecific polypeptide chains include polypeptide chains comprising one antigen binding domain. Another exemplary embodiment of a monospecific polypeptide chain comprises a polypeptide chain comprising more than one antigen binding domain, but the antigen binding domains have the same CDRs and framework regions. Another exemplary embodiment of a monospecific polypeptide chain comprises a polypeptide chain comprising more than one antigen binding domain, but the antigen binding domains have the same CDRs and different framework regions. Another exemplary embodiment of a monospecific polypeptide chain includes a polypeptide chain comprising an antigen binding domain whose amino acid sequence differs (e.g., conservative substitutions in one or more CDRs) without affecting its ability to bind to the same antigen or epitope.
In some aspects of the invention, the polypeptide chain may be multispecific. The polypeptide chain may encompass, for example, a bispecific polypeptide chain, a trispecific polypeptide chain, a tetraspecific polypeptide chain, a penta-specific polypeptide chain, a hexa-specific polypeptide chain, a double paratope polypeptide chain, a multi-paratope polypeptide chain, and the like.
In an exemplary configuration, one or more antigen binding domains can be located at the N-terminus, at the C-terminus, or on each side of the dimerization domain.
In another exemplary configuration, the polypeptide chain can comprise at least one antigen binding domain at the N-terminus of the dimerization domain and at least one antigen binding domain at the C-terminus of the dimerization domain.
In another exemplary configuration, the polypeptide chain may comprise one antigen binding domain at the N-terminus of the dimerization domain and at least two antigen binding domains at the C-terminus of the dimerization domain.
In yet another exemplary configuration, the polypeptide chain may comprise two antigen binding domains at the N-terminus of the dimerization domain and two antigen binding domains at the C-terminus of the dimerization domain.
The polypeptide chain may comprise the amino acid sequence of formula I in N-terminal to C-terminal manner:
X-[(Ab a )-(L b )] m -(DD)-[(L c )-(Ab d )] n -Y
wherein m is 0, 1, 2 or an integer greater than 2;
Wherein n is 0, 1, 2 or an integer greater than 2;
wherein m and n are not simultaneously 0;
wherein Ab a 、Ab d Each represents an antigen binding domain, wherein at least one of the antigen binding domains is antigen binding domain 1 (ABD 1), antigen binding domain 2 (ABD 2), or antigen binding domain 3 (ABD 3);
wherein X or Y is independently present or absent and comprises an amino acid sequence;
wherein L is b 、L c Each independently comprising one or more linkers; and is also provided with
Wherein DD represents the dimerization domain.
In some embodiments, L b May not be present.
In some embodiments, L c May not be present.
In some embodiments, L b L and L c May not be present.
In some embodiments, the polypeptide chain comprises more than one antigen binding domain.
In some embodiments wherein m is 2 or an integer greater than 2, [ (Ab) a )-(L b )]The units are identical.
In other embodiments wherein m is 2 or an integer greater than 2 [ (Ab) of the polypeptide chain or binding agent a )-(L b )]The cells are different.
In other embodiments, wherein m is an integer greater than 2, the polypeptide chain or binding agent [ (Ab) a )-(L b )]The units may comprise the same and different units.
In some embodiments wherein n is 2 or an integer greater than 2, [ (L) c )-(Ab d )]The units are identical.
In other embodiments wherein n is 2 or an integer greater than 2, [ (L) c )-(Ab d )]The cells are different.
In other embodiments wherein n is 2 or an integer greater than 2, [ (L) c )-(Ab d )]The units comprise the same and different units.
In some embodiments, when m is 2 or an integer greater than 2, each Ab a Are the same or different.
In some embodiments, when n is 2 or an integer greater than 2, each Ab d Are the same or different.
In some embodiments, ab a Representing ABD1.
In some embodiments, ab d Representing ABD2.
In some embodiments, ab d Representing ABD3.
In some embodiments, m is 1, and Ab a Representing ABD1.
In some embodiments, n is 2, and Ab d Represents ABD2.
In some embodiments, n is 2, and Ab d Represents ABD3.
In some embodiments, n is 2, and Ab d One of them represents ABD2 and the other Ab d Representing ABD3.
In some embodiments, m is 2, 3, 4, 5, or an integer greater than 5.
In some embodiments, m is 2.
In other embodiments, m is 3.
In still other embodiments, m is 4.
In a further embodiment, m is 5.
In other embodiments, m is an integer greater than 5.
In some embodiments, n is 2, 3, 4, 5, or an integer greater than 5.
In some embodiments, n is 2.
In other embodiments, n is 3.
In further embodiments, n is 4.
In a further embodiment, n is 5.
In other embodiments, n is an integer greater than 5.
In some embodiments, the one or more polypeptide chains further comprise a hinge region of an antibody or antigen binding fragment thereof.
In some embodiments, the hinge region is at the N-terminus of the dimerization domain.
In some embodiments, L b Is the hinge region of an antibody or antigen binding fragment thereof.
In some embodiments, the hinge region is a native human IgG1 hinge region. In other embodiments, the hinge region is a mutant human IgG1 hinge region.
In other embodiments, the hinge region is a native human IgG2 hinge region. In other embodiments, the hinge region is a mutant human IgG2 hinge region.
In other embodiments, the hinge region is a native human IgG3 hinge region. In other embodiments, the hinge region is a mutant human IgG3 hinge region.
In yet other embodiments, the hinge region is a native human IgG4 hinge region. In other embodiments, the hinge region is a mutant human IgG4 hinge region.
In some embodiments, each of the one or more linkers independently has a residue of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.
In some embodiments, each of the one or more connectors is independently a flexible connector, a screw connector, or a rigid connector.
In some embodiments, linker L c Is a rigid linker.
In some embodiments, lc comprises a non-cleavable linker. In other embodiments, lc consists of a non-cleavable linker.
In some implementations, the one or more connectors include a flexible connector.
In some implementations, the one or more linkers include a rigid linker.
In some embodiments, the flexible linker is a GS linker.
In some embodiments, the flexible linker comprises one or more GGGGS units.
In some embodiments, the flexible linker comprises at least 2, 3, 4, 5 or more GGGGS units.
In some embodiments, the rigid linker comprises a plurality of PA repeat sequences.
In some embodiments, the rigid linker is selected from PAPAPAPKA (SEQ ID NO: 105); APAPAPAPAPKA (SEQ ID NO: 106); APAPAPAPAPAPAPAPAPAPKA (SEQ ID NO: 107); or a combination thereof.
In some embodiments, the helical linker comprises one or more EAAAK units.
In some embodiments, the helical linker is selected from AEAAAKEAAAKA (SEQ ID NO: 109); AEAAAKEAAAKEAAAKA (SEQ ID NO: 110); AEAAAKEAAAKEAAAKEAAAKEAAAKA (SEQ ID NO: 111); or a combination thereof.
In some embodiments, the dimerization domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO. 116.
In some embodiments, the dimerization domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO. 117.
In other exemplary embodiments, the polypeptide chain comprises formula II:
X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 ) Y (formula II).
In yet other exemplary embodiments, the polypeptide chain comprises formula III:
X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 ) Y (formula III).
In further exemplary embodiments, the polypeptide chain comprises formula IV:
X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 ) Y (formula IV).
In further exemplary embodiments, the polypeptide chain comprises formula V:
X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 ) Y (formula V).
In further exemplary embodiments, the polypeptide chain comprises formula VI:
X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-(L c3 )-(Ab d3 ) Y (formula VI).
In yet a further exemplary embodiment, the polypeptide chain comprises formula VII:
X-(Ab a1 )-(L b3 )-(Ab a2 )-(L b2 )-(Ab a3 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 ) Y (formula VII).
In other exemplary embodiments, the polypeptide chain comprises formula VIII:
X-(Ab a1 )-(L b3 )-(Ab a2 )-(L b2 )-(Ab a3 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-(L c3 )-(Ab d3 ) Y (formula VIII).
In some embodiments, ab a1 、Ab a2 、Ab a3 、Ab d1 、Ab d2 、Ab d3 Each independently comprising an antigen binding domain.
In some embodiments, ab a1 、Ab a2 、Ab a3 、Ab d1 、Ab d2 、Ab d3 Each independently represents an antigen binding domain.
In some embodiments, at least one of the antigen binding domains is antigen binding domain 1 (ABD 1), antigen binding domain 2 (ABD 2), or antigen binding domain 3 (ABD 3).
In some embodiments, L b1 A hinge region comprising a linker or a plurality of linkers and/or an antibody or antigen binding fragment thereof. In some embodiments, L b1 Is L b 。
In some embodiments, L b2 、L b3 、L c1 、L c2 L and L c3 Each independently comprising a linker or a plurality of linkers.
In some embodiments, L b1 、L b2 And/or L b3 May not be present.
In some embodiments, L b2 And/or L b3 May not be present.
In some embodiments, L c1 、L c2 And/or L c3 May not be present.
In some embodiments, L c2 And/or L c3 May not be present.
In some embodiments, L c1 Is a rigid linker.
In some embodiments, L c2 Is a rigid linker.
In some embodiments, L c3 Is a rigid linker.
In some embodiments, L c1 L and L c2 Is a rigid linker.
In some embodiments, L c1 、L c2 L and L c3 Is a rigid linker.
In some embodiments, L c1 、L c2 And/or L c3 May not be present.
In some embodiments, L c2 And/or L c3 May not be present.
In some embodiments, L c1 And/or L c3 May not be present.
In some embodiments, L c2 And/or L c3 May not be present.
In some embodiments, L c1 、L c2 L and L c3 May not be present.
The polypeptide chains of the invention comprise an antigen binding domain that is single-chain or functionally active when part of a binding agent disclosed herein.
For example, the antigen binding domain of the polypeptide chain may bind to its target and may have biological activity.
In some embodiments, the biological activity of the antigen binding domain includes, for example and without limitation, blocking binding of the target to its natural receptor or ligand. Alternatively, the biological activity of the antigen binding domain includes its ability to sequester a target. Furthermore, the biological activity of the antigen binding domain includes its ability to induce signaling.
Polypeptide chains comprising more than one antigen binding domain are characterized as multivalent.
The polypeptide chains of the invention may comprise additional amino acid sequences (defined by X and Y, respectively, in the formulae disclosed herein) at their N-or C-terminus or both.
In some embodiments, the N-terminal amino acid sequence (defined by X) may include a signal peptide, exemplary embodiments of which are provided in SEQ ID NO: 133.
In some embodiments, the amino acid sequence of the N-terminus (defined by X) or the C-terminus (defined by Y) may independently include a linker, a cytokine, a chemokine, a tag (e.g., his tag (e.g., SEQ ID NO: 134), a masking domain, a phage capsid protein, an antigen binding domain, or a combination thereof.
Exemplary embodiments of multispecific polypeptide chains include polypeptide chains comprising at least two antigen-binding domains that differ in the amino acid sequence of one or more CDRs resulting in different binding specificities.
Polypeptide chains may be more specifically characterized as bispecific when they bind to two different epitopes or antigens. Polypeptide chains can be characterized as trispecific when they bind to three different epitopes or antigens. Polypeptide chains can be characterized as being four-specific when they bind to four different epitopes or antigens. When a polypeptide chain binds to five different epitopes or antigens, it can be characterized as being penta-specific. When a polypeptide chain binds to six different epitopes or antigens, it can be characterized as six-specific.
Polypeptide chains comprising two antigen binding domains that bind to two non-overlapping epitopes on the same target are characterized as being biparatopic. Polypeptide chains comprising antigen binding domains that bind to three, four or more epitopes on the same target are characterized as being multi-paratope.
The antigen binding domain of a given polypeptide chain will be selected based on the intended use, such as detection, diagnostic and/or therapeutic use. Each antigen binding domain of a particular polypeptide chain may be selected to produce an additive or synergistic effect.
In some embodiments, the antigen binding domain may be selected for its ability to specifically bind to a protein involved in a disease or condition.
For example, a polypeptide chain of the invention may comprise at least one antigen binding domain that specifically binds to an antigen expressed by a tumor cell or tumor cell environment (i.e., a tumor-specific antigen binding domain).
In other aspects and embodiments of the invention, the polypeptide chain may comprise at least one antigen binding domain that specifically binds to an immunomodulatory agent.
For example, the polypeptide chain can comprise one or more antigen binding domains (e.g., immunospecific antigen binding domains) that bind to an immune checkpoint protein, cytokine, chemokine, or immune receptor or co-receptor, or the like.
In some exemplary embodiments, the antigen binding domain may bind to dopamine receptor D2 (DR 2).
In some exemplary embodiments, the antigen binding domain may bind to PD 1.
In some exemplary embodiments, the antigen binding domain can bind to CD 47.
In exemplary embodiments, the polypeptide chains of the present invention may comprise at least one tumor-specific antigen binding domain and at least one immune-specific antigen binding domain.
In some embodiments, one or more tumor-specific antigen binding domains may be located at the N-terminus of the dimerization domain.
In some embodiments, one or more tumor-specific antigen binding domains may be located at the C-terminus of the dimerization domain.
In some embodiments, the tumor-specific antigen binding domain may be located at both the N-terminus and the C-terminus of the dimerization domain.
In some embodiments, further one or more immunospecific antigen-binding domains may be located at the N-terminus of the dimerization domain.
In some embodiments, one or more immunospecific antigen-binding domains may be located at the C-terminus of the dimerization domain.
In some embodiments, the immunospecific antigen-binding domain may be located at both the N-and C-terminus of the dimerization domain.
In exemplary and non-limiting embodiments, the polypeptide chain or binding agent may comprise two immunospecific antigen-binding domains at the C-terminus of the dimerization domain. In some embodiments, an immunospecific antigen-binding domain adjacent to the C-terminal portion of the dimerization domain may be linked via a non-cleavable linker.
Dimerization Domain (DD)
In some embodiments, the polypeptide chains of the invention comprise a dimerization domain. Thus, two polypeptide chains may assemble to form a binding agent. Exemplary embodiments of the binding agents include homodimers and heterodimers.
The dimerization domain may comprise, for example and without limitation, constant regions of an immunoglobulin, including, for example, the Fc, CH2, and/or CH3 domains of a heavy chain immunoglobulin.
In certain embodiments and aspects of the invention, the dimerization domain may have a sequence identical to a native IgG1, igG2, igG3 or IgG4 constant region or to its corresponding CH2 and/or CH3 domain.
The invention specifically encompasses dimerization domains having sequences identical to those of natural human antibodies. Exemplary embodiments of dimerization domains include, for example, the CH2-CH3 domain of a natural human heavy chain.
Thus, in some embodiments, the dimerization domain comprises a native constant region of an antibody, such as a native human IgG1 constant region, a native human IgG2 constant region, a native human IgG3 constant region, or a native human IgG4 constant region.
Thus, in some embodiments, the dimerization domain comprises a native CH3 domain.
In an exemplary embodiment, the dimerization domain comprises a native human CH3 domain.
In some embodiments, the dimerization domain comprises a native CH2 domain and a native CH3 domain.
In some embodiments, the native CH3 domain is a native IgG1 CH3 domain. In some embodiments, the native CH3 domain is native human IgG1 CH3 (e.g., SEQ ID NO: 116).
In some embodiments, the native CH3 domain is a native IgG2 CH3 domain. In other embodiments, the native CH3 domain is a native human IgG2 CH3 domain.
In some embodiments, the native CH3 domain is a native IgG3 CH3 domain. In other embodiments, the native CH3 domain is a native human IgG3 CH3 domain.
In some embodiments, the native CH3 domain is a native IgG4 CH3 domain. In some embodiments, the native CH3 domain is a native human IgG4 CH3 domain.
When two polypeptide chains of a binding agent are made up of the same amino acid sequence, the binding agent will form a homodimer. However, co-expression of polypeptide chains having CH2-CH3 domains of natural antibodies but differing in amino acid sequence may result in a mixture of homodimers and heterodimers. The different binders present in the mixture may be separated by methods known in the art, including, for example, size exclusion chromatography.
Thus, exemplary heterodimers of the invention include those having a CH3 domain or a CH2-CH3 domain of a native antibody and formed from two polypeptide chains having different sequences or conformations.
In some embodiments, the polypeptide chain may have a mutated dimerization domain comprising, for example, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3 amino acid substitutions compared to the native or wild-type sequence.
In exemplary embodiments, the mutated dimerization domain may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. Amino acid substitutions may be conservative or non-conservative, as described in table 2.
In exemplary embodiments, the polypeptide chain may have a mutated dimerization domain having a sequence 80% to 99% identical to a native IgG1, igG2, igG3, or IgG4 constant region or to a CH2 and/or CH3 domain. Polypeptide chains encompassed by the present invention include those comprising mutated dimerization domains which are 85% to 99% identical, 90% to 99% identical, 95% to 99% identical to the native IgG1, igG2, igG3 or IgG4 constant region or to the CH2 and/or CH3 domains.
In some embodiments, the polypeptide chains of the invention may comprise a mutated dimerization domain comprising amino acid substitutions that facilitate heterodimer formation. Heterodimers of the invention may thus be formed from polypeptide chains comprising such mutations.
Thus, in some embodiments, the dimerization domain comprises a mutated antibody constant region, such as a mutated human IgG1 constant region, a mutated human IgG2 constant region, a mutated human IgG3 constant region, or a mutated human IgG4 constant region. The mutated constant region can have one or more amino acid substitutions, amino acid insertions, or amino acid deletions compared to the native constant region.
Thus, in some embodiments, the dimerization domain comprises a mutant CH3 domain. The mutant CH3 domain may have one or more amino acid substitutions, amino acid insertions or amino acid deletions compared to the native CH3 domain.
In some embodiments, the dimerization domain comprises a native CH2 and a mutant CH3 domain.
In some embodiments, the dimerization domain comprises mutant CH2 and mutant CH3 domains. The mutant CH2 domain may have one or more amino acid substitutions, amino acid insertions or amino acid deletions compared to the native CH2 domain.
In some embodiments, the mutant CH3 domain is a mutant IgG1 CH3 domain. In other embodiments, the mutant CH3 domain is a mutant human IgG1 CH3 domain.
In some embodiments, the mutant CH3 domain is a mutant IgG2 CH3 domain. In other embodiments, the mutant CH3 domain is a mutant human IgG2 CH3 domain.
In some embodiments, the mutant CH3 domain is a mutant IgG3 CH3 domain. In other embodiments, the mutant CH3 domain is a mutant human IgG3 CH3 domain.
In some embodiments, the mutant CH3 domain is a mutant IgG4 CH3 domain. In other embodiments, the mutant CH3 domain is a mutant human IgG4 CH3 domain.
In some embodiments, the mutant CH2 domain is a mutant IgG1 CH2 domain. In other embodiments, the mutant CH2 domain is a mutant human IgG1 CH2 domain.
In some embodiments, the mutant CH2 domain is a mutant IgG2 CH2 domain. In other embodiments, the mutant CH2 domain is a mutant human IgG2 CH2 domain.
In some embodiments, the mutant CH2 domain is a mutant IgG3 CH2 domain. In other embodiments, the mutant CH2 domain is a mutant human IgG3 CH2 domain.
In some embodiments, the mutant CH2 domain is a mutant IgG4 CH2 domain. In other embodiments, the mutant CH2 domain is a mutant human IgG4 CH2 domain.
In some embodiments, the Fc region may be modified to prevent glycosylation, extend its half-life, modulate receptor binding, or effector function. Exemplary mutations are discussed in samundrs k.o. (front. Immunol.10:1296,2019, the entire contents of which are incorporated herein by reference) and include, for example, the mutation asparagine 297 (e.g., N297).
According to the invention, the mutant CH3 domain comprises one or more mutations compared to the native CH3 domain. Thus, in some embodiments, the dimerization domain comprises a mutant CH3 domain comprising one or more mutations compared to the native CH3 domain.
For example, a mutant CH3 domain may comprise one or more mutations compared to the native CH3 domain of human IgG 1. In another example, the mutant CH3 domain may comprise one or more mutations compared to the native CH3 domain of human IgG 2. In yet another example, the mutant CH3 domain may comprise one or more mutations compared to the native CH3 domain of human IgG 3. In another example, the mutant CH3 domain may comprise one or more mutations compared to the native CH3 domain of human IgG 4.
In some embodiments, the mutant CH3 domain may include amino acid substitutions at positions 356, 357, 370, 399 and/or 439 (according to the EU numbering system).
In some embodiments, the dimerization domain comprises a mutant CH3 domain comprising one or more mutations at positions corresponding to D399, D/E356 and/or K370 (according to the EU numbering system) as compared to the native CH3 domain of human IgG1 or IgG 4.
In other embodiments, the dimerization domain comprises a mutant CH3 domain comprising one or more mutations at positions corresponding to D399, E357 and/or K439 according to EU numbering (according to the EU numbering system) as compared to the native CH3 domain of human IgG1 or IgG 4.
In some embodiments, the mutant CH3 domain further comprises one or more mutations at positions corresponding to Y349, T350, L351, P352, S354, R/Q355, T394 and/or P395 according to EU numbering (according to the EU numbering system) as compared to the native CH3 domain of human IgG1 or IgG 4.
In an exemplary embodiment, one polypeptide chain of a given binding agent may comprise a mutant CH3 domain having amino acid substitutions at positions 357, 399 and 439, for example, while another polypeptide chain of a binding agent may comprise a mutant CH3 domain having amino acid substitutions at positions 356, 370 and 399 (according to the EU numbering system).
In an exemplary embodiment, one polypeptide chain of a given binding agent may comprise a mutant CH3 domain having amino acid substitutions at positions 357, 399 and 439, while the other polypeptide chain of the binding agent may comprise a mutant CH3 domain having amino acid substitutions at positions 356, 370 and 399 (according to the EU numbering system). One or both polypeptide chains of a given binding agent may optionally further comprise a mutation at a position selected from 349, 350, 351, 352, 354, 355, 394 and/or 395. One polypeptide chain of a given binding agent may thus comprise a first dimerization domain having an amino acid sequence disclosed herein (DD 1 ) And another polypeptide chain of a given binding agent may comprise a second dimerization domain having an amino acid sequence disclosed herein (DD 2 )。
In some embodiments, the amino acid at position 356 may be replaced with a neutral amino acid. In some embodiments, the amino acid at position 370 can be replaced with a positively charged amino acid. In some embodiments, the amino acid at position 399 may be replaced with a neutral amino acid. In some embodiments, the amino acid at position 357 may be replaced with a neutral amino acid. In some embodiments, the amino acid at position 439 can be replaced with a negatively charged amino acid.
For example, to favor heterodimer formation, one polypeptide chain may be mutated by: a) aspartic acid (D) or glutamic acid (E) at position 356 is replaced with a neutral amino acid, b) lysine (K) at position 370 is replaced with a positively charged amino acid, and c) aspartic acid (D) at position 399 is replaced with a neutral amino acid, and another polypeptide chain may be mutated by: a) substitution of glutamic acid (E) at position 357 with a neutral amino acid, b) substitution of aspartic acid (D) at position 399 with a neutral amino acid, c) substitution of lysine (K) at position 439 with a negatively charged amino acid.
An exemplary embodiment of a polypeptide chain of the invention may comprise a dimerization domain comprising a mutant CH3 domain, wherein aspartic acid (D) or glutamic acid (E) at position 356 may be changed to glutamine (Q), lysine (K) at position 370 may be changed to glutamic acid (E), and aspartic acid (D) at position 399 may be changed to asparagine (N).
Another exemplary embodiment of a polypeptide chain of the invention may comprise a dimerization domain comprising a mutant CH3 domain, wherein glutamic acid (E) at position 357 may be changed to glutamine (Q), aspartic acid (D) at position 399 may be changed to asparagine (N), and lysine (K) at position 439 may be changed to glutamic acid (E).
In some embodiments, the dimerization domain comprises a mutant CH3 domain wherein the amino acid substitution (as compared to the native CH3 domain) is selected from the group consisting of:
a. amino acid substitution at position D399;
b. amino acid substitution at position D/E356;
c. amino acid substitution at position E357;
d. amino acid substitution at position K370;
e. amino acid substitution at position K439;
f. amino acid substitution at position Y349;
g. amino acid substitution at position T350;
h. amino acid substitution at position L351;
i. amino acid substitution at position P352;
j. amino acid substitution at position S354;
k. an amino acid substitution at position R355;
amino acid substitution at position Q355;
amino acid substitution at position T394;
an amino acid substitution at position P395;
amino acid substitution at position Y349; a kind of electronic device with high-pressure air-conditioning system
p. wherein any combination of two or more of a-o.
In some embodiments, the mutant CH3 domain may comprise amino acid substitutions at positions D399, D/E356, K370, and Y349.
In some embodiments, the mutant CH3 domain may comprise amino acid substitutions at positions D399, D/E356, K370 and T350.
In some embodiments, the mutant CH3 domain may comprise amino acid substitutions at positions D399, D/E356, K370 and L351.
In some embodiments, the mutant CH3 domain may comprise amino acid substitutions at positions D399, D/E356, K370 and P352.
In some embodiments, the mutant CH3 domain may comprise amino acid substitutions at positions D399, D/E356, K370 and S354.
In some embodiments, the mutant CH3 domain may comprise amino acid substitutions at positions D399, D/E356, K370 and R355.
In some embodiments, the mutant CH3 domain may comprise amino acid substitutions at positions D399, D/E356, K370 and Q355.
In some embodiments, the mutant CH3 domain may comprise amino acid substitutions at positions D399, D/E356, K370, and T394.
In some embodiments, the mutant CH3 domain may comprise amino acid substitutions at positions D399, D/E356, K370, Y349, and S354.
In some embodiments, the mutant CH3 domain may comprise amino acid substitutions at positions D399, K439, E357, and Y349.
In some embodiments, the mutant CH3 domain comprises amino acid substitutions at positions D399, E357, K439, and T350.
In some embodiments, the mutant CH3 domain comprises amino acid substitutions at positions D399, E357, K439E and L351.
In some embodiments, the mutant CH3 domain may comprise amino acid substitutions at positions D399, K439, E357, and P352.
In some embodiments, the mutant CH3 domain may comprise amino acid substitutions at positions D399, K439, E357, and S354.
In some embodiments, the mutant CH3 domain may comprise amino acid substitutions at positions D399, K439, E357, and R355.
In some embodiments, the mutant CH3 domain may comprise mutations at positions D399, K439, E357, and Q355.
In some embodiments, the mutant CH3 domain may comprise amino acid substitutions at positions D399, K439, E357, and P395.
In some embodiments, the mutant CH3 domain comprises amino acid substitutions at positions D399, E357, K439, Y349, and S354.
It will be appreciated that other amino acid substitutions in the native CH3 domain or in the mutant CH3 domain (at other positions or at the same positions as the other amino acid residues) may be made without affecting the ability of the polypeptide chain to form dimers.
In some embodiments, the amino acid substitution at position Y349 is selected from Y349K, Y349D or Y349R. More specifically, in some embodiments, the amino acid substitution at position Y349 is Y349K. In other embodiments, the amino acid substitution at position Y349 is Y349D.
In some embodiments, the amino acid substitution at position S354 is selected from S354K, S354D, S W or S354M. More specifically, in some embodiments, the amino acid substitution at position S354 is S354K. In other embodiments, the amino acid substitution at position S354 is S354D. In other embodiments, the amino acid substitution at position S354 is S354M.
In some embodiments, the amino acid substitution at position L351 is L351Y, L351W, L351H, L351R, L351D, L351A, L351T. More specifically, in some embodiments, the amino acid substitution at position L351 is L351Y. In other embodiments, the amino acid substitution at position L351 is L351W. In other embodiments, the amino acid substitution at position L351 is L351R.
In some embodiments, the amino acid substitution at position T350 is T350L, T350I or T350V. More specifically, in some embodiments, the amino acid substitution at position T350 is T350I. In other embodiments, the amino acid substitution at position T350 is T350V.
In some embodiments, the amino acid substitution at position P352 is P352Y, P352V, P352R, P352T, P352L, P352G, P352E, P352C, P K or P352D. More specifically, in some embodiments, the amino acid substitution at position P352 is P352R. In other embodiments, the amino acid substitution at position P352 is P352E.
In some embodiments, the amino acid substitution at position T394 is T394N.
In some embodiments, the amino acid substitution at position P395 is P395I. In other embodiments, the amino acid substitution at position P395 is P395G. In other embodiments, the amino acid substitution at position P395 is P395E.
In some embodiments, the amino acid substitution at position R355 is R355K. In other embodiments, the amino acid substitution at position R355 is R355W.
In some embodiments, the amino acid substitution at position Q355 is Q355K. In other embodiments, the amino acid substitution at position Q355 is Q355W.
In some embodiments, the mutant CH3 domain comprises the mutations D399N, D/E356Q and K370E according to EU numbering.
In some embodiments, the mutant CH3 domain comprises the mutations D399Q, D/E356Q and K370E according to EU numbering.
In other embodiments, the mutant CH3 domain comprises mutations D399N, E357Q and K439E according to EU numbering.
In some embodiments, the mutant CH3 domain may comprise the mutations D399Q, D/E356Q, K370E, Y349K and S354K.
In some embodiments, the mutant CH3 domain may comprise the mutations D399N, D/E356Q, K370E and L351W.
In some embodiments, the mutant CH3 domain may comprise the mutations D399N, D/E356Q, K370E and S354M.
In some embodiments, the mutant CH3 domain may comprise the mutations D399N, D/E356Q, K370E and T350I.
In some embodiments, the mutant CH3 domain may comprise the mutations D399N, D/E356Q, K370E and T350V.
In some embodiments, the mutant CH3 domain may comprise the mutations D399N, D/E356Q, K370E and P352R.
In some embodiments, the mutant CH3 domain may comprise the mutations D399N, D/E356Q, K370E and P352E.
In some embodiments, the mutant CH3 domain may comprise the mutations D399Q, D/E356Q and K370E.
In some embodiments, the mutant CH3 domain may comprise the mutations D399N, D/E356Q, K370E and L351Y.
In some embodiments, the mutant CH3 domain may comprise the mutations D399N, D/E356Q, K370E and L351H.
In some embodiments, the mutant CH3 domain may comprise the mutations D399N, D/E356Q, K370E and R355K.
In some embodiments, the mutant CH3 domain may comprise the mutations D399N, D/E356Q, K370E and Q355K.
In some embodiments, the mutant CH3 domain may comprise the mutations D399N, D/E356Q, K370E and S354K.
In some embodiments, the mutant CH3 domain may comprise the mutations D399N, D/E356Q, K370E and T350L.
In some embodiments, the mutant CH3 domain may comprise the mutations D399N, D/E356Q, K370E and T394N.
In some embodiments, the mutant CH3 domain may comprise the mutations D399N, D/E356Q, K370E and P352Y.
In some embodiments, the mutant CH3 domain may comprise the mutations D399N, D/E356Q, K370E and P352V.
In some embodiments, the mutant CH3 domain may comprise the mutations D399N, D/E356Q, K370E and P352T.
In some embodiments, the mutant CH3 domain may comprise the mutations D399N, D/E356Q, K370E and P352L.
In some embodiments, the mutant CH3 domain may comprise the mutations D399N, D/E356Q, K370E and P352G.
In some embodiments, the mutant CH3 domain may comprise the mutations D399N, D/E356Q, K370E and P352C.
In some embodiments, the mutant CH3 domain may comprise the mutations D399N, D/E356Q, K370E and L351T.
In some embodiments, the mutant CH3 domain may comprise the mutations D399N, D/E356Q, K370E and L351A.
In some embodiments, the mutant CH3 domain comprises mutations D399Q, E357Q, K439E, Y349D and S354D.
In some embodiments, the mutant CH3 domain comprises the mutations D399N, E357Q, K439E and L351R.
In some embodiments, the mutant CH3 domain comprises mutations D399N, E357Q, K439E and L351Y.
In some embodiments, the mutant CH3 domain comprises mutations D399N, E357Q, K439E and T350I.
In some embodiments, the mutant CH3 domain may comprise the mutations D399N, E357Q, K439E and T350V.
In some embodiments, the mutant CH3 domain may comprise the mutation D399Q, K439E, E357Q.
In some embodiments, the mutant CH3 domain may comprise mutation D399N, K439E, E357Q, S354K.
In some embodiments, the mutant CH3 domain may comprise mutation D399N, K439E, E357Q, S354W.
In some embodiments, the mutant CH3 domain may comprise the mutation D399N, K439E, E357Q, Y349R.
In some embodiments, the mutant CH3 domain may comprise the mutation D399N, K439E, E357Q, T L.
In some embodiments, the mutant CH3 domain may comprise mutation D399N, K439E, E357Q, R355W.
In some embodiments, the mutant CH3 domain may comprise mutation D399N, K439E, E357Q, Q355W.
In some embodiments, the mutant CH3 domain may comprise mutation D399N, K439E, E357Q, P395I.
In some embodiments, the mutant CH3 domain may comprise mutation D399N, K439E, E357Q, P G.
In some embodiments, the mutant CH3 domain may comprise mutation D399N, K439E, E357Q, P395E.
In some embodiments, the mutant CH3 domain may comprise mutation D399N, K439E, E357Q, P352K.
In some embodiments, the mutant CH3 domain may comprise mutation D399N, K439E, E357Q, P352D.
In some embodiments, the mutant CH3 domain may comprise mutation D399N, K439E, E357Q, L351D.
In some embodiments, the binding agent comprises two polypeptide chains, wherein;
one of the two polypeptide chains comprises a first dimerization domain (DD 1 ) The first dimerization domain comprises a mutant CH3 domain, the mutant CH3 domain comprising one or more amino acid substitutions at positions corresponding to D399, D/E356 and/or K370 according to EU numbering, and
the other of the two polypeptide chains comprises a second dimerization domain (DD 2 ) The second dimerization domain comprises a mutant CH3 domain, the mutant CH3 domain comprising one or more amino acid substitutions at positions corresponding to D399, E357 and/or K439 according to EU numbering.
In some embodiments, the first dimerization domain (DD 1 ) Comprises the following EU codesMutant CH3 domain of mutations D399N, D/E356Q and K370E, and wherein the second dimerization domain (DD 2 ) Comprises a mutant CH3 domain comprising mutations D399N, E357Q and K439E according to EU numbering.
In some embodiments, the first dimerization domain (DD 1 ) And/or the second dimerization domain (DD 2 ) Comprises a mutant CH3 domain, which mutant CH3 domain further comprises an amino acid substitution at a position corresponding to Y349, T350, L351, P352 and/or S354 according to EU numbering.
In some embodiments, the first dimerization domain (DD 1 ) Comprises a mutant CH3 domain comprising mutations D399Q, D/E356Q, K370E, Y349K and S354K according to EU numbering, and wherein the second dimerization domain (DD 2 ) Comprises a mutant CH3 domain comprising the mutations D399Q, E357Q, K439E, Y349D and S354D according to EU numbering.
In some embodiments, the first dimerization domain (DD 1 ) Comprises a mutant CH3 domain comprising mutations D399N, D/E356Q, K E and L351W according to EU numbering, and wherein the second dimerization domain (DD 2 ) Comprises a mutant CH3 domain comprising mutations D399N, E357Q, K439E and L351R according to EU numbering.
In some embodiments, the first dimerization domain (DD 1 ) Comprises a mutant CH3 domain comprising mutations D399N, D/E356Q, K E and S354M according to EU numbering, and wherein the second dimerization domain (DD 2 ) Comprises a mutant CH3 domain comprising mutations D399N, E357Q, K439E and L351Y according to EU numbering.
In some embodiments, the first dimerization domain (DD 1 ) Comprises a mutant CH3 domain comprising mutations D399N, D/E356Q, K E and T350I according to EU numbering, and wherein the second dimerization domain (DD 2 ) Comprises a mutant CH3 domain comprising mutations D399N, E357Q, K439E and T350I according to EU numbering.
In some embodiments, the first dimerization domain (DD 1 ) Comprises a mutation D399N, D/E356Q according to EU numberingMutant CH3 domains of K370E and T350V, and wherein the second dimerization domain (DD 2 ) Comprises a mutant CH3 domain comprising mutations D399N, E357Q, K439E and T350V according to EU numbering.
In some embodiments, the first dimerization domain (DD 1 ) Comprises a mutant CH3 domain comprising mutations D399N, D/E356Q, K E and P352R according to EU numbering, and wherein the second dimerization domain (DD 2 ) Comprises a mutant CH3 domain comprising mutations D399N, E357Q, K439E and L351R according to EU numbering.
In some embodiments, the first dimerization domain (DD 1 ) Comprises a mutant CH3 domain comprising mutations D399N, D/E356Q, K E and P352E according to EU numbering, and wherein the second dimerization domain (DD 2 ) Comprises a mutant CH3 domain comprising mutations D399N, E357Q, K439E and L351R according to EU numbering.
According to the invention, the binding agent may be composed of two different polypeptides that associate to form a dimer, referred to herein as a heterodimer.
Heterodimers can be prepared by coexpression of two different polypeptide chains (e.g., chain a and chain B).
In some embodiments, the polypeptide chain may comprise a dimerization domain comprising wild-type human CH2 and mutated human CH3 that facilitates heterodimer formation. In some cases, the polypeptide chains may form homodimers when expressed alone, or heterodimers (or mixtures of homodimers and heterodimers) when expressed with complementary chains.
Heterodimers are prepared, inter alia, by transfecting a set of polypeptide chains comprising CH3 mutations (chains A and B are exemplified in Table 1).
Table 1 exemplary embodiments of chains A and B (native human IgG1 CH2 and mutant IgG1 CH 3)
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Depending on the ratio between chain a and chain B, it is also possible to form homodimers when two such polypeptide chains are co-expressed.
Thus, in some cases, the co-expression of polypeptide chain a with polypeptide chain B may thus result in heterodimers of chains a and B, homodimers of chain a, homodimers of chain B, and mixtures thereof. Residual monomers of chain a and/or chain B may also be present. Since monomers, heterodimers and homodimers all contain antigen binding domains, each component of the mixture may have a certain level of activity.
Thus, monomers, heterodimers and homodimers comprising the CH3 mutations disclosed herein, as well as mixtures of such monomers, heterodimers and/or homodimers, are also encompassed by the present invention.
In other embodiments, the polypeptide chains disclosed herein may comprise a mutated dimerization domain comprising mutations known in the art to facilitate heterodimer formation.
For example, the polypeptide chains of the invention may comprise the conformations set forth in formulas I, II, III, IIIa and IIIb, IV, V, VI, VII or VIII disclosed herein and mutations known in the art to favor heterodimer formation.
Exemplary embodiments of such mutations are disclosed, for example, in Ha, J-H et al (Front immunological), 2016; 7:394) or Godar M et al (expert opinion on therapeutic patents (Expert Opinion on Therapeutic patents), 2018;28 (3): 251-276), which is incorporated by reference in its entirety, and includes, for example, the Knob and socket (Knob-into-hole) (first CH3 domain mutation T366Y and second CH3 domain mutation Y407T, first CH3 domain mutation T366W and second CH3 domain mutation T366S, L368A, Y407V, or first CH3 domain mutation S354C, T366W and second CH3 domain mutation Y349 366S, L368A, Y V), DD/KK mutation (first CH3 domain mutation K409D, K D, second CH3 domain mutation D399K, E K), asymmetric re-engineering techniques (first CH3 domain mutation E K, E K, D399K and second CH3 domain mutation K E, K52409D), bib mutation (first CH3 domain mutation K249E, K E, the second CH3 domain mutation E236K, D278K), the XmAb mutation (the first CH3 domain mutation S364H, F405A, the second CH3 domain mutation Y349T, T F), the DuoBody mutation (the first CH3 domain mutation F405L, the second CH3 domain mutation K409R), the Azyme mutation (the first CH3 domain mutation T350V, L Y, S E, F405 35407V, the second CH3 domain mutation T350V, T366L, N390R, K392M, T394W), the bichonics mutation (the first CH3 domain mutation T366K (+L 351K), the second CH3 domain mutation L351D or E or D at Y349, L368 or Y349+R 355), ZW1 mutation (first CH3 domain mutation T350V, L351Y, F405A, Y V, second CH3 domain mutation T350V, T366L, K392L, T394W), 7.8.60 mutation (first CH3 domain mutation K360D, D399M, Y a, second CH3 domain mutation E345R, Q R, T366V, K V), EW-RVT mutation (first CH3 domain mutation K360E, K409W and second CH3 domain mutation Q347R, D399V, F T), EW-RVTs-s mutation (first CH3 domain mutation K360E, K W, Y C and second CH3 domain mutation Q347R, D399V, F399T, S C), SEED mutation (first CH3 domain mutation 45 residues on IgG1 CH3 and 57 residues on IgA CH3 domain mutation 1), a107 mutation (first CH3 domain mutation K360K 37W 6337W 357W).
Connector (L)
Different modules of the polypeptide chains disclosed herein may be associated with each other via a linker.
In some embodiments, the linker for joining one or more modules of a polypeptide chain is not a cleavable linker.
In one exemplary embodiment, the linker (Lc) located immediately adjacent to the C-terminus of the dimerization domain does not comprise a cleavable linker.
In another exemplary embodiment, at least one of the linkers located between the two antigen binding domains does not comprise a cleavable linker.
In other embodiments, the linker for joining one or more modules of the polypeptide chain may comprise a non-cleavable linker.
In one exemplary embodiment, the linker located immediately adjacent to the C-terminus of the dimerization domain is a non-cleavable linker.
In another exemplary embodiment, at least one of the linkers located between the two antigen binding domains is a non-cleavable linker.
In another exemplary embodiment, the linker located immediately adjacent to the C-terminus of the dimerization domain and the linker joining the first two antigen binding domains located at the C-terminus of the dimerization domain are non-cleavable linkers.
In some embodiments, the linker immediately adjacent to the N-terminus of the dimerization domain preferably comprises the hinge region of the antibody.
In some embodiments, the hinge region is a natural hinge region.
In some embodiments, the native hinge region is a native IgG1 hinge region. In some embodiments, the native hinge region is a native human IgG1 hinge region.
In some embodiments, the native hinge region is a native IgG2 hinge region. In other embodiments, the native hinge region is a native human IgG2 hinge region.
In some embodiments, the native hinge region is a native IgG3 hinge region. In other embodiments, the native hinge region is a native human IgG3 hinge region.
In some embodiments, the native hinge region is a native IgG4 hinge region. In some embodiments, the native hinge region is a native human IgG4 hinge region.
In some embodiments, the hinge region is a mutant hinge region.
In some embodiments, the mutant hinge region is a mutant IgG1 hinge region. In some embodiments, the mutant hinge region is a mutant human IgG1 hinge region.
In some embodiments, the mutant hinge region is a mutant IgG2 hinge region. In other embodiments, the mutant hinge region is a mutant human IgG2 hinge region.
In some embodiments, the mutant hinge region is a mutant IgG3 hinge region. In other embodiments, the mutant hinge region is a mutant human IgG3 hinge region.
In some embodiments, the mutant hinge region is a mutant IgG4 hinge region. In some embodiments, the mutant hinge region is a mutant human IgG4 hinge region.
In some embodiments, all of the modules of the polypeptide chain are linked via a non-cleavable linker.
Exemplary embodiments of non-cleavable linkers include those that remain substantially intact during protein expression or during the manufacturing process. As used herein, "substantially intact" means that linker cleavage occurs in 20% or less, 15% or less, 10% or less, 7.5% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less of the total polypeptide content of a given solution or composition.
Other exemplary embodiments of non-cleavable linkers also include linkers that do not comprise specific cleavage sites for one or more proteases present in human or animal blood or serum.
Other exemplary embodiments of non-cleavable linkers also include linkers that retain their integrity for at least one, two, three, four, five, six, twelve, twenty four, forty eight hours or more after administration of the binding agent to the individual.
In further exemplary embodiments, the linker comprises a non-cleavable linker and a cleavable linker.
In some embodiments, the linker is non-cleavable.
In some cases, cleavable linkers can be used to release a drug (e.g., cytostatic molecule, cytotoxic molecule, chemotherapeutic agent, etc.) or label attached to a polypeptide chain of the invention in vivo.
Exemplary embodiments of cleavable linkers are provided, for example, in US2019/0010242, and include linkers that are susceptible to cleavage by a protease, typically an extracellular protease, such as a protease produced by a tumor or activated immune effector cell, and include linkers having sites that can be specifically cleaved by a protease selected from ADAMS, ADAMTS, e.g., ADAMS; ADAMS; ADAM10; ADAM12; ADAM15; ADAM17/TACE; ADAMDEC1; ADAMTS1; ADAMTS4; ADAMTS5; aspartic proteases, such as BACE or renin; aspartic cathepsins, such as cathepsin D or cathepsin E; an apoptotic protease, such as apoptotic protease 1, apoptotic protease 2, apoptotic protease 3, apoptotic protease 4, apoptotic protease 5, apoptotic protease 6, apoptotic protease 7, apoptotic protease 8, apoptotic protease 9, apoptotic protease 10 or apoptotic protease 14; cysteine cathepsins, e.g., cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V/L2, cathepsin X/Z/P; cysteine proteases, such as Cruzipain (Cruzipain); asparagine endopeptidase (Legumain); otubain-2; KLKs, for example KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 or KLK14; metalloproteinases, such as transmembrane peptidase (Meprin); enkephalinase (Neprilysin); PSMA; BMP-1; MMPs, such as MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26 or MMP27, serine proteases, such as activated protein C, cathepsin a, cathepsin G, chymosin, coagulation factor proteases (e.g. FVIIa, FIXa, FXa, FXIa, FXIIa), elastase, granzyme B, guanidinobenzase, htrA1, human neutrophil elastase, lactoferrin, marapsin, NS3/4A, PACE4, plasmin, PSA, tPA, thrombin, neutral protease, uPA; type II transmembrane serine proteases (TTSPs), such as DESC1, DPP-4, FAP, heparin (Hepsin), matriase-2, matriase, TMPRSS2, TMPRSS3 or TMPRSS4; and any combination thereof. In some embodiments, the polypeptide chain of the invention does not include such a linker at a position corresponding to Lc.
Exemplary embodiments of the connectors include flexible connectors, rigid connectors, screw connectors, and combinations thereof. For example, the linker is discussed in Chen X et al (advanced Drug delivery review (Adv Drug Deliv Rev.) 2013;65 (10): 1357-1369), the entire contents of which are incorporated herein by reference.
In some embodiments, the hinge region or a portion thereof can be used to connect a module to a dimerization domain and is considered herein a linker. The hinge region may be derived from a natural antibody (of human or animal origin) or a synthetic antibody. The hinge region may be obtained, for example, from an IgG such as IgG1, igG2, igG3 or IgG 4. Exemplary embodiments of hinge regions are provided in SEQ ID NO. 98, SEQ ID NO. 121, SEQ ID NO. 125 and SEQ ID NO. 129.
In some cases, the hinge region can have one or more amino acid substitutions, amino acid insertions, and/or amino acid deletions as compared to the native hinge region. The mutant hinge region includes, for example, a sequence 80% to 99% identical to the sequence of the native IgG1, igG2, igG3 or IgG4 hinge region (mutant hinge region). Exemplary and non-limiting embodiments of the mutated hinge region include hinge regions of IgG4 wherein S228 is substituted with P (EU numbering) (Angal, S et al, molecular immunology (Mol Immunol) 30,105-108,1993). Other exemplary embodiments of mutant hinges are provided in SEQ ID NOS.118-120, 122-124, 126-128, and 130-132.
Flexible linkers are typically composed of small polar amino acids such as threonine or serine and glycine. Illustrative and non-limiting embodiments of flexible linkers include GS linkers (glycine/serine repeats), e.g., GGGS n (GGGGS) m 、(GS) n 、(G 4 S) n 、(GGS) n 、(GGGS) n 、(GGGGS) n 、(GGSG) n 、(GGGSS) n Wherein n and m may be integers such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater such as 15. 20 or 25.
Specific illustrative and non-limiting embodiments of flexible linkers include those comprising the amino acid sequences set forth in SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, or SEQ ID NO: 104.
It will be appreciated that SEQ ID NO 104 may be represented by the formula (GGGGS) n Wherein n is an integer selected from 1 to 10, or is represented by the formula GGGGSX 1 Represented by, wherein X 1 1 to 9 repeats of amino acid residues 1 to 5 of SEQ ID NO. 104, either absent or if present.
The rigid linker of the invention is typically composed of a proline-rich sequence (XP) n Composition, wherein X represents any amino acid, preferably Ala, lys or Glu, and n is an integer such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. (Chen X et al, 2013).
Specific illustrative and non-limiting embodiments of rigid linkers include those comprising the amino acid sequences set forth in SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, or SEQ ID NO: 108.
It will be appreciated that SEQ ID NO:108 may be represented by formula (X (PAPAAP)) n KA, wherein n is an integer selected from 1 to 10, wherein X is present or absent and, if present, A, or SEQ ID NO 108 may be represented by the formula (XPAPAP) X 2 KA represents, wherein X may be present or absent, and if present, is A; and wherein X is 2 1 to 9 repeats of amino acid residues 1 to 6 of SEQ ID NO. 108, either absent or if present.
Spiral linkers can sometimes be characterized as rigid, but are divided into different families of linkers herein. Exemplary embodiments of helical linkers are discussed in Chen X et al, 2013, and include repeats of alanine residues flanked by positively and negatively charged amino acid residues, for example.
Specific illustrative and non-limiting embodiments of the helical connector include those consisting of the amino acid sequences set forth in SEQ ID NO. 109, SEQ ID NO. 110, SEQ ID NO. 111, and SEQ ID NO. 112.
It will be appreciated that SEQ ID NO 112 may be represented by formula X (EAAAK) n X 2 Wherein n is an integer selected from 1 to 10, preferably 2-5, wherein X and X 2 Independently present or absent, and if present, is preferably a. Alternatively, SEQ ID NO 112 may be represented by the formula X (EAAAK) X 3 X 2 Wherein X and X are 2 Independently present or absent, and if present, is preferably a; and X is 3 1 to 9 repeats of amino acid residues 2 to 6 of SEQ ID NO. 112, either absent or if present.
In an exemplary embodiment, a linker immediately adjacent to the C-terminus of the dimerization domain (identified as L in formulas II through VIII c1 ) May comprise a flexible connector, a rigid connector or a screw connector. Linkers specifically selected to occupy this position include, for example and without limitation, linkers comprising or consisting of the amino acid sequences shown in SEQ ID NOS 100-109, SEQ ID NO 111, or SEQ ID NO 112, wherein n is 1.
In an exemplary embodiment, a linker (identified as L in formulas II through VIII) joining the first two antigen binding domains located C-terminal to the dimerization domain c2 ) May comprise a flexible connector, a rigid connector or a screw connector. Linkers that may be specifically selected to occupy this position include, for example and without limitation, linkers comprising or consisting of the amino acid sequences shown in SEQ ID NOS: 100-110 or SEQ ID NO:112, wherein n is 1.
The invention also provides an added linker having 1 to 10 amino acids at one or both of the N-or C-terminus of any one of SEQ ID NOs 100 to 112 (and any range or value comprised within 1 and 10, such as 1 to 5). These additional amino acid residues may each be independently selected from any amino acid residue. These additional amino acid residues preferably form a non-cleavable sequence.
The invention also provides a linker having a deletion of 1, 2, 3, 4 or 5 amino acids (and any value contained within 1 and 5) at one or both of the N-or C-terminus of any one of SEQ ID NOs 100 to 112.
Suitable linkers may comprise, for example, an amino acid sequence comprising from about 3 to about 50, from about 3 to about 40, from about 3 to about 30, from about 3 to about 25, from about 3 to about 20, from about 3 to about 15, from about 3 to about 10 amino acid residues.
In exemplary embodiments, each linker can independently have a length in the range of about 5 to about 50 amino acid residues, including, for example, about 5 to about 40 amino acid residues, about 10 to about 40 amino acid residues, about 20 to about 35 amino acid residues, about 25 to about 30 amino acid residues, and any subrange that includes and encompasses such ranges.
In some embodiments, a linker comprising the amino acid sequence shown in SEQ ID NO. 104, SEQ ID NO. 108 or SEQ ID NO. 112 may have an "n" value, preferably 1 to 10, more preferably 2-5, including 2, 3, 4 or 5.
Binding agent
Binding agents of the invention encompass, for example, the antigen binding domains disclosed herein.
Binding agents of the invention encompass, for example, polypeptide chains disclosed herein.
Binding agents of the invention encompass, for example, dimers of the polypeptide chains disclosed herein.
Binding agents of the invention encompass multimers of polypeptide chains such as those disclosed herein.
The binding agents of the invention may have the following pattern: antibodies and antigen binding fragments thereof, antibody-like molecules (Fc-, CH 3-fusions, etc.), fusions with protein scaffolds, immune cell modulators, etc.
In some embodiments, the binding agent comprises an antibody or antigen binding fragment thereof.
In some embodiments, the binding agent comprises an antibody-like molecule.
In some embodiments, the binding agent may be fused to a protein scaffold.
In some embodiments, the binding agent comprises an immune cell modulating agent.
Thus, in some embodiments, the binding agent includes antibodies and antigen binding fragments thereof, such as, and not limited to, single domain antibodies from camelidae or shark; human antibodies, including IgG (including human IgG1, human IgG2, human IgG3, human IgG 4), human IgM, human IgA (including human IgA1 and human IgA 2), human IgE, human IgD; animal antibodies, including, for example, igG (IgG 1, igG2a, igG2b, igG2c, igG3, igG 4), igM, igA, igE, and IgD.
In other embodiments, the binding agent includes antigen binding fragments, such as, but not limited to, fab ', F (ab') 2 Complementarity determining regions, variable regions, including VH, VHH, VL, and the like.
Binding agents also include immune cell modulators, such as dual affinity retargeting molecules (DART), chimeric Antigen Receptor (CAR) constructs, bispecific T cell cement constructs (BiTE), bispecific killer cell cement (BiKE), trispecific killer cell cement (TriKE) containing scFv or VHH.
Binding agents also include fusions with protein scaffolds, including ankyrin repeat proteins, Z-domains of staphylococcal protein-A, fibronectin type III, kinking mycotins (knottins), and the like. Exemplary embodiments of binding agents include monospecific, bispecific (symmetrical or asymmetrical) trispecific or multispecific antibodies as well as monovalent, bivalent, trivalent or multivalent antibodies, single chain FV (scFV) and derivatives, such as bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, tandem di-scFV vs, tandem tri-scFV vs, scFV-Fc, minibodies (scFV-CH 3), tandem diabodies, di-bifunctional antibodies, diabodies, and the like, VH or VHH and derivatives, such as tandem bispecific or multispecific VHH, bivalent VHH-Fc fusions, VHH-hinge-CH 2-CH3 fusions, bivalent CH3 fusions, VHH pentabodies, decabodies, and the like.
In some embodiments, the binding agents of the present invention may have a form of formula Ia, formula Ib, formula Ic, formula II, formula III, formula IV, formula V, formula VI, formula VII or formula VIII and the like as disclosed in PCT/CA2020/051753 filed on 18 of 12 months 2020 or formula I, formula II, formula III, formula IIIa and formula IIIb, formula IV, formula V, formula VI, formula VII or formula VIII as disclosed herein.
The binding agents of the invention may be formed by assembling two polypeptide chains having the same configuration (having the same or different amino acid sequences) or having different configurations, wherein the same or different configurations include the configurations described in formula I, formula II, formula III, formula IIIa and formula IIIb, formula IV, formula V, formula VI, formula VII, or formula VIII disclosed herein.
The binding agents of the invention may comprise, for example, one or more polypeptide chains independently comprising in N-terminal to C-terminal fashion an amino acid sequence of formula I:
X-[(Ab a )-(L b )] m -(DD)-[(L c )-(Ab d )] n -Y
wherein m is 0, 1, 2 or an integer greater than 2;
wherein n is 0, 1, 2 or an integer greater than 2;
wherein m and n are not simultaneously 0;
wherein Ab a 、Ab d Each represents an antigen binding domain, wherein at least one of the antigen binding domains is antigen binding domain 1 (ABD 1), antigen binding domain 2 (ABD 2), or antigen binding domain 3 (ABD 3);
Wherein X or Y is independently present or absent and comprises an amino acid sequence;
wherein L is b 、L c Each independently comprising one or more linkers; and is also provided with
Wherein DD represents the dimerization domain.
In some embodiments, L b May not be present. In some embodiments, L b2 And/or L b3 May not be present.
In some embodiments, L c May not be present. In some embodiments, L c1 、L c2 And/or L c3 May not be present.
In some embodiments, L b L and L c May not be present.
In some embodiments, the polypeptide chain comprises more than one antigen binding domain.
In some embodiments, the polypeptide chain comprises two or more antigen binding domains.
In some embodiments, the polypeptide chain comprises three or more antigen binding domains.
In some embodiments, the polypeptide chain comprises four or more antigen binding domains.
In some embodiments, the polypeptide chain comprises five or more antigen binding domains.
In some embodiments, the polypeptide chain comprises six or more antigen binding domains.
In some embodiments, the polypeptide chain comprises between one and twelve antigen binding domains.
In some embodiments, the binding agent may comprise an antigen binding domain between one and twelve, such as between one and two, between one and three, between one and four, between one and five, between one and six, between one and seven, between one and eight, between one and nine, between one and ten, between one and eleven, between one and twelve, between two and three, between two and four, between two and five, between two and six, between two and seven, between two and eight, between two and ten, between two and eleven, between two and twelve, between three and seven, between three and nine, between three and ten, between three and eleven, between three and twelve, between four and five, between four and six, between four and seven, between four and eight, between four and nine, between four and ten, between four and eleven, between four and twelve, between five and six, between five and seven, between five and eight, between five and nine, between five and twelve, between seven and nine, between seven and ten, between seven and eleven, between seven and twelve, between eight and nine, between eight and ten, between eight and eleven, between eight and twelve, between nine and ten, between nine and eleven, between nine and twelve, between ten and eleven, between ten and twelve, or between eleven and twelve.
In some embodiments, the binding agent comprises one polypeptide chain.
In some embodiments, the binding agent comprises two polypeptide chains.
In some embodiments, the binding agent comprises three polypeptide chains.
In some embodiments, the binding agent comprises four polypeptide chains.
In some embodiments, the binding agent comprises five polypeptide chains.
In some embodiments, the binding agent comprises six polypeptide chains.
In some embodiments, the binding agent comprises seven polypeptide chains.
In some embodiments, the binding agent comprises eight polypeptide chains.
In some embodiments, the binding agent comprises nine polypeptide chains.
In some embodiments, the binding agent comprises ten polypeptide chains.
In some embodiments, the binding agent comprises more than ten polypeptide chains.
In some embodiments, the binding agent comprises at least one polypeptide chain having formula II: x- (Ab) a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 ) Y (formula II).
In some embodiments, the binding agent comprises at least one polypeptide chain having formula III: x- (Ab) a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 ) Y (formula III).
In some embodiments, the binding agent comprises at least one ofA polypeptide chain having formula IV: x- (Ab) a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 ) Y (formula IV).
In some embodiments, the binding agent comprises at least one polypeptide chain having formula V: x- (Ab) a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 ) Y (formula V).
In some embodiments, the binding agent comprises at least one polypeptide chain having formula VI: x- (Ab) a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-(L c3 )-(Ab d3 ) Y (formula VI).
In some embodiments, the binding agent comprises at least one polypeptide chain having formula VII: x- (Ab) a1 )-(L b3 )-(Ab a2 )-(L b2 )-(Ab a3 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 ) Y (formula VII).
In some embodiments, the binding agent comprises at least one polypeptide chain having formula VIII: x- (Ab) a1 )-(L b3 )-(Ab a2 )-(L b2 )-(Ab a3 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-(L c3 )-(Ab d3 ) Y (formula VIII).
In some embodiments, the binding agent comprises one or more polypeptide chains, each of which independently comprises in N-terminal to C-terminal fashion an amino acid sequence of formula III:
X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 ) -Y (formula III);
wherein Ab a1 、Ab d1 Or Ab d2 Represents antigen binding domain 1 (ABD 1).
In some embodiments, the binding agent comprises one or more polypeptide chains, each of which independently comprises in N-terminal to C-terminal fashion an amino acid sequence of formula III:
X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 ) -Y (formula III);
wherein Ab a1 、Ab d1 Or Ab d2 Represents antigen binding domain 2 (ABD 2).
In some embodiments, the binding agent comprises one or more polypeptide chains, each of which independently comprises in N-terminal to C-terminal fashion an amino acid sequence of formula III:
X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 ) -Y (formula III);
wherein Ab a1 、Ab d1 Or Ab d2 Represents antigen binding domain 3 (ABD 3).
In some embodiments, the binding agent comprises one or more polypeptide chains, each of which independently comprises in N-terminal to C-terminal fashion an amino acid sequence of formula III:
X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 ) -Y (formula III);
wherein Ab a1 、Ab d1 Or Ab d2 One of them represents antigen binding domain 1 (ABD 1), ab a1 、Ab d1 Or Ab d2 One of them represents antigen binding domain 2 (ABD 2), and Ab a1 、Ab d1 Or Ab d2 Represents antigen binding domain 3 (ABD 3).
In some embodiments, the binding agent comprises one or more polypeptide chains, each of which independently comprises in N-terminal to C-terminal fashion an amino acid sequence of formula III:
X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 ) -Y (formula III);
wherein Ab a1 Representing antigen binding domain 1 (ABD 1), ab d1 Represents antigen binding domain 2 (ABD 2), and Ab d2 Representing antigen binding domain 3 (ABD 3).
In some embodiments, the binding agent comprises one or more polypeptide chains each independently comprising an amino acid sequence of formula III in N-to C-terminal fashion, wherein Ab a1 Is an antigen binding domain that binds DR2, ab d1 Is an antigen binding domain that binds to PD-1 and Ab d2 Is an antigen binding domain that binds CD 47.
In some embodiments, the binding agent comprises one or more polypeptide chains each independently comprising an amino acid sequence of formula III in N-to C-terminal fashion, wherein Ab a1 Is an antigen binding domain that binds DR2, ab d1 Is an antigen binding domain that binds to CD47 and Ab d2 Is an antigen binding domain that binds to PD-1.
In some embodiments, the binding agent comprises one or more polypeptide chains, each of which independently comprises in N-terminal to C-terminal fashion an amino acid sequence of formula IIIa:
X-(ABD1)-(L b1 )-(DD)-(L c1 )-(ABD2)-(L c2 ) - (ABD 3) -Y (formula IIIa).
In some embodiments, the binding agent comprises one or more polypeptide chains, each of which independently comprises in N-terminal to C-terminal fashion an amino acid sequence of formula IIIb:
X-(ABD1)-(L b1 )-(DD)-(L c1 )-(ABD3)-(L c2 ) - (ABD 2) -Y (formula IIIb).
In some embodiments, the binding agent comprises two identical polypeptide chains.
In some embodiments, the two polypeptide chains of the binding agent may have the conformation shown in formula II (with the same or different amino acid sequences).
In some embodiments, the two polypeptide chains of the binding agent may have the conformation shown in formula III (with the same or different amino acid sequences).
In some embodiments, the two polypeptide chains of the binding agent may have the conformation shown in formula IIIa (with the same or different amino acid sequences).
In some embodiments, the two polypeptide chains of the binding agent may have the conformation shown in formula IIIb (with the same or different amino acid sequences).
In some embodiments, one polypeptide chain can have the conformation shown in formula IIIa and one polypeptide chain can have the conformation shown in formula IIIb (wherein the antigen-binding domains have the same or different amino acid sequences).
In some embodiments, one of the polypeptide chains may have the conformation shown in formula II, while the other may have the conformation shown in formula III.
In some embodiments, one of the polypeptide chains may have the conformation shown in formula II, while the other has the conformation shown in formula IV.
In some embodiments, one of the polypeptide chains may have the conformation shown in formula III, while the other has the conformation shown in formula IV.
In some embodiments, one of the polypeptide chains may have the conformation shown in formula IV, while the other has the conformation shown in formula IV.
In some embodiments, the binding agents of the invention are monospecific or multispecific.
Monospecific binders encompass binders that are specific for a single epitope of a given antigen.
Exemplary embodiments of monospecific binders include binders comprising one antigen binding domain. Another exemplary embodiment of a monospecific binding agent comprises a binding agent comprising more than one antigen binding domain, but the antigen binding domains have the same CDRs and framework regions. Yet another exemplary embodiment of a binding agent chain includes a binding agent comprising more than one antigen binding domain, but the antigen binding domains have the same CDRs and different framework regions. Another exemplary embodiment of a monospecific binding agent includes a binding agent comprising an antigen binding domain whose amino acid sequence differs (e.g., conservative substitutions in one or more CDRs) without affecting its ability to bind to the same antigen or epitope.
Multispecific binders encompass binders that are specific for more than one epitope (epitopes of the same antigen or different antigens) or more than one antigen. For example, a multispecific polypeptide chain or binding agent may thus have more than one antigen-binding domain, at least two of which bind to different antigens or epitopes.
The binding agents of the present invention may thus be bispecific, trispecific, tetraspecific, penta-specific, hexa-specific, and the like. In some embodiments, each antigen binding domain may be specific for a given antigen. In some embodiments, two or more antigen binding domains of a given binding agent may be specific for the same or different antigens. In some embodiments, three or more antigen binding domains of a given binding agent may be specific for the same or different antigens. In some embodiments, four or more antigen binding domains of a given binding agent may be specific for the same or different antigens. In some embodiments, five or more antigen binding domains of a given binding agent may be specific for the same or different antigens. In some embodiments, six or more antigen binding domains of a given binding agent may be specific for the same or different antigens. The specificity may depend on the number of antigen binding domains present in a given binding agent.
Exemplary non-limiting embodiments of multispecific binders include those composed of multispecific polypeptide chains. Other exemplary non-limiting embodiments of multispecific binders include those having two antigen-binding domains of different specificities. Other exemplary non-limiting embodiments of multispecific binders include those having more than two antigen binding domains that bind to two different antigens, proteins, or to two different epitopes on the same antigen or protein.
In some embodiments, the binding agents of the invention are monovalent or multivalent.
Exemplary non-limiting embodiments of multivalent binding agents include binding agents that are comprised of multivalent polypeptide chains. Other non-limiting exemplary embodiments of multivalent binding agents include binding agents that are comprised of more than one monovalent polypeptide chain.
According to the invention, the bispecific binding agent may be bivalent or multivalent, depending on the number of antigen binding domains it contains. Exemplary non-limiting embodiments of bispecific binding agents include those comprising two identical bispecific polypeptide chains that form a dimer.
Exemplary embodiments of monospecific binders
As shown herein, the binding agents of the invention may be monospecific.
Exemplary and non-limiting exemplary embodiments of monospecific binders are provided herein.
For example, the binding agent may comprise only one antigen binding domain. Alternatively, the binding agent may comprise more than one identical antigen binding domain.
Exemplary embodiments of monospecific binders include antigen binding domains disclosed herein, including ABD1, ABD2, ABD3, or heavy chains disclosed herein.
In exemplary embodiments, a monospecific binding agent may comprise an antigen binding domain and a dimerization domain as disclosed herein.
Exemplary embodiments of dimerization domains are provided throughout the specification and include, but are not limited to, CH3 domains (including native CH3 domains and native human CH3 domains, mutant CH3 domains) may also include CH2 domains (native CH2 domains or mutant CH2 domains).
In some embodiments, the dimerization domain is the Fc region of an antibody or a portion thereof.
In some embodiments, the dimerization domain may also comprise a hinge region of an antibody or a portion thereof. In some embodiments, the hinge region or portion thereof is a human hinge region or portion thereof.
In some embodiments, the binding agent may comprise ABD1 and a dimerization domain.
In some embodiments, the binding agent may comprise ABD2 and a dimerization domain.
In some embodiments, the binding agent may comprise ABD3 and a dimerization domain.
In some exemplary embodiments, the binding agents of the invention may comprise heavy chains and dimerization domains having amino acid sequences at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequences set forth in SEQ ID NO. 7.
In some exemplary embodiments, the binding agents of the invention may comprise heavy chains and dimerization domains having amino acid sequences at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequences set forth in SEQ ID NO. 14.
In some exemplary embodiments, the binding agents of the invention may comprise heavy chains and dimerization domains having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 21.
In some exemplary embodiments, the binding agents of the invention may comprise heavy chains and dimerization domains having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 28.
In some exemplary embodiments, the binding agents of the invention may comprise heavy chains and dimerization domains having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 35.
In some exemplary embodiments, the binding agents of the invention may comprise heavy chains and dimerization domains having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 42.
In some exemplary embodiments, the binding agents of the invention may comprise heavy chains and dimerization domains having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 49.
In some exemplary embodiments, the binding agents of the invention may comprise heavy chains and dimerization domains having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 56.
In some exemplary embodiments, the binding agents of the invention may comprise heavy chains and dimerization domains having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 63.
In some exemplary embodiments, the binding agents of the invention may comprise heavy chains and dimerization domains having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 70.
In some exemplary embodiments, the binding agents of the invention may comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 71.
In some exemplary embodiments, the binding agents of the invention may comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 72.
In some exemplary embodiments, the binding agents of the invention may comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 73.
In some exemplary embodiments, the binding agents of the invention may comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 74.
In some exemplary embodiments, the binding agents of the invention may comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 75.
Other exemplary embodiments of monospecific binders are provided herein.
Exemplary embodiments of multispecific binding agents
As described herein, the binding agents of the invention may be multispecific.
Exemplary and non-limiting exemplary embodiments of multispecific binders are provided herein.
For example, in some embodiments, the binding agent may comprise more than one antigen binding domain, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) as described herein and at least one antigen binding domain is an antigen binding domain capable of binding to an immune checkpoint protein.
In other embodiments, the binding agent may comprise more than one antigen binding domain, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) as described herein, at least one antigen binding domain is an antigen binding domain capable of binding to an immune checkpoint protein, and at least one antigen binding domain is an antigen binding domain capable of binding to a protein expressed on the surface of an immune cell.
In other embodiments, the binding agent may comprise more than one antigen binding domain, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) as described herein, at least one antigen binding domain is an antigen binding domain capable of binding to an immune checkpoint protein, and at least one antigen binding domain is an antigen binding domain capable of binding to a protein expressed on the surface of a tumor cell.
In some embodiments, the binding agent comprises at least one antigen binding domain 1 (ABD 1) and at least one antigen binding domain 2 (ABD 2).
In some embodiments, the binding agent comprises at least one antigen binding domain 1 (ABD 1) and at least one antigen binding domain 3 (ABD 3).
In some embodiments, the binding agent comprises at least one antigen binding domain 2 (ABD 2) and at least one antigen binding domain 3 (ABD 3).
In some embodiments, the binding agent comprises at least one antigen binding domain 1 (ABD 1), at least one antigen binding domain 2 (ABD 2), and at least one antigen binding domain 3 (ABD 3).
In some embodiments, the binding agent comprises one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 7, at least one antigen binding domain is antigen binding domain 2 (ABD 2) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 63, and at least one antigen binding domain is antigen binding domain 3 (ABD 3) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 70.
In some embodiments, the binding agent comprises one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 14, at least one antigen binding domain is antigen binding domain 2 (ABD 2) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 63, and at least one antigen binding domain is antigen binding domain 3 (ABD 3) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 70.
In some embodiments, the binding agent comprises one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 21, at least one antigen binding domain is antigen binding domain 2 (ABD 2) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 63, and at least one antigen binding domain is antigen binding domain 3 (ABD 3) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 70.
In some embodiments, the binding agent comprises one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 28, at least one antigen binding domain is antigen binding domain 2 (ABD 2) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 63, and at least one antigen binding domain is antigen binding domain 3 (ABD 3) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 70.
In some embodiments, the binding agent comprises one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 35, at least one antigen binding domain is antigen binding domain 2 (ABD 2) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 63, and at least one antigen binding domain is antigen binding domain 3 (ABD 3) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 70.
In some embodiments, the binding agent comprises one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 42, at least one antigen binding domain is antigen binding domain 2 (ABD 2) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 63, and at least one antigen binding domain is antigen binding domain 3 (ABD 3) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 70.
In some embodiments, the binding agent comprises one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 49, at least one antigen binding domain is antigen binding domain 2 (ABD 2) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 63, and at least one antigen binding domain is antigen binding domain 3 (ABD 3) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 70.
In some embodiments, the binding agent comprises one or more antigen binding domains, wherein at least one antigen binding domain is antigen binding domain 1 (ABD 1) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 56, at least one antigen binding domain is antigen binding domain 2 (ABD 2) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 63, and at least one antigen binding domain is antigen binding domain 3 (ABD 3) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 70.
In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one polypeptide chain has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO 77. In some embodiments, the binding agent comprises a polypeptide chain having the amino acid sequence set forth in SEQ ID NO. 77. In other embodiments, the binding agent comprises two polypeptide chains having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 77. In other embodiments, the binding agent comprises two polypeptide chains having the amino acid sequence set forth in SEQ ID NO. 77.
In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one polypeptide chain has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 78. In some embodiments, the binding agent comprises a polypeptide chain having the amino acid sequence set forth in SEQ ID NO. 78. In other embodiments, the binding agent comprises two polypeptide chains having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 78. In other embodiments, the binding agent comprises two polypeptide chains having the amino acid sequence set forth in SEQ ID NO. 78.
In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one polypeptide chain has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 79. In some embodiments, the binding agent comprises a polypeptide chain having the amino acid sequence set forth in SEQ ID NO. 79. In other embodiments, the binding agent comprises two polypeptide chains having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 79. In other embodiments, the binding agent comprises two polypeptide chains having the amino acid sequence set forth in SEQ ID NO. 79.
In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one polypeptide chain has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 80. In some embodiments, the binding agent comprises a polypeptide chain having the amino acid sequence set forth in SEQ ID NO. 80. In other embodiments, the binding agent comprises two polypeptide chains having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 80. In other embodiments, the binding agent comprises two polypeptide chains having the amino acid sequence set forth in SEQ ID NO. 80.
In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one polypeptide chain has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 81. In some embodiments, the binding agent comprises a polypeptide chain having the amino acid sequence set forth in SEQ ID NO. 81. In other embodiments, the binding agent comprises two polypeptide chains having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 81. In other embodiments, the binding agent comprises two polypeptide chains having the amino acid sequence set forth in SEQ ID NO. 81.
In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one polypeptide chain has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 82. In some embodiments, the binding agent comprises a polypeptide chain having the amino acid sequence set forth in SEQ ID NO. 82. In other embodiments, the binding agent comprises two polypeptide chains having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 82. In other embodiments, the binding agent comprises two polypeptide chains having the amino acid sequence set forth in SEQ ID NO. 82.
In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one polypeptide chain has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO 83. In some embodiments, the binding agent comprises a polypeptide chain having the amino acid sequence set forth in SEQ ID NO. 83. In other embodiments, the binding agent comprises two polypeptide chains having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 83. In other embodiments, the binding agent comprises two polypeptide chains having the amino acid sequence set forth in SEQ ID NO. 83.
In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one polypeptide chain has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 85. In some embodiments, the binding agent comprises a polypeptide chain having the amino acid sequence set forth in SEQ ID NO. 85. In other embodiments, the binding agent comprises two polypeptide chains having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 85. In other embodiments, the binding agent comprises two polypeptide chains having the amino acid sequence set forth in SEQ ID NO. 85.
In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one polypeptide chain has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 86. In some embodiments, the binding agent comprises a polypeptide chain having the amino acid sequence set forth in SEQ ID NO. 86. In other embodiments, the binding agent comprises two polypeptide chains having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 86. In other embodiments, the binding agent comprises two polypeptide chains having the amino acid sequence set forth in SEQ ID NO. 86.
In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one polypeptide chain has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 87. In some embodiments, the binding agent comprises a polypeptide chain having the amino acid sequence set forth in SEQ ID NO. 87. In other embodiments, the binding agent comprises two polypeptide chains having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 87. In other embodiments, the binding agent comprises two polypeptide chains having the amino acid sequence set forth in SEQ ID NO. 87.
In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one polypeptide chain has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 88. In some embodiments, the binding agent comprises a polypeptide chain having the amino acid sequence set forth in SEQ ID NO. 88. In other embodiments, the binding agent comprises two polypeptide chains having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 88. In other embodiments, the binding agent comprises two polypeptide chains having the amino acid sequence set forth in SEQ ID NO. 88.
Other exemplary embodiments of multispecific binders are provided herein, including in table 7.
In some embodiments, the binding agents of the invention comprise an antigen binding domain that binds DR2, an antigen binding domain that binds PD1, and an antigen binding domain that binds CD 47. In some embodiments, the binding agents of the invention may be capable of bringing T cells into proximity with tumor cells expressing DR2, may be capable of restoring T cell function and/or may be capable of enhancing macrophage function via blocking sirpa/CD 47 interactions. In some embodiments of the invention, the binding agent may exert its anti-tumor activity via antibody-dependent cell-mediated cytotoxicity (ADCC).
Variants
Variants of the sequences disclosed herein are also encompassed by the present invention.
Variants encompassed by the present invention include variants (conservative or non-conservative substitutions) which may comprise insertion of one or more amino acid residues at one or more positions, deletion of one or more amino acid residues at one or more positions, or substitution of one or more amino acid residues at one or more positions.
For example, naturally occurring residues are grouped into groups based on common side chain characteristics. Conservative substitutions may be made by substituting an amino acid from one of the following groups (groups 1 to 6) with another amino acid of the same group. Non-conservative substitutions will require the replacement of one member of the group with another.
(group 1) hydrophobicity: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile)
(group 2) neutral hydrophilicity: cysteine (Cys), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln),
(group 3) acidity: aspartic acid (Asp), glutamic acid (Glu)
(group 4) basicity: histidine (His), lysine (Lys), arginine (Arg)
(group 5) residues affecting chain orientation: glycine (Gly), proline (Pro); a kind of electronic device with high-pressure air-conditioning system
(group 6) aromatic: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe)
Other exemplary embodiments of conservative substitutions are shown in table 2 under the heading "preferred substitutions". If such substitutions result in undesirable properties, more substantial changes may be introduced, designated as "exemplary substitutions" in Table 2, or as described further below with reference to amino acids, respectively, and the products screened.
Those skilled in the art will recognize that certain amino acids are less positively charged, neutral, negatively charged, or have a reduced charge compared to other amino acids. Amino acids can be classified according to their net charge as indicated by their isoelectric point. Isoelectric point is the pH at which the average net charge of an amino acid molecule is zero. Amino acids have a net negative charge when pH > pI, and a net positive charge when pH < pI. In some embodiments, the measured pI value of the antibody is between about 3 and 9 (e.g., 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, and 9) and any value therebetween. In some embodiments, the measured pI value of the antibody is between about 4 and 7 (e.g., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0) and any value in between. Exemplary isoelectric points of amino acids are shown in table 2 below. Amino acids typically having positively charged side chains include, for example, arginine (R), histidine (H), and lysine (K). Amino acids having negatively charged side chains include, for example, aspartic acid (D) and glutamic acid (E). Amino acids having polar properties include, for example, serine (S), threonine (T), asparagine (N), glutamine (Q), and cysteine (C), tyrosine (Y), and tryptophan (W). Nonpolar amino acids include, for example, alanine (a), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), glycine (G), and proline (P).
In some embodiments, the isoelectric point of the antibody is altered via amino acid substitution. See, for example, US20110076275. In some embodiments, modifying the isoelectric point of the polypeptide comprising the antibody results in a change in the half-life of the antibody.
TABLE 2 exemplary amino acid substitutions
In general, the degree of similarity and identity between variable strands is determined herein using the Blast 2sequence program (Tatiana A. Tatusova, thomas L. Madden (1999), "Blast2 sequence-a new tool for comparing protein and nucleotide sequences (Blast 2sequences-a new tool for comparing protein and nucleotide sequences)", FEMS Microbiol Lett. Report (FEMS Microbiol Lett.)) 174:247-250, using preset settings, namely the blastp program, BLOSUM62 matrix (open gap 11 and extended gap penalty 1; gap. Times. Decay 50, expected 10.0, word length 3) and activated filters.
The percent identity will therefore be indicative of amino acids that are identical and likely occupy the same or similar positions as compared to the original peptide.
Percent similarity will indicate amino acids that are identical and amino acids that are substituted with conservative amino acid substitutions as compared to the original peptide at the same or similar positions.
Thus, a variant of the invention may comprise a sequence that is at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the original or reference sequence or a portion of the original sequence.
In some embodiments, a polypeptide chain having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% identical or less than 100% identical to a given amino acid sequence may have amino acid substitutions, additions or deletions that are typically located outside of the complementarity determining regions.
In some embodiments, the variant may have at least 80% sequence identity to a sequence disclosed herein. In other embodiments, the variant may have at least 85% sequence identity to a sequence disclosed herein. In yet other embodiments, the variants may have at least 90% sequence identity to the sequences disclosed herein. In further embodiments, the variants may have at least 95% sequence identity to the sequences disclosed herein. In other embodiments, the variants may have at least 99% sequence identity to the sequences disclosed herein.
Exemplary embodiments of variants include polypeptide chains or binders comprising a hinge region, fc, CH3, CH2/CH3 region derived from a natural antibody but comprising one, two, three, four, five, six, seven, eight, nine, ten or more amino acid differences (including amino acid substitutions, insertions or deletions).
In some embodiments, the polypeptide chains of the invention may thus comprise a mutant hinge region that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical to the hinge region of the native antibody.
In some embodiments, the polypeptide chains of the invention may thus comprise a mutant Fc portion that is at least 80% identical to the Fc of a native antibody.
In some embodiments, the polypeptide chain of the invention may thus comprise a mutant CH2 domain that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical to the CH2 domain of the native antibody.
In some embodiments, the polypeptide chain of the invention may thus comprise a mutant CH3 domain that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical to the CH3 domain of the native antibody.
In some embodiments, the polypeptide chain of the invention may thus comprise a mutant CH2/CH3 domain that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical to the CH2/CH3 domain of the native antibody.
Nucleic acid, vector, kit, cell and method for producing polypeptide chain
The nucleic acid molecules of the invention may be single-stranded or double-stranded. The nucleic acid molecules disclosed herein may comprise deoxyribonucleotides, ribonucleotides, modified deoxyribonucleotides, or modified ribonucleotides. The nucleic acid molecules of the invention may comprise, for example, DNA.
In particular, DNA segments and vectors encoding one or more modules or the entire polypeptide chain are provided.
The DNA segments and/or vectors may be provided in separate vials and sold as a kit.
Of particular concern are cloning vectors that contain groups of DNA segments that allow for the sequence of the assembly module to be oriented, and that incorporate DNA segments or the entire polypeptide chain.
The DNA segments and vectors may be provided as part of a kit for assembling a DNA construct capable of expressing the polypeptide chains or binding agents disclosed herein.
The kit may comprise at least one or more DNA segments or vectors that allow a user to generate a polypeptide chain (according to the EU numbering system) comprising a mutant dimerization domain having amino acid substitutions at positions 356, 357, 370, 399 and/or 439 as disclosed herein.
Because of the inherent degeneracy of the genetic code, DNA sequences encoding identical, substantially identical or functionally equivalent amino acid sequences may be produced and used. The nucleotide sequences of the present invention may be engineered using methods well known in the art to alter the nucleotide sequence for a variety of purposes, including, but not limited to, cloning, processing and/or modification of expression products of genes. DNA shuffling by random fragmentation and PCR recombination of gene fragments and synthetic oligonucleotides can be used to engineer nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, alter codon preference, create splice variants, and the like. The present invention encompasses codon optimized nucleic acids encoding the polypeptide chains described herein.
The polypeptide chains and binding agents disclosed herein may be prepared by a variety of methods familiar to those skilled in the art, including by recombinant DNA methods or by in vitro transcription/translation.
In general, the polypeptide chains described herein are expressed from a nucleic acid sequence inserted into an expression vector, i.e., a vector containing components for transcriptional and translational control of an inserted coding sequence in a particular host. These modules may include regulatory sequences such as enhancers, constitutive and inducible promoters, and 5 'and 3' untranslated regions.
A variety of expression vector/host cell systems known to those of skill in the art may be used to express the polypeptide chains described herein. Where the binding agent is composed of different polypeptide chains, each such polypeptide chain may be provided by a separate expression vector or by a unique expression vector. According to the invention, both strands of the binding agent may be encoded by a single vector or by separate vectors (vector sets).
The polypeptide is typically expressed in mammalian cells. For long-term production of recombinant proteins, stable expression systems can be used in which the DNA segment is integrated into the host cell genome or maintained in episomal form by the use of selectable markers. The host cell type may be selected for its ability to modulate the expression of the inserted sequence or process the expressed polypeptide in a desired manner. Different host cells (e.g., CHO, heLa, MDCK, HEK293 and W138) with specific cellular mechanisms and mechanisms characteristic of post-translational activity are commercially available from the American Type Culture Collection (ATCC) and can be selected to ensure proper modification and processing of expressed polypeptides.
Other types of expression systems may be used. These types include, for example, microorganisms, such as bacteria transformed with recombinant phage, plasmid, or cosmid DNA expression vectors; yeast transformed with a yeast expression vector; insect cell systems infected with baculovirus vectors; a plant cell system transformed with a viral or bacterial expression vector; or an animal cell system.
Thus, the present invention relates to isolated cells transformed or transfected with a vector, nucleic acid, set of vectors, or set of nucleic acids encoding at least one of the polypeptide chains described herein. Thus, the invention also relates to isolated cells capable of expressing the polypeptide chains or binding agents disclosed herein.
The invention also relates to a method of manufacturing the binding agent. The method can comprise providing a vector or set of vectors encoding one or more polypeptide chains disclosed herein to a cell (e.g., a mammalian cell), and allowing expression.
The method may further comprise purifying the binding agent from the cells or cell debris.
In some embodiments, the manufacturing process allows a purity level of at least 80%, at least 85%, at least 90%, at least 99% (dimer) to be achieved.
In some embodiments, the purity level of the binding agent is between 80.0% and 99.9%.
In some embodiments, the purity level of the binding agent is between 95.0% and 99.9%.
In some embodiments, the purity level of the binding agent is at least 90.0+/-5.0%.
In some embodiments, the purity level of the binding agent is at least 95.0+/-5.0%.
In some embodiments, the purity level of the binding agent is equal to or greater than 95%.
In some embodiments, the purity level of the binding agent is 97.0+/-1.0%.
In some embodiments, the purity level of the binding agent is equal to or greater than 97%.
In some embodiments, the purity level of the binding agent is 99.0+/-1.0%.
In some embodiments, the purity level of the binding agent is equal to or greater than 99%.
In some embodiments, the titer of the binding agent produced by the cells may be 0.1g/L or greater. In some cases, the titer of the binding agent produced by the cells may be 0.5g/L or greater. In some cases, the titer of the binding agent produced by the cells may be 1g/L or greater. In some cases, the titer of the binding agent produced by the cells may be 2g/L or greater. In some cases, the titer of the binding agent produced by the cells may be 3g/L or greater. In some cases, the titer of the binding agent produced by the cells may be 4g/L or greater. In some cases, the titer of the binding agent produced by the cells may be 5g/L or greater. In some cases, the titer of the binding agent produced by the cells may be 6g/L or greater. Typically, homodimers are made by transfecting cells with a vector comprising a nucleic acid sequence encoding one of the polypeptide chains disclosed herein. The collected supernatant may contain homodimers or a mixture of monomers and/or homodimers.
Typically, heterodimers are prepared by co-transfecting cells with at least two types of vectors (vector sets), each comprising nucleic acid sequences encoding two different polypeptide chains. The appropriate ratio of chain a to chain B generally depends on the protein expression level obtained from each individual plasmid and may vary from, for example, about 1:10 to about 10:1. For some of the constructs disclosed herein, a DNA ratio of about 1:1 is particularly preferred.
Heterodimers can also be prepared by transfecting cells with a single vector encoding two polypeptide chains. The collected supernatant may contain heterodimers or mixtures of monomers, heterodimers and/or homodimers.
The method of preparing a polypeptide chain of the present invention may further comprise the step of separating or isolating the monomers, homodimers and heterodimers from the mixture comprising. The homodimers or heterodimers can be purified and isolated, for example, by size exclusion chromatography or with the aid of a label or by other methods known to those skilled in the art.
The method may further comprise the step of separating and/or purifying the binding agent from the impurities.
Thus, the process of the present invention will produce a composition comprising a homodimer, a heterodimer or a mixture of monomeric heterodimers and/or homodimers.
In some exemplary embodiments, the composition may comprise predominantly homodimers. In an exemplary embodiment, the composition may comprise homodimers in a ratio of at least about 80%, at least 85%, at least 90%, at least 99%, or 100%.
In other exemplary embodiments, the composition may comprise predominantly heterodimers. In an exemplary embodiment, the composition may comprise heterodimers in a ratio of at least about 80%, at least 85%, at least 90%, at least 99%, or 100%.
Conjugate(s)
The polypeptide chains or binding agents of the invention may be conjugated, for example, to a therapeutic moiety (for therapeutic purposes) or to a detectable moiety (i.e., for detection or diagnostic purposes) or to a protein that allows for an extended half-life or attached to a nanoparticle. In some cases, the therapeutic or detectable moiety may be attached to at least one amino acid residue of the polypeptide chain.
In an exemplary embodiment, the polypeptide chains or binding agents of the invention are conjugated to a therapeutic moiety, such as, but not limited to, a chemotherapeutic agent, a cytokine, a cytotoxic agent, an anticancer agent (e.g., a small molecule), and the like.
The therapeutic moiety may include, for example and without limitation, yttrium-90, scandium-47, rhenium-186, iodine-131, iodine-125, and those of skill in the art Many other materials that are approved (e.g., lutetium (e.g., lu 177 ) Bismuth (e.g., bi) 213 ) Copper (e.g. Cu 67 ) 5-fluorouracil, adriamycin (adriamycin), anticancer toxan (irinotecan), taxane (taxane), pseudomonas endotoxin (pseudomonas endotoxin), ricin (ricin), auristatin (auristatin) (e.g., monomethyl auristatin E, monomethyl auristatin F), maytansine (e.g., maytansine), and other toxins.
In another exemplary embodiment, the polypeptide chains or binding agents of the present invention are conjugated to a detectable moiety, including for example and without limitation, moieties that are detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical and/or other physical means. The detectable moiety may be conjugated to the polypeptide chain or binding agent directly and/or indirectly (e.g., via a linkage such as, but not limited to, DOTA or NHS linkage) using methods well known in the art. A variety of detectable moieties may be used, the choice depending on the sensitivity desired, ease of conjugation, stability requirements, and available instrumentation. Suitable detectable moieties include, but are not limited to, fluorescent labels, radioactive labels (e.g., and without limitation 125 I、In 111 、Tc 99 、I 131 And include positron emitting isotopes for PET scanners, etc.), nuclear magnetic resonance active labels, luminescent labels, chemiluminescent labels, chromophore labels, enzyme labels (such as, but not limited to, horseradish peroxidase, alkaline phosphatase, etc.), quantum dots, and/or nanoparticles. The detectable moiety may cause and/or generate a detectable signal, allowing detection of the signal from the detectable moiety.
Pharmaceutical composition
Pharmaceutical compositions comprising the polypeptide chains or binding agents of the invention are also encompassed by the invention. The pharmaceutical composition may also comprise a pharmaceutically acceptable carrier.
In some embodiments, the pharmaceutical composition comprises a conjugated polypeptide chain or conjugated binding agent as disclosed herein. In some embodiments, the pharmaceutical composition comprises a polypeptide chain or binding agent conjugated to a therapeutic moiety. In some embodiments, the pharmaceutical composition comprises a polypeptide chain or binding agent conjugated to a detectable label.
In addition to the active ingredient, the pharmaceutical compositions may contain pharmaceutically acceptable carriers, including water, PBS, saline solutions, gelatin, oils, alcohols, and other excipients, and adjuvants that facilitate the processing of the active compound into a pharmaceutically acceptable formulation. In other cases, such formulations may be sterilized.
As used herein, "pharmaceutical composition" refers to a therapeutically effective amount of an agent as well as pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. As used herein, "therapeutically effective amount" refers to an amount that provides a therapeutic effect for a given condition and dosing regimen. Such compositions are liquid or lyophilized or otherwise dried formulations and include diluents of various buffer contents (e.g., tris-HCl, acetate, phosphate), pH, and ionic strength; additives that prevent surface absorption, such as albumin or gelatin; detergents (e.g., tween 20, tween 80, pluronic F68, bile acid salts). Solubilizing agents (e.g., glycerol, polyethylene glycerol); antioxidants (e.g., ascorbic acid, sodium metabisulfite); preservatives (e.g., thimerosal, benzyl alcohol, parabens); fillers or tonicity modifiers (e.g. lactose, mannitol); covalent attachment of polymers such as polyethylene glycol to proteins; complexing with metal ions; or incorporating the material into or onto a polymeric compound microparticle formulation such as polylactic acid, polyglycolic acid, hydrogel, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte pannus, or spherical plasmids. Such compositions will affect physical state, solubility, stability, in vivo release rate, and in vivo clearance rate. Controlled release or sustained release compositions include formulations in lipophilic reservoirs (e.g., fatty acids, waxes, oils). The invention also encompasses microparticle compositions coated with a polymer, such as poloxamer (poloxamer) or poloxamer (poloxameramine). Other embodiments of the compositions of the present invention incorporate protective coatings, protease inhibitors or permeation enhancers in particulate form for various routes of administration, including parenteral, pulmonary, nasal, oral, vaginal, rectal routes. In one embodiment, the pharmaceutical composition is administered parenterally, paracancerous, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitoneally, intraventricularly, intracranially, and intratumorally.
Furthermore, as used herein, a "pharmaceutically acceptable carrier" or "pharmaceutical carrier" is known in the art and includes, but is not limited to, 0.01-0.1M or 0.05M phosphate buffer or 0.8% saline. Furthermore, such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions and emulsions. An example of a nonaqueous solvent is propylene glycol; polyethylene glycol; vegetable oils such as olive oil; and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol/water solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, ringer's dextrose, sodium chloride, lactated ringer's or non-volatile oils. Intravenous carriers include fluid and nutritional supplements, electrolyte supplements, such as those based on ringer's dextrose, and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, finishing agents, inert gases and the like.
For any compound, a therapeutically effective dose may be estimated initially in a cell culture assay or in an animal model such as mouse, rat, rabbit, dog or pig. Animal models can also be used to determine the concentration range and route of administration. Such information can then be used to determine useful dosages and routes for human administration. These techniques are well known to those skilled in the art, and a therapeutically effective dose refers to the amount of active ingredient that improves symptoms or conditions. Therapeutic efficacy and toxicity can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by calculating and comparing ED 50 (therapeutically effective dose in 50% of population) and LD 50 (dose lethal to 50% of the population). Any of the above pharmaceutical compositions may be applied to any individual in need of treatment, including but not limited to mammals such as dogs, cats, cattle, horses, rabbits, monkeys, especially humans.
The pharmaceutical compositions described herein may be administered by a variety of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
In some embodiments, the pharmaceutical composition is made from a binding agent having a purity level between 80.0% and 99.9%. Thus, in some embodiments, the pharmaceutical composition comprises a binding agent that is substantially free of impurities. For example, the pharmaceutical composition comprises the binding agent in a purity of at least 90.0 +/-5.0%. In other examples, the pharmaceutical composition comprises the binding agent in a purity of at least 95.0 +/-5.0%. In other embodiments, the pharmaceutical composition comprises the binding agent in a purity of at least 99.0 +/-1.0%.
In some embodiments, the pharmaceutical composition comprises a binding agent that is stable in solution under one or more pressure conditions as described herein.
Application method
The polypeptide chains and binding agents of the invention are useful in the treatment of disorders or diseases.
In some embodiments, the polypeptide chains and binding agents may be used to target therapeutic and/or diagnostic agents to a target cell, circulating protein, or tissue.
In some embodiments, the polypeptide chain and binding agent may be conjugated to a therapeutic moiety and used in a method of treatment.
In some embodiments, the polypeptide chain and binding agent may be conjugated to a detectable moiety and used in a detection or diagnostic method.
In some embodiments, the polypeptide chains and binding agents of the invention may be used to target tumors in vivo.
In some embodiments, the polypeptide chains and binding agents are used to promote tumor regression and/or reduce tumor volume in vivo.
Thus, the polypeptide chains and binding agents of the invention may be used in cancer treatment.
The methods of the invention may comprise the step of administering to an individual in need thereof a polypeptide chain, a binding agent or a mixture as disclosed herein or a pharmaceutical composition comprising the polypeptide chain, the binding agent or the mixture.
In some embodiments, the polypeptide chain and binding agent are used in combination with a chemotherapeutic agent.
According to the invention, the individual in need thereof may be a human. Further in accordance with the present invention, the individual in need thereof may be an animal.
In some embodiments, particular consideration is given to treating a condition or disease caused by or associated with expression of a neoantigen.
In certain embodiments, treatment of a condition or disease caused by or associated with overexpression of an antigen is particularly contemplated.
In some embodiments, the condition or disease may be cancer.
In other embodiments, the condition or disease may be an infection.
In other embodiments, the condition or disease may be an immune disorder.
In other embodiments, the condition or disease may be a metabolic disorder.
In some embodiments, the binding agent is administered intravenously.
In some embodiments, the binding agent is administered by infusion.
The polypeptide chains and binding agents of the invention may be used for detection purposes.
Detection of a particular target can be performed in vitro by: a sample containing or suspected of containing a target is contacted with a polypeptide chain or binding agent comprising an antigen binding domain for such target and a detection device is used to quantify the signal associated with positive or negative binding.
The sample may be of mammalian (e.g., human) origin. The sample may be a tissue sample obtained from a mammal or a cell culture supernatant.
In some embodiments, the sample may be a serum sample, a plasma sample, a blood sample, semen, or ascites fluid obtained from a mammal.
Detection of a particular target can be performed in vivo by administering a polypeptide chain or binding agent comprising an antigen binding domain of such target to an individual and quantifying a signal associated with positive or negative binding using a detection device.
Upon detection of the presence of a target in a sample or individual, a drug (e.g., an antibody, small molecule, polypeptide chain, or binding agent disclosed herein) may be administered to the individual.
In addition to the embodiments described and provided in the present invention, the following non-limiting embodiments are specifically contemplated.
Tables 3, 4, 5 and 6 illustrate that VHH itself (e.g., as a fusion with a dimerization domain) has biological activity as part of a multimeric, multivalent and/or multispecific binding agent as exemplified in table 7.
Examples
EXAMPLE 1 animal immunization and immune library construction
Single domain antibodies are produced by immunizing alpaca (Vicugna pacos) or llama (llama glama) with a human antigen, a cDNA plasmid encoding the human antigen, and/or cells overexpressing the human antigen.
For example, anti-DR 2 single domain antibodies are generated by immunization of alpaca with DR2 antigens including human DR2 protein, human DR2 cDNA plasmid and human DR2 overexpressing cell lines, i.e., SH-SY5Y-D2 cells. anti-PD-1 single domain antibodies were generated by immunization of alpaca with PD-1 antigen including human PD-1 protein, human PD-1cDNA plasmid and human PD-1 overexpressing cell lines, i.e., CHO-PD1 cells. In the case of anti-CD 47, single domain antibodies are generated by immunizing llamas with CD47 antigen, including human CD47 protein, human CD47 cDNA and human PBMCs (activated).
After phage display panning of the recombinant immune library against the antigen, the sequence of the single domain antibody was found from the clone selection.
The DNA fragments encoding VHHs are typically subcloned into constructs for expression of the polypeptide chains (see e.g., table 6 or table 7) and in vitro and in vivo testing.
Controls were also generated by replacing one or more VHHs with HEWL-specific VHHs.
Example 2 transfection, expression and purification of binding agents
Transfection and expression of polypeptide chains in ExpiCHO or Expi293 cells
Using expifer TM Expression systems (Thermo Fisher, catalog number A29133) or Expi293 TM The expression system (Thermo Fisher, catalog No. a 14635) expresses protein dimers (e.g., homodimers or heterodimers) from the DNA construct in a culture volume of 2.5mL or 400 mL.
Briefly, freshly thawed CHO cells were allowed to recover two or more passages in culture prior to transfection. Next, the cells were passaged every 3-4 days until the cells reached 4X 10 6 -6×10 6 Individual cells/ml, at this time at the ExpiCHO pre-heated to 37 ℃ TM Diluting it to 2X 10 in expression Medium 5 -3×10 5 Individual cells/ml. The day prior to transfection, the cells were diluted to 3X 10 6 -4×10 6 Individual cells/ml, and on the day of transfection, the cells were further diluted to 6.×10 6 Individual cells/ml. A culture volume of 1. Mu.g DNA/mL was incubated with cold OptiPRO TM Culture medium (100. Mu.L for a culture volume of 2.5 mL; 16mL for a culture volume of 400 mL) was diluted. Expifectamine is prepared TM CHO reagent (8. Mu.L for a culture volume of 2.5 mL; 1280. Mu.L for a culture volume of 400 mL) was added to the DNA-containing medium and incubated with ExpiFectamine at room temperature TM Incubation of the DNA complex for 1-5 min. Next, the DNA complex was transferred to the culture while vortexing (6X 10) 6 Individual cells/ml). Cells were incubated at 37℃with 8% CO 2 And incubation was performed under shaking (INFORS HT shaker, 125 rpm) at 80% humidity. 18-22 hours after transfection was initiated, expiCHO was added to the cells TM Feeder (0.6 mL for 2.5mL culture volume; 96mL for 400mL culture volume) and ExpiCHO TM Enhancer (15. Mu.L for a culture volume of 2.5 mL; 2.4mL for a culture volume of 400 mL). Cells were returned to the INFORS HT incubator, set at 37℃at 8% CO 2 And 80% humidity, shaking at 125rpm (25 mm orbit). 8 days after transfection, by centrifugation at 4000 XgThe supernatant was clarified for 30 minutes. The supernatant was treated with Nalgene TM Rapid-Flow TM Sterile disposable filtration unit 1000mL filtration unit (Thermo Scientific, catalog No. 567-0020) was filter sterilized and stored at 4 ℃ or frozen for later analysis.
The newly thawed HEK293 cells were allowed to recover two or more passages in culture prior to transfection. Next, the cells were passaged every 3-4 days until the cells reached 3X 10 6 -5×10 6 Individual cells/ml, at this point, expi293, pre-warmed to 37 °c TM Dilution to 3X 10 in expression Medium 5 -5×10 5 Individual cells/ml. The day before transfection, cells were diluted to 2.5X10 6 -3×10 6 And on the day of transfection, the cells were further diluted to 3X 10 6 Viable cells per milliliter. By Opti-MEM TM I reduced serum medium dilution of 1 u g DNA/mL culture volume, get 150 u L final volume for 2.5mL culture volume, and 24mL for 400mL culture volume. Expifectamine is prepared TM 293 reagent (8. Mu.L for a culture volume of 2.5 mL; 1.3mL for a culture volume of 400 mL) was added to the culture medium Opti-MEM TM Reduced serum medium (140. Mu.L for 2.5mL culture volume; 22.5mL for 400mL culture volume) was incubated at room temperature for 5 min. Dilute Expifectamine TM Added to the diluted DNA and incubated for 15 minutes at room temperature. Vortex while vortex the epiFectamine TM The DNA solution was transferred drop by drop to the culture (3X 10 6 Individual cells/ml). Cell at 37℃and 8% CO 2 And incubated at 80% humidity and shaken overnight (INFORS HT shaker, 125 rpm). 18-22 hours after transfection was initiated, the cells were added with Expifectamine TM 293 transfection enhancer 1 (15. Mu.L for a culture volume of 2.5 mL; 2.4mL for a culture volume of 400 mL) and Expifectamine TM 293 transfection enhancer 2 (50. Mu.L for a culture volume of 2.5 mL; 24mL for a culture volume of 400 mL). Cells were returned to the INFORS HT incubator, set at 37℃at 8% CO 2 And 80% humidity, shaking at 125rpm (25 mm orbit). 5 days after transfection, the supernatant was clarified by centrifugation at 4000 Xg for 30 minutes. The supernatant was treated with Nalgene TM Rapid-Flow TM Sterile polishingDisposable filtration unit 1000mL filtration unit (Thermo Scientific, catalog No. 567-0020) was filter sterilized and stored at 4 ℃ or frozen for later analysis.
Purification
Based on the volume of supernatant, 3mL MabSelect was used TM SuRe TM Resin (GE Healthcare, catalog number 17-5438-02) with gravity column or 40mL MabSelect TM SuRe TM The protein was purified with AKTA PURE (GE Healthcare, piscataway, NJ). The resin was incubated overnight with 0.5M NaOH and equilibrated with Tris base buffer (50 mM Tris-HCl,150mM NaCl,pH 7.4) pH 7.4 prior to injection. The supernatant was applied to a gravity column or to a 40mL column at a rate of 5 mL/min. The resin column was washed with Tris base buffer pH 7.4 at a flow rate of 10 ml/min at 3CV (column volume). The protein was eluted with 3CV of 0.1M pH 3 citric acid at 10 ml/min. Fractions determined to have protein from the visual output of the chromatogram (absorbance at 280 nm) were pooled together. Pooled fractions were neutralized with 1M Tris-HCl, pH 9.0, to reach pH about 5-6, then transferred to Phosphate Buffered Saline (PBS) pH 6.0 buffer prepared from PBS10 XpH 7.2 (15 mM potassium dihydrogen phosphate, 1552mM sodium chloride, 27mM disodium hydrogen phosphate, thermoFisher, cat. 70013073).
Buffer exchange was performed by a sample concentrator for proteins purified from a gravity column, or by dialysis or by a desalting column for proteins purified from AKTA PURE. Proteins purified from a gravity column were concentrated by centrifugation at 3,500-4,000 Xg at 4℃with a sample concentrator Vivaspin 2,50kDa MWCO (GE Healthcare, cat. No. 28932257), followed by dilution with PBS at pH 6 to 4-fold, and repeated until the sample reached 200-fold. Dialysis was performed overnight at 4℃in 4L of PBS pH 6 using a dialysis tube (ThermoFisher, cat. No. 68799) with a molecular weight cut-off of 7 kDa. On the other hand, the desalting column was incubated overnight with 0.5M NaOH and equilibrated with PBS pH 6. A15 mL volume of the neutralized protein sample was loaded into a HiPrep 26/10 desalting column (GE Healthcare, catalog No. 17-5087-02) at 0.5 mL/min, followed by elution of the protein with 2CV of PBS at pH 6. The loading and elution steps are repeated until there is no neutralized protein sample eluted from the affinity column. Fractions determined to have protein from the visual output of the chromatogram (absorbance at 280 nm) were pooled together.
Using Nalgene TM Rapid-Flow TM Sterile disposable filtration unit 150mL filtration unit (Thermo Scientific, catalog nos. 565-0010) filter-sterilizes the sample. Pierce for final protein samples TM Double caprylic acid protein analysis kit (ThermoFisher, catalog number 23227) for quantification and useLAL reagent cartridge (Charles River, catalog PTS 2005) tested endotoxin levels. The final protein samples were analyzed on SDS-PAGE gels under reducing or non-reducing conditions.
Production of binding agents in CHO cells followed by conventional two-step purification using protein a affinity chromatography followed by Size Exclusion Chromatography (SEC) yields purities ranging from 95.5% to 99.5% (dimer).
After Size Exclusion Chromatography (SEC) and SDS clamp, KC020 was found to be more than 97% pure. Verifying the DNA sequences of the first three clones; the charge profile and glycosylation profile of KC020 were found to be similar to those of monoclonal antibodies. The following table represents KC020 titers after 14 days for three representative clones selected for cell line development.
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SDS-PAGE and Western blotting
Using NuPAGE TM LDS sample buffer (ThermoFisher, catalog NP 0007) and NuPAGE TM Sample reducing agent (thermo fisher, cat.no. NP 0004) or no reducing agent was used, and samples were prepared under reducing or non-reducing conditions for SDS-PAGE analysis. The sample under reducing conditions was denatured by heating at 70 ℃ for 10 minutes. Samples (16. Mu.L) were loaded onto 3-8% Tris-acetate minigel (1.5 mm,15 wells) together with BSA standard. Make the following steps With X-Cell SureLock TM The mini gel device was run for about 1 hour at 125 volts. Gel use of GelCode TM Staining was performed with staining reagents (Thermo Fisher, catalog No. 24594).
For Western blot analysis, according to manufacturer's instructions, iBlot was used TM The system (Thermo Fisher, catalog IB 301031) transfers proteins to nitrocellulose membranes.
His tag detection was performed with anti-5 XHis-HRP antibody. Briefly, the membrane is closed by: incubation was performed in 20mL of Qiagen blocking buffer (Qiagen, cat. No. 1018862) with shaking at room temperature for 1 hour, followed by 20mL of Starting Blocking TM T20 (PBS) blocking solution (Thermo Fisher, cat. No. 37528) was incubated with shaking for 1 hour at room temperature. With 1 XTBS Tween TM -20 washing the membrane three times, 10 minutes each. The membrane was incubated with anti-pentaHis-HRP (Qiagen, cat. 1014992) previously diluted 1:2000 in blocking buffer for 1 hour with shaking at room temperature. With 1 XTBS Tween TM -20 washing the membrane three times, 10 minutes each. According to the manufacturer's instructions, super Signal is used TM West Pico PLUS (Thermo Fisher, catalog number 34080) visualizes the signals. Images were recorded using an Azure Biosystem imaging system.
These experimental results demonstrate that the binding agents (homodimers or heterodimers) of the invention can be efficiently expressed, purified in cells, and that the pattern of polypeptide chains disclosed herein can bring the yield of binding agent (homodimer or heterodimer) to the g/l range (data not shown).
Example 3-in vitro and in vivo testing of binding agents comprising one or more anti-D2 antigen binding domains
Cell lines
The OCI-AML3 cells are acute myeloid leukemia cells of human origin and are used as target cells in PBMC-dependent cytotoxicity assays. THP-1 is a monocytic lineage derived from acute monocytic leukemia patients. THP-1 cells were used as target cells in PBMC-dependent cytotoxicity assays.
NCI-H82, NCI-H69 and NCI-H510A are human Small Cell Lung Carcinoma (SCLC) cell lines used in SCLC xenograft tumor models of immunodeficient mice. NCI-H82, NCI-H69 and NCI-H510A overexpress the dopamine receptor D2 and are used herein, inter alia, as models for D2 targeting studies.
NCI-H727 cells are human non-small cell lung cancer (NSCLC) cells used in a NSCLC xenograft tumor model of an immunodeficient mouse model. D2 has been shown to be up-regulated in NCI-H727 cells.
PANC1 is a human pancreatic cell line used in xenograft tumor models in immunodeficient mouse models.
Production of proteoliposomes
To determine the binding specificity of the anti-D2 binding agent, proteoliposomes exhibiting human D2 were generated according to the complementary protocol using the ProteoLiposome BD kit of CellFree Science (CellFree Science, cat. CFS-CPLE-BD).
Briefly, the transcription reaction is established by: the required volumes of nuclease-free water, 5 Xtranscription buffer, NTP mixture, ribonuclease inhibitor, SP6 RNA polymerase and plasmid DNA were mixed in 1.5mL tubes and incubated for six hours at 37 ℃. To establish the translation reaction, a mixture of mRNA mixtures, feeding buffer, WEPRO7240, creatine kinase and soybean phospholipid (asectrin) liposomes was prepared as indicated. The translation reaction is established by: the translation reaction mixture was added to a slide-A-Lyzer microdialysis device (Thermo Scientific, catalog number 69570) and incubated at 15℃for 72 hours.
Proteoliposomes were purified by washing with PBS and centrifugation at 15000rpm,4℃for 10 min. After repeating the washing step 3 times, the harvested proteoliposomes were resuspended in an appropriate amount of PBS and stored at-80 ℃.
Binding of anti-D2 binding agents (e.g., table 6) to proteoliposomes presenting human D2 (fig. 1, 25, 26) was assessed by ELISA. Briefly, protein liposomes or empty liposomes containing the target protein are plated on 96-well plates. The plates were covered and left overnight at 4 ℃. The next day, the plates were washed once with PBS and blocked with blocking buffer for 1 hour at room temperature. The binding agent is tested at the indicated concentrations, diluted in blocking buffer, and Incubate at 37℃for 1 hour. After incubation, the plates were washed three times with wash buffer. Plates were incubated with anti-human Fc-HRP (Sigma-Aldrich, catalog number AP 113P) at 1:5000 dilution for 1 hour at room temperature, followed by three washes with PBS-T wash buffer. By SuperSignal TM ELISA Pico chemiluminescent substrate (Thermofish, cat. 37069) visualized the signal. In SpectraMax TM The plate is read on an i3x multimode microplate reader (Molecular Devices).
Figure 1 presents representative results of such experiments, which demonstrate that the tested anti-D2 binding agent specifically binds to D2 protein liposomes (EC 50 is 0.1488 nM) and has very high affinity.
GPCR BRET assay
The effect of various anti-D2 binding agents on D2 downstream signaling was evaluated (fig. 2). Prior to transfection, HEK293 cells were maintained in Du Erbei Kou Gailiang Igor medium (Dulbecco's Modified Eagle Medium, DMEM) supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum. All transfections used Polyethylenimine (PEI) transfection reagents. HEK293 cells were co-transfected with hDR2 and one bioreceptor. The total amount of transfected DNA remained unchanged, 1. Mu.g per ml of cell culture to be transfected. The ratio of PEI to DNA (μg: μg) was fixed at 3:1. The DNA and PEI were first diluted in 150mM NaCl, respectively. The volume of diluent in each tube corresponds to 5% of the cell culture volume to be transfected. Once the DNA and PEI were diluted, the solution containing PEI was added to the DNA solution and the DNA/PEI mixture was immediately vortexed for 5 seconds. The DNA/PEI mixture is incubated at room temperature for at least 20 minutes to allow the formation of DNA/PEI complexes. During incubation, HEK293 cells were detached, counted and resuspended in culture medium. At the end of the incubation period, the DNA/PEI mixture was added to the cells. Finally, cells were distributed in 96-well plates at a density of 35000.
At 48 hours post-transfection, BRET experiments were performed to detect downstream signaling of D2 in the agonist test mode. Media was aspirated from the 96-well plate and replaced with 50. Mu.l Hank's Balanced Salt Solution buffer. When analyzed in HEK239 cells, 10. Mu.l of 10. Mu. M e-Coelenterazine Prolume Purple were added per well at a final concentration of 1. Mu.M. Dopamine was added to each well as a positive control using an HP D300 digital dispenser. Dopamine was analyzed at 16 concentrations per photoreceptors. Antibodies were manually added to each well (final concentration of 1 μm per well). anti-D2 binding agents were assayed in duplicate at 8 concentrations with each biosensor. Cells were incubated with dopamine for 10 min at room temperature and antibodies for 60 min at room temperature. The BRET signal was collected on a Synergy NEO plate reader with an integration time of 0.4 seconds.
The EC50 s of various anti-D2 binding agents in GPCR BRET assays transfected with various G protein receptors are presented in figure 2. Dopamine was used as a positive control to show the reaction of GPCRs with G proteins including gαz, gαi2, gαoa and barre 2. Unexpectedly, all anti-D2 binders including KC011, KC012, KC013, and KC014 showed specific GPCR signaling via gαz. The results indicate that the anti-D2 binding agent binds to a unique epitope on D2 and initiates a highly specific signaling pathway.
RNAseq and qRT-PCR validation
The mechanism of action of KC013 was studied in NCI-H69 cells in vitro by evaluating the overall transcriptional profile after treatment using next generation sequencing techniques. Suspension NCI-H69 cells were collected and grown at 5X 10 6 The individual cells/ml concentration was resuspended. 1000 ten thousand cells per 2mL of complete medium were transferred to wells of a 6-well plate. KC013 and negative control KC018 (in which the VHH moiety is replaced by the anti-4 HEM VHH shown in SEQ ID NO: 114) were added to the appropriate wells at a final concentration of 1.0. Mu.M. Cells were returned to 37 ℃,5% co2 incubator, and treated for 3 hours. After the incubation period, the cells were homogenized with a qiashreder column and total RNA was isolated from the disrupted cell lysate using the RNeasy RNA isolation kit according to the manufacturer's protocol. A total of 500ng of total RNA was flash frozen and transferred to Genome Quebec for analysis.
The integrity of the RNA was assessed using an Agilent bioanalyzer. Libraries were prepared using the NEB mRNA standard nebnet Ultra II kit (NEB, catalog No. E7645S), combined with the mRNA enrichment step and Illumina library QC evaluation. Samples were sequenced on a NovaSeq 6000PE100 instrument (Illumina), 2500 ten thousand reads per sample. Reads were mapped to human reference genome sequences and annotations version 86 of ENSEMBL GRCh38 (Chile). This produced count data for 58,051 genes. Genes of no more than 1 Count Per Million (CPM) in at least 3 samples (i.e., the smallest population size) were filtered out, leaving 14,983 genes for downstream analysis.
A gene list was constructed using a maximum false discovery rate (False Discovery Rate, FDR) of 0.05 between KC013 treatment versus KC018 negative control. Using an Enrichr (Icahn School of Medicine, mount Sinai;http://amp.pharm.mssm.edu/Enrichr/) Enrichment analysis was performed. Using the panher database (University of Southern California;http://pantherdb.org) The over-representation analysis was performed using Fischer's exact test (FDR) and FDR multiplex test corrections, with emphasis on analyzing Gene Ontology (GO) biological pathways and protein classes. The gene list used consisted of: 191 up-regulated genes and 634 down-regulated genes, totaling 825 significantly altered genes (FDR)<0.05 (fig. 3). Pathway analysis was performed using the Ingeny pathway analysis (IPA, qiagen) (FIGS. 4A and B). Transcript-translated read counting (FDR) using Log2<0.05 825 genes) was constructed, and full-linked clustered and pearson distance measurements were performed (Pearson distance measurement).
Pathway analysis of the RNA-Seq data showed that several cancer-associated pathways, including CREB, wnt and mTOR signaling pathways, were down-regulated relative to negative control (KC 018) treated cells, including NCI-H69 and NCI-H82 SCLC, and activated p53 via multiple signaling pathways.
Pathway analysis of RNA-Seq data was validated by quantitative reverse transcriptase-PCR (qRT-PCR) using three different cell lines, including NCI-H69 (FIG. 5A), NCI-H82 (FIG. 5B) and HEK-D2 (FIG. 5C). Briefly, suspension cells were grown in complete medium in T175 flasks. Cells at 5X 10 per well 6 The individual cells/ml were seeded in three 6-well plates and treated in triplicate with either 1.0. Mu.M KC013 or 1.0. Mu.M KC018 for 3 hours and 6 hours. After a defined incubation period, replicators were scraped off at each time point and transferredTo 15mL fire Kang Guan (falcon tube). Cells were washed twice with 1 XPBS and cell pellets were flash frozen in liquid nitrogen and stored at-80℃for downstream analysis. Total RNA was extracted using the Quick-RNA Miniprep Plus kit (Cedarlane, catalog number R1057) according to the manufacturer's instructions. The amount of RNA recovered was quantified for each sample by measuring absorbance at 260 and 280nm using a NanoPhotometer. According to the instruction of the manufacturer, iScript is used TM Reverse transcription super mix (Reverse Transcription Supermix) reverse transcription was performed using 1 μg RNA. Next, the resulting cDNA was diluted 1:20 with ddH2O containing 40ng/mL glycogen.
Gene expression analysis was performed using the relative standard curve method in the iQTM5 real-time PCR system using SsoAdvanced Universal IT SYBR Green. The final reaction volume was 10. Mu.l per well on a 96-well plate containing 1 XSYBR Green mixture, 200nM primer concentration, 2. Mu.l cDNA and ddH2O. Raw data of qRT-PCR of target genes are collected as the number of cycles required for fluorescence signal to cross background levels (i.e., threshold). These values are referred to as cycle threshold or Ct values. In order to determine the relative gene expression levels of the target genes after treatment with KC013 and KC018, a geometric mean method of a plurality of internal control genes was employed, followed by a ΔΔct calculation method described below. Vandesompele J. Et al, genome Biology,2002 and Hellemans et al, genome Biology,2007 describe this method in detail. The final data are expressed as fold change in gene expression normalized to the selected common endogenous reference gene and to the control binding agent. The data are presented as an average of 3 biological replicates performed in 3 technical replicates. The result is representative data in at least 3 assays performed in triplicate.
Changes in gene expression of NCI-H69 cells after treatment with KC013 were visualized by thermography (fig. 3), with changes in color and intensity representing changes in individual gene expression. The results of this experiment show that after treatment with KC013, a total of 825 significantly altered genes were observed (FDR < 0.05) with 634 genes down-regulated.
Figure 4 shows down-regulated signaling pathways and protein classes derived from the panher analysis of RNAseq results. The most down-regulated signaling pathways include the Wnt pathway, gonadotropin releasing hormone (GnRH) pathway, and the cadherin signaling pathway (fig. 4A). The most down-regulated protein class by panher analysis was the gene-specific transcriptional regulator (fig. 4B). The Wnt pathway and the cadherin signaling pathway are thought to play important roles in tumor cell survival, proliferation, tumor formation and progression. Aberrant Wnt signaling pathways have been shown to play a key role in the initiation of many cancer cells by modulating Cancer Stem Cells (CSCs). Downregulation of these pathways and transcriptional regulators suggests an anti-tumor mechanism for KC 013.
Like the RNAseq profile, specific signaling components of the Wnt pathway, mTOR pathway, and cAMP pathway were shown to be down-regulated in NCI-H69 cells (fig. 5A), NCI-H82 cells (fig. 5B), and HEK-D2 cells (fig. 5C) after treatment with KC 013.
Single dose PK in CB-17Fox Chase SCID mice
CB-17Fox Chase SCID mice (6-8 weeks old, female) were purchased from University Health Network (UHN, toronto, ON) for PK studies. Mice were housed in a pathogen-free environment of the animal facility of UHN. Animal work was performed according to guidelines of the canadian animal care committee (Canadian Council on Animal Care, CCAC) and animal care committee (Animal Care Committee) approved animal use protocol (Animal Use Protocol) of UHN. Briefly, mice were divided into 2 experimental groups (6/group), each group received either Trastuzumab (UHN pharmacy) or KC013 in a single dose of 30mg/kg by intravenous injection (fig. 6A) or intraperitoneal injection (fig. 6B). In each study, blood samples were collected at 0.083 hours (for PK intravenous study only), 1 hour (for PK intraperitoneal study only), 3 hours, 6 hours, 1 day, 3 days, 7 days, 10 days, 15 days, 21 days, and 28 days post-injection. The blood samples were centrifuged and serum samples were collected and stored at-80 ℃ until they were analyzed. The concentration of trastuzumab and KC013 in mouse serum was measured by quantitative enzyme-linked immunosorbent assay (ELISA), as described below. Pharmacokinetic data analysis was performed using the non-compartmental method using Phoenix WinNonlin software (Certara).
Antibodies in mouse serum were quantified by quantitative ELISA (fig. 6). Briefly, 96-well plates were coated overnight at 4℃with mouse anti-human IgG antibodies (Bio-rad, cat. No. MCA878G, used at 1/200 dilution). The next day, the plates were washed three times and blocked with 2% BSA for 1 hour at room temperature. At the same time, standards (STD, bio-rad, cat. No. MCA 6092), quality Control (QC) and each serum sample were diluted in wash buffer containing 1% mouse serum. Mu.l of STD, QC and diluted serum samples were added to 96-well plates and incubated for 2 hours at room temperature. After three washes, the plates were incubated with secondary antibody (goat anti-human Fc antibody, 1/5000 dilution, cedarlane, catalog number 109-035-098) for 1 hour at room temperature. Plates were washed three times before adding 100 μl of detection substrate per well. When the highest STD reading reached 0.9-1.1 at 650nm wavelength, 50. Mu.l stop solution was added to each well to stop the reaction. Plates were read at 450nm wavelength to measure antibodies.
The results of these experiments showed that the IV injection of KC013 (FIG. 6A) showed T1/2 of 214 hours, while the IP injection (FIG. 6B) showed T1/2 of 159 hours. The Cmax obtained for KC013 was similar to the full-size human antibody trastuzumab. This data shows that the half-life of the anti-D2 binding agent in SCID mice is long.
Tumor prevention model in CB-17 Fox Chase SCID mice
CB-17 Fox Chase SCID mice (6-8 weeks old, female) were purchased from Charles river laboratory (Charles River Laboratories) (St. Constant, QC) or UHN (Toronto, ON). The guidelines for animal feeding and animal work are the same as described above.
The efficacy of anti-D2 binding agents was evaluated in various tumor prevention models, namely NCI-H510A (fig. 7C, fig. 8), NCI-H727 (fig. 7A, fig. 8), PANC1 (fig. 8) and NCI-H69 (fig. 7B, fig. 8). For each model, tumor-containing cells (800 ten thousand cells/mouse for NCI-H510A, 600 ten thousand cells/mouse for NCI-H727, 500 ten thousand cells/mouse for PANC1, or 300 ten thousand cells/mouse for NCI-H69) were subcutaneously injected (s.c.) into the right abdomen of the mice. One day after cell inoculation, mice vaccinated with each cell line were randomly divided into 2 experimental groups (10 or 5/group) and each group received 16mg/kg ip.kc018 (negative control) or KC013Twice a week for eight weeks. Tumor volumes were measured with a cursor caliper and mice were weighed once or twice a week. Tumor volume was calculated using the following formula: 1/2 (length. Times. Width) 2 ). To calculate the percent Tumor Growth Inhibition (TGI), KC013 treated groups were compared to their respective negative controls. TGI is calculated as:
TV day z represents tumor volume of individual animals on the determined study day (day z), while TV day x represents tumor volume of individual animals on the staging day (day x).
Statistical tests were performed by student t-test (two-tailed).
As shown in fig. 7, KC013 showed 49% tumor growth inhibition in the NCI-H727 tumor prevention model (fig. 7A), 46% tumor growth inhibition in the NCI-H69 tumor prevention model (fig. 7B), and 77% tumor growth inhibition in the NCI-H510A tumor prevention model (fig. 7C) compared to the isotype control.
As shown in fig. 8, KC013, KC012 and KC014 expressed a general tumor growth inhibition in all tumor-preventing SCLC tumor models, with KC013 achieving the best in vivo efficacy than other anti-D2 binding agents. Since all of these anti-D2 binding agents initiate unique gαz signals in BRET assays, the in vivo efficacy achieved in SCLC tumor models may be related to specific gαz signals in tumor cells. In vivo efficacy can also be explained by down-regulated signaling pathways that are important for tumor progression.
Example 4-in vitro and in vivo testing of binding agents comprising anti-PD 1 and anti-CD 47 antigen binding domains
ELISA binding assay
Binding of binding agents comprising anti-PD-1 and anti-CD 47 antigen binding domains to recombinant human or cynomolgus monkey PD-1 (Cedarlane, catalog number HPLC-10377-H08H-100) or recombinant human or cynomolgus monkey CD47 (Cedarlane, catalog numbers 12283-H08H-200 and 90869-C08H) was assessed by ELISA (FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, FIG. 22, FIG. 23). Palbociclib was used as a positive control for PD1 binding assays. Monoclonal anti-CD 47 antibody (clone B6H 12) was used as a positive control for CD47 binding assay (Biolegend, cat. 401401).
Briefly, recombinant proteins were coated on 96-well plates. The plates were covered and left overnight at 4 ℃. The next day, the plates were washed once with PBS and blocked with blocking buffer for 1 hour at room temperature. The binders were tested at the indicated concentrations, diluted in blocking buffer and incubated for 1 hour at 37 ℃. After incubation, the plates were washed three times with wash buffer. Plates were incubated with anti-human Fc-HRP (Sigma-Aldrich, catalog number AP 113P) at 1:5000 dilution for 1 hour at room temperature, followed by three washes with PBS-T wash buffer. SuperSignal for signal TM ELISA Pico chemiluminescent substrate (Thermofish, cat. No. 37069) was visualized. In SpectraMax TM The plate is read on an i3x multimode microplate reader (Molecular Devices).
As shown in fig. 9A, the anti-PD 1 binding agent KC009 efficiently bound to human, cynomolgus monkey and rat recombinant PD1 protein. The cross-species binding activity of KC009 implies the feasibility of using cynomolgus macaques and rats as a toxicity model for KC 009.
The binding of KC036 was compared to palbociclib mab. KC036 and Pabo Li Zhushan antibodies were both diluted 1/5, starting at 500 nM. As shown in fig. 9B, KC036 was similar to binding of palbociclib by ELISA. The negative control KC036 did not bind to PD-1 recombinant protein.
KC015 was compared to the anti-CD 47 antibody B6H12 (ThermoFisher, catalog number 14-0479-82). Both KC015 and B6H12 antibodies were diluted 1/5, starting at 50 nM. As shown in fig. 9C, KC015 showed higher binding affinity than clone B6H12 by ELISA. The negative control KC016 did not bind to CD47 recombinant protein.
As shown in fig. 9C and 9D, the anti-CD 47 binding agent effectively bound CD47 recombinant proteins of human and cynomolgus macaques, whereas the negative control did not bind to both CD47 recombinant proteins.
Determination of blood cell binding of binding agents by flow cytometry
To assess the binding activity of KC015 on various blood cell subtypes, fresh blood was collected from two healthy donors to Becton Dicinson Vacutainer. For the preparation of leukocytes, 10mL of 1 Xerythrocyte lysis buffer (eBioscence, 00-4300-54) was added to each 1mL of fresh blood and incubated for 15 minutes at room temperature. The cell suspension was then centrifuged at 300g for 5 min at room temperature, followed by another wash with PBMC medium ((RPMI+10% heat-inactivated FBS). The number of leukocytes was counted before staining with the binding agent.
For preparation and staining of Red Blood Cells (RBCs), fresh blood was transferred to 15mL conical bottom polypropylene tubes and mixed with PBS wash buffer (pbs+2% heat inactivated FBS). The tube was centrifuged at 800g for 10 minutes at room temperature and the brake was closed. Red blood cells were collected from the bottom of the tube, resuspended in PBS wash buffer (pbs+2% heat-inactivated FBS) and the cell numbers were counted prior to staining with binding agent.
For preparation of platelet cells, fresh blood was transferred to 15mL conical bottom polypropylene tubes and mixed with PBS wash buffer (pbs+2% heat-inactivated FBS). The tube was centrifuged at 800g for 10 minutes at room temperature and the brake was closed. Platelet rich plasma (2/3 of the plasma volume) was collected from the top of the tube. Platelet rich plasma was transferred to 9mL of PBS wash buffer and centrifuged at 1250g for 15 min at room temperature and the brake was turned off. Platelet cell numbers were counted prior to staining with binding agent.
The Fc receptor blocking antibodies were added to the cell suspension at a dilution of 1/100 and incubated on ice for 10 minutes. Primary binders were added to the cell suspension at a range of antibody concentrations and incubated on ice for 30 minutes. Cells were washed once with PBS wash buffer followed by staining with secondary detection antibody. T cells were also stained with CD3 marker antibody (Biolegend, 557705). Monocytes were stained with CD14 antibody (Biolegend, 301814). Granulocyte populations are gated according to FSC and SSC. Erythrocytes were stained with CD235ab antibody (Biolegend, 306620). Platelets were stained with CD41 antibody (Biolegend, 303704). All cells were stained with 7-AAD (BD, 559925), diluted 1/100, and subsequently analyzed by a cell counter FACSCantoII.
As shown in fig. 9E, KC015 binds effectively to human T cells, but binds less efficiently to RBCs and platelets.
FACS binding analysis of cells
Binding of the binding agent to the cells was assessed by flow cytometry. To determine the binding specificity of the binding agent comprising the anti-PD-1 antigen binding domain, CHO parental and CHO-PD-1 cell lines were used in the binding assay (fig. 10). Briefly, 1X 10 5 Individual cells were incubated with Fc receptor blocking antibodies (1/100 dilution, cedarlane, cat. 422302) for 10 minutes at room temperature. Binding agents comprising anti-PD-1 antigen binding domains were diluted to 20 μg/mL in FACS staining buffer and added to cells and incubated with the cells on ice for 30 minutes. PD-1 antibody (clone EH12.2H7, biolegend) was used as a positive control. After three washes with FACS staining buffer, APC conjugated secondary antibodies recognizing the human Fc region ((Cedarlane, cat. No. 409306) were mixed with the cells after 30 min staining, the cells were washed three times and then resuspended in 100. Mu.l 7-amino-actinomycin D (7-AAD) solution (1/500 dilution, BD Bioscience, cat. No. 559925.) using BD FACSCanto TM II flow cytometry (BD Bioscience) analyzed cells. Data were analyzed with FlowJo via gating on single cells and living cells.
As shown in fig. 10, KC036 binds only CHO-PD1 cells, but not to cells that do not express PD1 (CHO parent cells), demonstrating the high specificity of anti-PD-1 module KC 009.
PBMC mediated cytotoxicity assay
PBMC mediated cytotoxicity was assessed by flow cytometry. Briefly, tumor cells (THP-1 or OCI-AML 3) were stained with CellTrace violet for 10 min at 37 ℃. Cells were washed and mixed with human PBMCs (Cedarlane, cat# 70025.1) at a ratio of 5:1. Adding anti-CD 3 antibody or CD33 bispecific T cell cement (BiTE, G)&P Bioscience, catalog No. FLC 2032) to activate T cells. A specified concentration of binding agent was added to the corresponding wells to determine the effect of the anti-PD-1 module on cytotoxicity. After 48 or 72 hours of incubation, the cells were washed three times, followed byResuspension was followed by 100 μl of 7-AAD solution (1/100 dilution, BD Bioscience, catalog number 559925). Using BD FACSCanto TM II flow cytometry (BD Bioscience) analyzed cells. Data were analyzed with FlowJo by gating only single cells and living cells.
As shown in FIG. 11, KC036 (SEQ ID NO: 94) showed checkpoint inhibition (CPI) activity by increasing T cell dependent cytotoxicity to a level similar to that of positive controls palbociclizumab and nivolumab.
PD-1/PD-L1 blocked bioassays
In the luminescent NFAT-RE reporter system, PD-1/PD-L1 blocking assay (Promega, cat No. J1255) was used to evaluate the blocking activity of binding agents comprising an anti-PD-1 antigen binding domain and compared to a positive control. PD-L1 aAPC/CHO-K1 (target) cells were thawed and seeded into 96-well plates at the recommended density and allowed to adhere overnight on the plates. The next day, the binders were diluted to 350nM in assay buffer (Ham's F12 medium with 10% low IgG FBS) and serially diluted eight times 2.5. The 96-well plate medium was poured and 40. Mu.l of diluted binding agent and 40. Mu.l of Jurkat cells were added to the plate. Plates were incubated for 6 hours at 37 ℃. The Bio-Glo luciferase assay buffer and substrate were combined and 80 μl of solution was transferred to each well. The plates were incubated for 5 minutes at room temperature and luminescence was measured using a plate reader.
These experimental results indicate that KC036 achieved anti-similar in vitro CPI activity as pambo Li Zhushan, suggesting that the binding agent may induce anti-tumor activity by restoring T cell function of "depleted" T cells (fig. 12).
The anti-PD 1 antigen-binding domain of KC036 was replaced with other anti-PD-1 antigen-binding domains listed in table 4 in a manner similar to that described herein.
Example 5-in vitro and in vivo testing of binding agents comprising one or more anti-CD 47 antigen binding domains
Binding in HEK293T or HEK293T-CD47KO cells (CD 47 knockdown) (FIG. 13) or in primary human T cells (PBMC, stemcell) (FIG. 14) was tested for binding to binding agents comprising anti-D2 and anti-CD 47 antigen binding domains (KC 040) or anti-D2, anti-PD-1 and anti-CD 47 antigen binding domains (SEQ ID NO: 70).
As shown in fig. 13, the binding agent comprising the anti-CD 47 module binds to the parental HEK293T cells, but not to CD47 knockout cells (HEK 293T-CD47 KO).
As shown in fig. 14, the binding agent comprising an anti-CD 47 module binds to primary human T cells.
T cell activation assay
The ability of the binding agent comprising the anti-CD 47 module to induce activation of polyclonal T cells was performed with (fig. 16) or without concanavalin a (ConA, cedarlane, 14951-250) (fig. 15A). Briefly, PBMC were incubated with indicated concentrations of binding agent for 24 hours with or without 10 μg/mL ConA. Plates were centrifuged and cells incubated with Fc receptor blocking antibodies (Biolegend, 1/200 dilution) for 10 min on ice. Plates were again centrifuged and cells were stained with APC conjugated anti-CD 3 (Cedarlane, cat# 300312) and FITC conjugated anti-CD 69 antibody (bioleged, cat# 310904,1/500 diluted) for 30 min on ice. The cells were washed three times and then resuspended in 100. Mu.l of 7-AAD solution (BD Bioscience,1/100 dilution). Using BD FACSCanto TM II flow cytometry (BD Bioscience) analyzed cells. Data were analyzed with FlowJo by gating only single cells and living cells.
PBMC mediated cytotoxicity assay
PBMC mediated cytotoxicity was assessed by flow cytometry (fig. 15B). Briefly, tumor cells (THP-1 or OCI-AML 3) were stained with CellTrace violet for 10 min at 37 ℃. Cells were washed and mixed with PBMCs at a ratio of 5:1. Adding anti-CD 3 antibody or CD33 bispecific T cell cement (BiTE, G)&P Bioscience, catalog No. FLC 2032) to activate T cells. A specified concentration of binding agent was added to the corresponding wells to determine the effect of CD47 on cytotoxicity. After 48 or 72 hours incubation, the cells were washed three times, followed by resuspension with 100 μl of 7-AAD solution (1/100 dilution, BD Bioscience, catalog number 559925). Using BD FACSCanto TM II flow cytometry (BD Bioscience) analyzed cells. Data were analyzed with FlowJo by gating only single cells and living cells.
As shown in fig. 15, the binding agent comprising the anti-CD 47 module did not activate T cells (fig. 15A), nor did it mediate T cell-dependent cytotoxicity against THP-1 tumor cells (fig. 15B). Although the binding agent is shown to bind HEK293T and primary T cells, it does not activate T cells in vitro, which may lead to better safety in redirecting immune cells without activating T cells.
As shown in fig. 16, concanavalin a mediated T cell activation was not affected by the binding agent comprising an anti-CD 47 module. Concanavalin a is a superantigen, which leads to activation of T cells via the TCR-MHC complex. These results indicate that the anti-CD 47 module does not interfere with T cell activation induced via TCR-MHC complexes.
CD 47/SIRPalpha blocking bioassay
Blocking activity of binders comprising anti-CD 47 antigen binding domains was assessed using a CD 47/sirpa blocking assay in a luminescence reporter system and compared to B6H12 antibody (Promega, catalog No. CS 316013) as a positive control. CD47 CHO-K1 target cells were thawed and resuspended in Ham's F12 medium containing 10% FBS. The cell suspension was dispensed in 96-well plates with 100. Mu.L of CD47 CHO-K1 cells per well and at 37℃with 5% CO 2 Incubate overnight. The following day, 1.5-fold serial dilutions of test molecules were prepared in assay buffer (RMPI-1640 medium with 2% FBS). Cell culture medium was removed and 50 μl of serial dilutions of antibody were added to the corresponding wells. Sirpa effector cells were thawed and resuspended in assay buffer. The cell suspension was dispensed as 75 μl of sirpa cell suspension per well. At 37℃with 5% CO 2 The plates were incubated for four hours. At the end of the 4 hour incubation, 75. Mu.L of Bio-Glo-NL luciferase assay reagent was added to each well and luminescence was measured after 5-10 minutes using a Spectra Max 3 plate reader.
As shown in FIG. 17, KC015 (SEQ ID NO: 75) exhibited checkpoint inhibitory (CPI) activity by blocking the interaction between CD47 and SIRPalpha. The negative control (KC 016: SEQ ID NO: 76) did not show any CPI activity.
Example 6-in vitro and in vivo testing of binding agents comprising anti-DR 2, PD-1 and CD47 antigen binding domains
Established tumor model of NCG mice
To determine the antitumor efficacy of multivalent and multispecific binders, established tumor models of humanized NCG mice were used in the study (fig. 18, fig. 22). Briefly, NCG mice (5-6 weeks old, female) were purchased from Charles river laboratory (St. Constat, QC). Tumor cells (NCI-H82 of 800 ten thousand cells/mouse) in DPBS were injected subcutaneously (s.c.) into the right abdomen of mice. Once the tumor grows to 100mm 3 Mice were randomly divided into experimental groups (10 or 9 mice/group). Human PBMCs (1000 ten thousand cells) were injected intravenously (i.v.). The following day, each group of mice received treatment with binder (28 mg/kg) twice weekly for a total of eight doses by intraperitoneal (i.p.). Tumor volumes were measured with a cursor calliper and mice were weighed twice a week. The calculation method of tumor volume and tumor growth inhibition was the same as described above (in the "tumor prevention tumor model in CB-17 Fox Chase SCID mice in methods section"). Statistical tests were performed by Student t-test (two-tailed).
As shown in fig. 18, the binding agent comprising anti-D2, anti-PD-1 and anti-CD 47 antigen binding domains showed a TGI of 52%. The results suggest that the trispecific binding agent can inhibit tumor growth in vivo. The binding agents disclosed herein comprising an anti-CD 47 antigen binding domain are expected to provide better safety than other activating antibodies against immune cell redirection.
In vitro T cell recruitment assay
To test whether a binding agent targeting tumor antigen (D2) and T cells can recruit T cells and bridge T cells to tumor cells, T cells were co-cultured with NCI-H69 cell clusters in the presence of the binding agent.
Briefly, the levels of T cell recruitment into NCI-H69SCLC cell clusters were quantified for the changes in various binders containing anti-D2, anti-PD 1 and/or anti-CD 47 modules, as well as combinations of such modules (fig. 19). Suspended NCI-H69 cells were collected and transferred into 12-well plates and returned to 37 ℃, 5% CO 2 The incubator was incubated for 72 hours to allow for the growth of larger clusters of cells. After 72 hours, 5.0X10 were added to NCI-H69 cells 5 Individual PBMCs and knotsMixture (final concentration 25. Mu.g/mL). The plate was returned to 37℃with 5% CO 2 For 24 hours to recruit T cells to the NCI-H69 cell cluster.
After 24 hours, the medium from each well was transferred to a microcentrifuge tube and each tube was briefly centrifuged using a bench top microcentrifuge for 5 seconds of pulse rotation. The supernatant was separated and the pellet resuspended in 150. Mu.L of running buffer (PBS containing 5mM EDTA). The pellet and supernatant fractions were evaluated using a bright field microscope to ensure efficient isolation of large cell clusters. The resuspended pellet in the microcentrifuge tube was transferred to a 96-well plate. Plates were centrifuged at 1200RPM to pellet cells, the plates were inverted to remove supernatant, and 2 μ L Human TruStain FcX Block (BD Bioscience, cat No. 559925) was added per 100 μl of flow buffer per well and incubated on ice for 10 minutes. Plates were centrifuged at 1200RPM to pellet cells and APC conjugated anti-human CD3 (Cedarlane, cat# 300312) and FITC conjugated anti-human CD69 antibody (Cedarlane, cat# 310904) were added at 0.5 μl per 100 μl of flow buffer per well, including appropriate isotype controls in individual wells. After addition of the fluorescently labeled antibody, the plate was covered and incubated on ice for 30 minutes. After the incubation period was completed, the plates were centrifuged at 1200RPM to pellet the cells and washed with 150 μl of running buffer. The plate was centrifuged at 1200RPM to pellet the cells and resuspended in 1.0. Mu.L of 7-AAD in 7-AAD solution per 100. Mu.L of flow buffer per well.
The cell populations were analyzed by flow cytometry. Raw readings were collected on a BD FACSCanto II machine. A total of 100,000 events were collected for data analysis. The total viable cell population was assessed by gating on 7-AAD negative cells (PerCP-Cy5.5). The total T cell population was assessed by gating on CD3 positive cells (APC), and the activated T cell population was assessed by gating on CD3 (APC) and CD69 (FITC) biscationic cells. The average fluorescence intensities (MFI) of CD3 (APC) and CD69 (FITC) were quantified and compared between treatment groups. All samples described in this report were tested in triplicate. Results represent mean +/-standard deviation.
As shown in fig. 19, the binding agent was found to be effective in recruiting T cells into the NCI-H69 cell cluster in vitro.
Tumor prevention tumor model of CB-17Fox Chase SCID mice
Binding agents comprising an anti-D2 module were tested in a tumor prevention model as described in example 3.
Briefly, the binding agents (KC 020; SEQ ID NO:77,KC022;SEQ ID NO:79,KC023;SEQ ID NO:80,KC024;SEQ ID NO:81,KC025;SEQ ID NO:82,KC026;SEQ ID NO:83) were tested in a tumor preventative model vaccinated with NCI-H82 cells, similar to NCG mice, except that human PBMC and NCI-H82 cells were co-implanted at a ratio of 1:5 (FIGS. 20A-B and 24C).
As shown in fig. 20B, binders containing KC001, KC009, and KC010 (SEQ ID NO: 77) showed 74% of optimal efficacy compared to other D2 modules with KC009 and KC010 (fig. 20a & B). The superiority of KC001 efficacy data was also consistent with D2 efficacy data as VHH-hinge-CH 2-CH3 (FIG. 20), where KC001 (KC 013; SEQ ID NO: 73) showed the best efficacy in several tumor models including NCI-H69, NCI-H510A and NCI-H727.
In vivo efficacy of KC015 in PBMC pre-implant model
The anti-tumor activity of KC015 was evaluated in NCG mice using PBMC pre-implantation NCI-H82 xenograft tumor model (fig. 21A). The antitumor activity of KC020 was also studied in the NCI-H69, RKO and SCLC-21H xenograft tumor model pre-implantation of PBMC (FIG. 21B). Briefly, NCG mice (5-6 weeks old, female) were purchased from Charles river laboratory (St. Constat, QC). 1000 ten thousand PBMC were implanted into NCG mice 3 days prior to tumor cell implantation. Treatment was started 10 days after PBMC implantation twice weekly for 8 doses. The negative control used in the study was PBS. Tumor volume was calculated using the following formula: 1/2 (Length. Times. Width) 2 ). To calculate the percent Tumor Growth Inhibition (TGI), the treated group was compared to the respective negative control group. The calculation of TGI is described herein. Statistical tests were performed by Student t-test (two-tailed).
At the end of the study, animals were sacrificed and tumors and livers were collected for metastasis analysis.
The results of these experiments showed that KC015 (SEQ ID NO: 75) inhibited NCI-H82 tumor growth in a PBMC pre-implant model. KC020 (SEQ ID NO: 77) inhibited NCI-H69, RKO, SCLC-21H xenograft tumor growth in a human PBMC pre-implanted cancer model.
In vivo efficacy of KC015 in established tumor model of SCID mice
The antitumor efficacy of KC015 binding agents was evaluated in an established MDA-MB-231 tumor model in SCID mice (figure 21C). Tumor cells (500 tens of thousands of cells/mouse) in PBS were subcutaneously (s.c.) injected into the right abdomen of SCID mice (5-6 weeks old, female; charles river laboratory (st. Constant, QC)). Once the tumor grows to 100mm 3 Mice were randomly divided into experimental groups (9 mice/group). The following day, each group of mice received a treatment with binder (8 mg/kg) by intraperitoneal (i.p.), once a week for a total of eight doses. Tumor volumes were measured with a cursor calliper and mice were weighed twice a week. The calculation method of tumor volume and tumor growth inhibition was the same as described above. Statistical tests were performed by Student t-test (two-tailed).
As shown in FIG. 21C, the established MDA-MB-231 tumor model did not grow after treatment with KC015, indicating potent antitumor activity in such tumor model.
In vivo efficacy of IgG4 and IgG1 versions of the binding agent.
IgG4 is commonly used in therapeutic antibodies to avoid ADCC effects, which in this case may lead to T cell killing and cytokine release storms. To determine the antitumor efficacy of binding agents comprising an IgG4 CH3 domain, an established tumor model in humanized NCG mice was used as described above.
As shown in FIG. 22, there was NO significant difference between the IgG4 and IgG1 versions of the binding agents (compare KC021 (SEQ ID NO: 78) and KC027: SEQ ID NO: 85).
Micro PET/CT imaging of biodistribution of binding agent in tumor-bearing mouse model radiolabeling of binding agent and micro PET/CT imaging of its biodistribution was performed (fig. 23). First useAn Ultra-4 centrifugal filter (MWCO 30kDa, UFC 803008) was buffer exchanged with 0.1M sodium carbonate, pH 9.0 to prepare a binding agent. The binding agent was then modified with Desferrioxamine (DFO) by reaction with a 14-fold molar excess of p-isothiocyanatobenzyl desferrioxamine (p-NCS-Bz-DFO, macrocirculations). Briefly, 20-40. Mu.L of a solution of 20mM (15. Mu.g/. Mu.L) p-NCS-Bz-DFO in anhydrous DMSO was reacted with 4-7mg of binding agent (3-4 mg/mL in 0.1M NaHCO3 buffer, pH 9.0) at 37℃for 30 minutes and mixed on an Excella E24 mixer (New Brunswick Scientific) at 300 rpm. The reactants were split into small aliquots to avoid cross-linking and aggregation of the antibodies. After 30 minutes of reaction, excess p-NCS-Bz-DFO was removed by ultrafiltration and repeated 4 times on an Amicon Ultra-4 centrifuge filter (Millipore, 30kDa cut-off) using an excess of 0.25M sodium acetate buffer, pH 5.5, and centrifuged at 5000 Xg for 5 minutes. The purity and homogeneity of the DFO binding agent was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 4-20% tris-HCl gel (Bio-Rad) under reducing (2-mercaptoethanol) and non-reducing conditions.
After DFO conjugation, the following protocol was used 89 Zr radiolabels the binding agent. As oxalate salt 89 Zr was obtained from Washington university (Washington University) (St. Louis, MO) and was combined with 2M NaCO 3 The pH 10.0 was incubated for 3 minutes to neutralize the solution. Next, 0.25M sodium acetate buffer, pH7.0, containing 32mM gentisic acid (5 mg/mL) and 0.5M 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid (HEPES buffer) was added followed by the addition of a binding agent (KC 020; SEQ ID NO: 77) (1-5.0 mg/mL) comprising D2, PD-1 and CD47 specific antigen binding domains. Radiolabelling was carried out at room temperature for 60-90 minutes with a specific activity of 0.28mCi/mg. Radiochemical purity was measured by real-time thin-layer silica gel chromatography (ITLC-SG) on 20mM Na with 22mM citric acid 2 CO 3 And developed at pH 5.0.
After radiolabeling [ 89 Zr]DFO binders were injected into mice with 2 different types of xenografts (NCI-H69 and NCI-H82). Mice were anesthetized (5% induction) with isoflurane (Fresenius Kabi Canada ltd.)1.5-2% maintenance), followed by intravenous injection [ i.v. ] 89 Zr]DFO binding agent and received 200. Mu.L, 2.86.+ -. 0.2MBq and 30mg/Kg. Using MedisoThe SPECT/CT/PET 82S system images animals 24 hours, 96 hours, and 144 hours after injection.
As shown in fig. 23A and 23B, radiolabeled binding agents were found to accumulate in tumors of both NCI-H69 and NCI-H82 xenograft models, indicating that binding agents were recruited via tumor antigens.
EXAMPLE 7 in vivo testing of binding Agents in hCD34+ humanized NCG mouse model
hcd34+ humanized NCG mice were purchased from charles river laboratories. HSC engraftment levels were assessed by flow cytometry the day prior to tumor engraftment. Mice were randomly divided into 2 groups and NCI-H69 cells were transplanted subcutaneously (s.c) into the right abdomen of each animal (800 ten thousand cells per animal). Once the tumor reached an average of 100mm 3 The negative control and test article were administered by IP injection twice weekly. Tumor volume, percent Tumor Growth Inhibition (TGI) and statistical analysis were performed as described in example 3.
At the end of the study, animals were sacrificed and tumors and livers were collected for metastasis analysis.
The results of these experiments presented in FIGS. 24A-B show that KC020 (SEQ ID NO: 77) reduced the transfer of NCI-H69 cells to the liver in the hCD34+ humanized NCG mouse model. The results of FIG. 24C also show the efficacy of KC020 (SEQ ID NO: 77) in NCI-H82/PBMC co-implantation model.
Example 8 testing binding Agents in other solid tumors
To determine the anti-tumor efficacy of anti-D2 binders including KC013, KC011, KC012 and KC014 in NCI-H510A and NCI-H727 s.c. prophylactic xenograft models of human small cell lung cancer, female SCID mice were purchased from Charle River laboratories and used for study at 4-6 weeks of age. For NCI-H510a cells, cells were implanted subcutaneously (s.c) in the right abdomen of each animal, 800 ten thousand cells per mouse. For NCI-H727 cells, cells were implanted subcutaneously (s.c) in the right abdomen of each animal, 100 tens of thousands and 600 tens of thousands of cells per mouse. The next day, control and test articles were administered by intravenous injection, once a week. Tumor volume, percent Tumor Growth Inhibition (TGI) and statistical analysis were performed as described in example 3.
To evaluate the efficacy of anti-D2 binding agents in PANC1 s.c. prophylactic xenograft models of human pancreatic cancer, female SCID mice were purchased from charles river laboratories and used for research at 4-6 weeks of age. Cells were implanted subcutaneously (s.c) in the right abdomen of each animal, 500 tens of thousands each. Treatment was started one day after cell implantation. The negative control antibodies and test articles were administered by intraperitoneal injection, once a week. Tumor volume, percent Tumor Growth Inhibition (TGI) and statistical analysis were performed as described in example 3.
EXAMPLE 9 stability Studies
A purified binding agent solution was prepared at a concentration of 20mg/mL (+/-0.5) and isolated in two batches. One batch was stored at-80 ℃ (no pressure conditions) and the other batch was subjected to various pressure conditions including stirring for three days (25 ℃), five cycles of freeze thawing, two weeks at 40 ℃, or low pH (3.5) treatment for 4 hours and 48 hours. Two batches were tested for binding to D2-containing proteoliposomes (FIGS. 25A and 26A), to recombinant PD-1 (Cedarlane, catalog No. 10377-H08H-50) (FIGS. 25B and 26B) or to recombinant CD47 (Cedarlane, catalog No. 12283-H08H-200) (FIGS. 25C and 26C) as described in examples 3 and 4.
The results presented in fig. 25 and 26 indicate that the KC020 binding to its target is not affected by the pressure conditions tested.
Furthermore, under these conditions, the biophysical properties of the solution (solubility and aggregation) did not change significantly.
Similar results were obtained for several other binders (data not shown).
EXAMPLE 10 testing of binding Agents in the NCI-H82 tail vein transfer model of SCLC
NCG mice (5-6 weeks old, female) were purchased from charles river laboratory (st. Constant, QC). NCG mice were vaccinated with 50,000 NCI-H82 cells via tail vein injection. Mice received KC020 treatment 24 hours prior to tumor cell injection by intraperitoneal injection. KC020 was administered every two weeks for a total of 8 doses. Throughout the duration of the study, animals were monitored for weight loss and overall health. Overall, KC020 treatment significantly prolonged the survival of mice with iv-transplanted NCI-H82 SCLC compared to vehicle-treated negative control group (fig. 27).
In an in vivo bioluminescence imaging study, a total of 20 NCG mice were randomly divided into 4 groups including PBS group, KC020 3mg/kg group, KC020 10mg/kg group and KC020 30mg/kg group. On day 0, all mice received one KC020 or PBS treatment. For the KC020 3mg/kg group, mice received 10mg/kg of human IgG once a day before KC020 treatment. NCI-H82-luciferase cells were inoculated into NCG mice via tail vein injection, 100,000 cells per mouse. All mice were treated twice weekly with PBS or KC020 for a total of 8 doses. Bioluminescence imaging was performed three weeks after tumor cell inoculation, once a week. Prior to imaging, mice were given 200. Mu.L of firefly luciferase substrate in DPBS at 15mg/mL via intraperitoneal injection. The results of this experiment show that KC020 treatment prevented tumor progression even at all doses tested, including the lowest dose of 3 mg/kg.
The results presented herein show, inter alia, that the binding agents of the invention bind to each of their targets and exhibit in vitro CPI activity, exhibit in vivo tumor suppression efficacy in prophylactic and established xenograft models of human cancer, prevent metastatic progression and prolong mouse survival in tail vein models of human cancer. In addition, the binding agents of the invention are produced in very high yields in the preparation of cell lines and exhibit good stability in accelerated stability tests.
The implementations and examples described herein are illustrative and are not intended to limit the scope of the claims. The inventors intend that variations of the above embodiments, including alternatives, modifications, and equivalents, be included within the scope of the claims. Citations listed in this application are incorporated herein by reference.
Reference to the literature
All patents, patent applications, and publications mentioned throughout this disclosure are incorporated herein by reference.
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Sequence listing
TABLE 3 anti-D2 VHH sequences
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TABLE 4 anti-PD-1 VHH
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TABLE 5 anti-CD 47 VHH
TABLE 6-exemplary embodiment of polypeptide chain comprising an antigen binding domain (VHH-hinge-CH 2-CH 3) (with or without His tag)
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Table 7-exemplary embodiment of a polypeptide chain comprising three antigen binding domains (which may or may not comprise a His tag)
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SEQ ID NO. 98 (Natural human IgG1 hinge region)
EPKSCDKTHTCPPCP
SEQ ID NO. 99 (linker-HL 1)
EPKIPQPQPKPQPQPQPGGSGSAEAAAKAPKAP
SEQ ID NO. 100 (Flexible connector-FL 2)
GGGGSGGGGS
SEQ ID NO. 101 (Flexible connector-FL 18)
GGGGSGGGGSGGGGS
SEQ ID NO. 102 (Flexible connector-FL 4)
GGGGSGGGGSGGGGSGGGGSGGGGS
SEQ ID NO. 103 (Flexible connector-FL 5)
GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS
SEQ ID NO. 104 (Flexible connector)
(GGGGS) n Wherein n is an integer selected from 1 to 10
SEQ ID NO. 105 (rigid linker-RL 5)
PAPAPKA
SEQ ID NO. 106 (rigid linker-RL 7)
APAPAPAPAPKA
SEQ ID NO. 107 (rigid linker-RL 12)
APAPAPAPAPAPAPAPAPAPKA
SEQ ID NO. 108 (rigid linker)
(X(PAPAP)) n KA, wherein n is an integer selected from 1 to 10, wherein X is present or absent and is A
SEQ ID NO. 109 (spiral connector-RL 1)
AEAAAKEAAAKA
SEQ ID NO. 110 (spiral linker-RL 2)
AEAAAKEAAAKEAAAKA
SEQ ID NO. 111 (spiral linker-RL 4)
AEAAAKEAAAKEAAAKEAAAKEAAAKA
SEQ ID NO. 112 (spiral linker)
X(EAAAK) n Y, wherein n is an integer selected from 1 to 10, preferably 2-5, wherein X and Y are independently present or absent and are preferably A.
SEQ ID NO. 113 anti-HEWL VHH (KC 017)
XVQLVESGGGSVQAGGSLRLSCAASGSTDSIEYMTWFRQAPGKAREGVAALYTHTGNTYYTDSVKGRFTISQDKAKNMAYLRMDSVKSEDTAIYTCGATRKYVPVRFALDQSSYDYWGQGTQVTVSS
Wherein X is E or Q
SEQ ID NO. 114 anti-4 HEM VHH (VHH part of KC 018)
XVQLVESGGGLVQAGGSLRLSCAASESTFSNYAMGWFRQAPGPEREFVATISQTGSHTYYRNSVKGRFTISRDNAKNTVYLQMNNMKPEDTAVYYCAAGDNYYYTRTYEYDYWGQGTQVTVSS
Wherein X is E or Q
SEQ ID NO. 115 anti-CD 3 VHH (KC 019)
XVQLVESGGGLVQPGGSLRLSCAASGDIYKSFDMGWYRQAPGKQRDLVAVIGSRGNNRGRTNYADSVKGRFTISRDGTGNTVYLLMNKLRPEDTAIYYCNTAPLVAGRPWGRGTLVTVSS
Wherein X is E or D
116 natural human IgG1 CH3
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
117 Natural human IgG1 CH2
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
118 mutant human IgG1 hinge region of SEQ ID NO
EPKSSDKTHTCPPCP
SEQ ID NO. 119 mutant human IgG1 hinge region
EPKSSDKTHTSPPSP
SEQ ID NO. 120 mutant human IgG1 hinge region
DKTHTCPPC
SEQ ID NO. 121 native human IgG2 hinge region
ERKCCVECPPCP
SEQ ID NO. 122 mutant human IgG2 hinge region
ERKSSVECPPCP
123 mutant human IgG2 hinge region of SEQ ID NO
ERKSSVESPPCP
SEQ ID NO. 124 mutant human IgG2 hinge region
ERKSSVESPPSP
125 natural human IgG3 hinge region of SEQ ID NO
ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPP CPRCPEPKSCDTPPPCPRCP
SEQ ID NO. 126 mutant human IgG3 hinge region
EPKSSDTPPPCPRCP
SEQ ID NO. 127 mutant human IgG3 hinge region
EPKSSDTPPPSPRCP
128 mutant human IgG3 hinge region of SEQ ID NO. 128
EPKSSDTPPPSPRSP
SEQ ID NO. 129 native human IgG4 hinge region
ESKYGPPCPSCP
130 mutant human IgG4 hinge region of SEQ ID NO
ESKYGPPCPPCP
SEQ ID NO. 131 mutant human IgG4 hinge region
ESKYGPPSPSCP
SEQ ID NO. 132 mutant human IgG4 hinge region
ESKYGPPSPSSP
SEQ ID NO. 133 Signal peptide
MEWSWVFLFFLSVTTGVHS
SEQ ID NO 134 epitope tag
HHHHHH
SEQ ID NO. 135 Natural human IgG1 constant region
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO. 136 mutant CH3 domain (chain A-mutation D399N; K370E; E356Q)
GQPREPQVYTLPPSRQEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYKTTPPVLNSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO. 137 mutant CH3 domain (chain B-mutation D399N; K439E; E357Q)
GQPREPQVYTLPPSREQMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLNSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQESLSLSPGK
SEQ ID NO. 138 mutant human IgG1 constant regions (chain A-mutant D399N; K370E; E356Q)
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRQEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYKTTPPVLNSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO. 139 mutant human IgG1 constant region (chain B-mutation D399N; K439E; E357Q)
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREQMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLNSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQESLSLSPGK
140 mutant human IgG4 constant region APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK of SEQ ID NO. 140

Claims (183)

1. A binding agent comprising one or more antigen binding domains, wherein at least one of the antigen binding domains is antigen binding domain 1 (ABD 1) and comprises:
a. heavy chain complementarity determining region 1 (CRDH 1) having the amino acid sequence shown in SEQ ID NO. 1, heavy chain complementarity determining region 2 (CDRH 2) having the amino acid sequence shown in SEQ ID NO. 2, and heavy chain complementarity determining region 3 (CDRH 3) having the amino acid sequence shown in SEQ ID NO. 3;
b. CRDH1 having the amino acid sequence shown in SEQ ID NO. 4, CDRH2 having the amino acid sequence shown in SEQ ID NO. 5, and CDRH3 having the amino acid sequence shown in SEQ ID NO. 6;
c. CRDH1 having the amino acid sequence shown in SEQ ID NO. 8, CDRH2 having the amino acid sequence shown in SEQ ID NO. 9, and CDRH3 having the amino acid sequence shown in SEQ ID NO. 10;
d. CRDH1 having the amino acid sequence shown in SEQ ID NO. 11, CDRH2 having the amino acid sequence shown in SEQ ID NO. 12, and CDRH3 having the amino acid sequence shown in SEQ ID NO. 13;
e. CRDH1 having the amino acid sequence shown in SEQ ID NO. 15, CDRH2 having the amino acid sequence shown in SEQ ID NO. 16, and CDRH3 having the amino acid sequence shown in SEQ ID NO. 17;
f. CRDH1 having the amino acid sequence shown in SEQ ID NO. 18, CDRH2 having the amino acid sequence shown in SEQ ID NO. 19, and CDRH3 having the amino acid sequence shown in SEQ ID NO. 20;
g. CRDH1 having the amino acid sequence shown in SEQ ID NO. 22, CDRH2 having the amino acid sequence shown in SEQ ID NO. 23, and CDRH3 having the amino acid sequence shown in SEQ ID NO. 24;
h. CRDH1 having the amino acid sequence shown in SEQ ID NO. 25, CDRH2 having the amino acid sequence shown in SEQ ID NO. 26, and CDRH3 having the amino acid sequence shown in SEQ ID NO. 27;
i. CRDH1 having the amino acid sequence shown in SEQ ID NO. 29, CDRH2 having the amino acid sequence shown in SEQ ID NO. 30, and CDRH3 having the amino acid sequence shown in SEQ ID NO. 31;
j. CRDH1 having the amino acid sequence shown in SEQ ID NO. 32, CDRH2 having the amino acid sequence shown in SEQ ID NO. 33, and CDRH3 having the amino acid sequence shown in SEQ ID NO. 34;
k. CRDH1 having the amino acid sequence shown in SEQ ID NO. 36, CDRH2 having the amino acid sequence shown in SEQ ID NO. 37, and CDRH3 having the amino acid sequence shown in SEQ ID NO. 38;
CRDH1 having the amino acid sequence shown in SEQ ID NO. 39, CDRH2 having the amino acid sequence shown in SEQ ID NO. 40, and CDRH3 having the amino acid sequence shown in SEQ ID NO. 41;
m. CRDH1 having the amino acid sequence shown in SEQ ID NO. 43, CDRH2 having the amino acid sequence shown in SEQ ID NO. 44, CDRH3 having the amino acid sequence shown in SEQ ID NO. 45;
n. CRDH1 having the amino acid sequence shown in SEQ ID NO. 46, CDRH2 having the amino acid sequence shown in SEQ ID NO. 47, CDRH3 having the amino acid sequence shown in SEQ ID NO. 48;
CRDH1 having the amino acid sequence shown in SEQ ID NO. 50, CDRH2 having the amino acid sequence shown in SEQ ID NO. 51, and CDRH3 having the amino acid sequence shown in SEQ ID NO. 52;
CRDH1 having the amino acid sequence shown in SEQ ID NO. 53, CDRH2 having the amino acid sequence shown in SEQ ID NO. 54, and CDRH3 having the amino acid sequence shown in SEQ ID NO. 55;
a heavy chain having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 7;
a heavy chain having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID No. 14;
s. a heavy chain having an amino acid sequence which is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO. 21;
a heavy chain having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 28;
a heavy chain having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 35;
a heavy chain having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 42;
a heavy chain having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID No. 49; or (b)
x.a heavy chain having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 56.
2. A binding agent comprising one or more antigen binding domains, wherein at least one of the antigen binding domains is antigen binding domain 2 (ABD 2) and comprises:
a. heavy chain complementarity determining region 1 (CRDH 1) having the amino acid sequence shown in SEQ ID NO. 57, heavy chain complementarity determining region 2 (CDRH 2) having the amino acid sequence shown in SEQ ID NO. 58, and heavy chain complementarity determining region 3 (CDRH 3) having the amino acid sequence shown in SEQ ID NO. 59;
b. CRDH1 having the amino acid sequence shown in SEQ ID NO. 60, CDRH2 having the amino acid sequence shown in SEQ ID NO. 61, and CDRH3 having the amino acid sequence shown in SEQ ID NO. 62; or (b)
c. A heavy chain having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 63.
3. A binding agent comprising one or more antigen binding domains, wherein at least one of the antigen binding domains is antigen binding domain 3 (ABD 3) and comprises:
a. heavy chain complementarity determining region 1 (CRDH 1) having the amino acid sequence shown in SEQ ID NO. 64, heavy chain complementarity determining region 2 (CDRH 2) having the amino acid sequence shown in SEQ ID NO. 65, and heavy chain complementarity determining region 3 (CDRH 3) having the amino acid sequence shown in SEQ ID NO. 66;
b. CRDH1 having the amino acid sequence shown in SEQ ID NO. 67, CDRH2 having the amino acid sequence shown in SEQ ID NO. 68 and CDRH3 having the amino acid sequence shown in SEQ ID NO. 69, or
c. A heavy chain having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 70.
4. A binding agent comprising one or more antigen binding domains, wherein at least one of the antigen binding domains is selected from the group consisting of:
a. an antigen binding domain 1 (ABD 1) according to claim 1,
b. An antigen binding domain 2 (ABD 2) or according to claim 2
c. An antigen binding domain 3 (ABD 3) according to claim 3.
5. A binding agent, comprising:
a. heavy chain complementarity determining region 1 (CRDH 1) having the amino acid sequence shown in SEQ ID NO. 1, heavy chain complementarity determining region 2 (CDRH 2) having the amino acid sequence shown in SEQ ID NO. 2, and heavy chain complementarity determining region 3 (CDRH 3) having the amino acid sequence shown in SEQ ID NO. 3;
b. CRDH1 having the amino acid sequence shown in SEQ ID NO. 4, CDRH2 having the amino acid sequence shown in SEQ ID NO. 5, and CDRH3 having the amino acid sequence shown in SEQ ID NO. 6;
c. CRDH1 having the amino acid sequence shown in SEQ ID NO. 8, CDRH2 having the amino acid sequence shown in SEQ ID NO. 9, and CDRH3 having the amino acid sequence shown in SEQ ID NO. 10;
d. CRDH1 having the amino acid sequence shown in SEQ ID NO. 11, CDRH2 having the amino acid sequence shown in SEQ ID NO. 12, and CDRH3 having the amino acid sequence shown in SEQ ID NO. 13;
e. CRDH1 having the amino acid sequence shown in SEQ ID NO. 15, CDRH2 having the amino acid sequence shown in SEQ ID NO. 16, and CDRH3 having the amino acid sequence shown in SEQ ID NO. 17;
f. CRDH1 having the amino acid sequence shown in SEQ ID NO. 18, CDRH2 having the amino acid sequence shown in SEQ ID NO. 19, and CDRH3 having the amino acid sequence shown in SEQ ID NO. 20;
g. CRDH1 having the amino acid sequence shown in SEQ ID NO. 22, CDRH2 having the amino acid sequence shown in SEQ ID NO. 23, and CDRH3 having the amino acid sequence shown in SEQ ID NO. 24;
h. CRDH1 having the amino acid sequence shown in SEQ ID NO. 25, CDRH2 having the amino acid sequence shown in SEQ ID NO. 26, and CDRH3 having the amino acid sequence shown in SEQ ID NO. 27;
i. CRDH1 having the amino acid sequence shown in SEQ ID NO. 29, CDRH2 having the amino acid sequence shown in SEQ ID NO. 30, and CDRH3 having the amino acid sequence shown in SEQ ID NO. 31;
j. CRDH1 having the amino acid sequence shown in SEQ ID NO. 32, CDRH2 having the amino acid sequence shown in SEQ ID NO. 33, and CDRH3 having the amino acid sequence shown in SEQ ID NO. 34;
k. CRDH1 having the amino acid sequence shown in SEQ ID NO. 36, CDRH2 having the amino acid sequence shown in SEQ ID NO. 37, and CDRH3 having the amino acid sequence shown in SEQ ID NO. 38;
CRDH1 having the amino acid sequence shown in SEQ ID NO. 39, CDRH2 having the amino acid sequence shown in SEQ ID NO. 40, and CDRH3 having the amino acid sequence shown in SEQ ID NO. 41;
m. CRDH1 having the amino acid sequence shown in SEQ ID NO. 43, CDRH2 having the amino acid sequence shown in SEQ ID NO. 44, CDRH3 having the amino acid sequence shown in SEQ ID NO. 45;
n. CRDH1 having the amino acid sequence shown in SEQ ID NO. 46, CDRH2 having the amino acid sequence shown in SEQ ID NO. 47, CDRH3 having the amino acid sequence shown in SEQ ID NO. 48;
CRDH1 having the amino acid sequence shown in SEQ ID NO. 50, CDRH2 having the amino acid sequence shown in SEQ ID NO. 51, and CDRH3 having the amino acid sequence shown in SEQ ID NO. 52;
CRDH1 having the amino acid sequence shown in SEQ ID NO. 53, CDRH2 having the amino acid sequence shown in SEQ ID NO. 54, and CDRH3 having the amino acid sequence shown in SEQ ID NO. 55;
a heavy chain having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 7;
A heavy chain having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID No. 14;
s. a heavy chain having an amino acid sequence which is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO. 21;
a heavy chain having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 28;
a heavy chain having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 35;
a heavy chain having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 42;
a heavy chain having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID No. 49;
x. a heavy chain having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO. 56;
y. heavy chain complementarity determining region 1 (CRDH 1) having the amino acid sequence set forth in SEQ ID NO. 57, heavy chain complementarity determining region 2 (CDRH 2) having the amino acid sequence set forth in SEQ ID NO. 58, and heavy chain complementarity determining region 3 (CDRH 3) having the amino acid sequence set forth in SEQ ID NO. 59;
z. CRDH1 having the amino acid sequence shown in SEQ ID NO. 60, CDRH2 having the amino acid sequence shown in SEQ ID NO. 61, and CDRH3 having the amino acid sequence shown in SEQ ID NO. 62;
aa. a heavy chain having an amino acid sequence which is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO. 63；
bb. heavy chain complementarity determining region 1 (CRDH 1) having the amino acid sequence set forth in SEQ ID NO. 64, heavy chain complementarity determining region 2 (CDRH 2) having the amino acid sequence set forth in SEQ ID NO. 65, and heavy chain complementarity determining region 3 (CDRH 3) having the amino acid sequence set forth in SEQ ID NO. 66;
cc. CRDH1 having the amino acid sequence shown in SEQ ID NO. 67, CDRH2 having the amino acid sequence shown in SEQ ID NO. 68, and CDRH3 having the amino acid sequence shown in SEQ ID NO. 69;
dd. has a heavy chain with an amino acid sequence which is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO. 70; or (b)
A combination of any of ee.a. to dd..
6. The binding agent of any one of the preceding claims, wherein the binding agent comprises more than one antigen binding domain.
7. The binding agent of any one of the preceding claims, wherein the binding agent comprises two or more antigen binding domains.
8. The binding agent of any one of the preceding claims, wherein the binding agent comprises three or more antigen binding domains.
9. The binding agent of any one of the preceding claims, wherein the binding agent comprises four or more antigen binding domains.
10. The binding agent of any one of the preceding claims, wherein the binding agent comprises five or more antigen binding domains.
11. The binding agent of any one of the preceding claims, wherein the binding agent comprises six or more antigen binding domains.
12. The binding agent of any one of the preceding claims, wherein the binding agent comprises between one and twelve antigen binding domains.
13. The binding agent of any one of the preceding claims, wherein at least one of the antigen binding domains is capable of binding to a tumor cell.
14. The binding agent of any one of the preceding claims, wherein at least one of the antigen binding domains is capable of binding to an immunomodulatory agent.
15. The binding agent of claim 14, wherein the immunomodulatory agent is an immune checkpoint protein.
16. The binding agent of any one of the preceding claims, wherein at least one of the antigen binding domains is capable of binding to an immune cell.
17. The binding agent of any one of the preceding claims, wherein at least one of the antigen binding domains is capable of binding to a protein expressed at the surface of an immune cell.
18. The binding agent of claim 16 or 17, wherein the immune cells comprise T cells, NK cells, monocytes or macrophages.
19. A binding agent comprising more than one antigen binding domain, wherein at least one of the antigen binding domains is an antigen binding domain 1 (ABD 1) according to claim 1, and at least one of the antigen binding domains is an antigen binding domain capable of binding to an immune checkpoint protein.
20. A binding agent comprising more than one antigen binding domain, wherein at least one of the antigen binding domains is an antigen binding domain 1 (ABD 1) according to claim 1, at least one of the antigen binding domains is an antigen binding domain capable of binding to an immune checkpoint protein, and at least one of the antigen binding domains is an antigen binding domain capable of binding to a protein expressed at the surface of an immune cell.
21. The binding agent of any one of claims 15 to 20, wherein the immune checkpoint protein is PD-1.
22. The binding agent of any one of claims 17, 18, 20 or 21, wherein the protein expressed at the surface of an immune cell is CD47.
23. The binding agent of any one of claims 17, 18, 20 or 21, wherein the protein expressed at the surface of an immune cell is CD3.
24. The binding agent of any one of claims 4 to 23, wherein the binding agent comprises at least one antigen binding domain 1 (ABD 1) and at least one antigen binding domain 2 (ABD 2).
25. The binding agent of any one of claims 4 to 23, wherein the binding agent comprises at least one antigen binding domain 1 (ABD 1) and at least one antigen binding domain 3 (ABD 3).
26. The binding agent of any one of claims 4 to 23, wherein the binding agent comprises at least one antigen binding domain 2 (ABD 2) and at least one antigen binding domain 3 (ABD 3).
27. The binding agent of any one of claims 4 to 23, wherein the binding agent comprises at least one antigen binding domain 1 (ABD 1), at least one antigen binding domain 2 (ABD 2), and at least one antigen binding domain 3 (ABD 3).
28. A binding agent comprising one or more antigen binding domains, wherein at least one of the antigen binding domains is antigen binding domain 1 (ABD 1) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 7, at least one of the antigen binding domains is antigen binding domain 2 (ABD 2) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 63, and at least one of the antigen binding domains is antigen binding domain 3 (ABD 3) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 70.
29. A binding agent comprising one or more antigen binding domains, wherein at least one of the antigen binding domains is antigen binding domain 1 (ABD 1) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 14, at least one of the antigen binding domains is antigen binding domain 2 (ABD 2) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 63, and at least one of the antigen binding domains is antigen binding domain 3 (ABD 3) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 70.
30. A binding agent comprising one or more antigen binding domains, wherein at least one of the antigen binding domains is antigen binding domain 1 (ABD 1) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 21, at least one of the antigen binding domains is antigen binding domain 2 (ABD 2) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 63, and at least one of the antigen binding domains is antigen binding domain 3 (ABD 3) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 70.
31. A binding agent comprising one or more antigen binding domains, wherein at least one of the antigen binding domains is antigen binding domain 1 (ABD 1) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 28, at least one of the antigen binding domains is antigen binding domain 2 (ABD 2) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 63, and at least one of the antigen binding domains is antigen binding domain 3 (ABD 3) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 70.
32. A binding agent comprising one or more antigen binding domains, wherein at least one of the antigen binding domains is antigen binding domain 1 (ABD 1) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 35, at least one of the antigen binding domains is antigen binding domain 2 (ABD 2) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 63, and at least one of the antigen binding domains is antigen binding domain 3 (ABD 3) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 70.
33. A binding agent comprising one or more antigen binding domains, wherein at least one of the antigen binding domains is antigen binding domain 1 (ABD 1) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 42, at least one of the antigen binding domains is antigen binding domain 2 (ABD 2) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 63, and at least one of the antigen binding domains is antigen binding domain 3 (ABD 3) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 70.
34. A binding agent comprising one or more antigen binding domains, wherein at least one of the antigen binding domains is antigen binding domain 1 (ABD 1) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 49, at least one of the antigen binding domains is antigen binding domain 2 (ABD 2) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 63, and at least one of the antigen binding domains is antigen binding domain 3 (ABD 3) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 70.
35. A binding agent comprising one or more antigen binding domains, wherein at least one of the antigen binding domains is antigen binding domain 1 (ABD 1) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 56, at least one of the antigen binding domains is antigen binding domain 2 (ABD 2) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 63, and at least one of the antigen binding domains is antigen binding domain 3 (ABD 3) and comprises a heavy chain having an amino acid sequence as set forth in SEQ ID No. 70.
36. The binding agent of any one of claims 1 to 35, wherein the binding agent comprises a dimerization domain.
37. The binding agent of claim 36, wherein the dimerization domain comprises a constant region of an antibody or a portion thereof.
38. The binding agent of claim 36, wherein the dimerization domain comprises a CH3 domain.
39. The binding agent of claim 38, wherein the CH3 domain is a native CH3 domain or a mutant CH3 domain.
40. The binding agent of claim 38 or 39, wherein the dimerization domain further comprises a CH2 domain.
41. The binding agent of claim 40, wherein the CH2 domain is a native CH2 domain or a mutant CH2 domain.
42. The binding agent of claim 41, wherein the dimerization domain comprises a native CH2 domain and a native CH3 domain.
43. The binding agent of claim 41, wherein the dimerization domain comprises a native CH2 domain and a mutant CH3 domain.
44. The binding agent of claim 41, wherein the dimerization domain comprises a mutant CH2 domain and a native CH3 domain.
45. The binding agent of claim 41, wherein the dimerization domain comprises a mutant CH2 domain and a mutant CH3 domain.
46. The binding agent of any one of claims 36 to 45, wherein the binding agent further comprises a hinge region.
47. The binding agent of claim 46, wherein the hinge region is a natural hinge region or a mutated hinge region.
48. The binding agent of any one of the preceding claims, wherein the antigen binding domain is located on one or more polypeptide chains.
49. The binding agent of any one of the preceding claims, wherein the antigen binding domain antigens are on the same polypeptide chain.
50. The binding agent of any one of the preceding claims, wherein the binding agent comprises at least two polypeptide chains.
51. The binding agent of any one of the preceding claims, wherein the binding agent comprises at least two polypeptide chains capable of assembling to form a dimer, and wherein each polypeptide chain comprises one or more antigen binding domains.
52. The binding agent of any one of the preceding claims, wherein the binding agent comprises at least two polypeptide chains capable of assembling to form a dimer, and wherein each polypeptide chain comprises a different antigen binding domain.
53. The binding agent of any one of the preceding claims, wherein the binding agent comprises at least two polypeptide chains capable of assembling to form a dimer, and wherein each polypeptide chain comprises the same antigen binding domain.
54. The binding agent of any one of claims 4 to 53, wherein the binding agent comprises at least two polypeptide chains that assemble to form a dimer, and wherein each polypeptide chain comprises the same antigen binding domain 1 (ABD 1).
55. The binding agent of any one of claims 4 to 53, wherein the binding agent comprises at least two polypeptide chains that assemble to form a dimer, and wherein each polypeptide chain comprises the same antigen binding domain 2 (ABD 2).
56. The binding agent of any one of claims 4 to 53, wherein the binding agent comprises at least two polypeptide chains that assemble to form a dimer, and wherein each polypeptide chain comprises the same antigen binding domain 3 (ABD 3).
57. The binding agent of any one of claims 4 to 53, wherein the binding agent comprises at least two polypeptide chains that assemble to form a dimer, and wherein each polypeptide chain comprises the same antigen binding domain 1 (ABD 1) and the same antigen binding domain 2 (ABD 2).
58. The binding agent of any one of claims 4 to 53, wherein the binding agent comprises at least two polypeptide chains that assemble to form a dimer, and wherein each polypeptide chain comprises the same antigen binding domain 1 (ABD 1) and the same antigen binding domain 3 (ABD 3).
59. The binding agent of any one of claims 4 to 53, wherein the binding agent comprises at least two polypeptide chains that assemble to form a dimer, and wherein each polypeptide chain comprises the same antigen binding domain 2 (ABD 2) and the same antigen binding domain 3 (ABD 3).
60. The binding agent of any one of claims 4 to 53, wherein the binding agent comprises at least two polypeptide chains that assemble to form a dimer, and wherein each polypeptide chain comprises the same antigen binding domain 1 (ABD 1), the same antigen binding domain 2 (ABD 2), and the same antigen binding domain 2 (ABD 3).
61. The binding agent of any one of the preceding claims, wherein the binding agent comprises at least two polypeptide chains capable of assembling to form a dimer, and wherein each polypeptide chain is identical.
62. The binding agent of any one of claims 48 to 61, wherein each of the polypeptide chains independently comprises the amino acid sequence of formula I in N-terminal to C-terminal fashion:
X-[(Ab a )-(L b )] m -(DD)-[(L c )-(Ab d )] n -Y
wherein m is 0, 1, 2 or an integer greater than 2; wherein n is 0, 1, 2 or an integer greater than 2;
wherein m and n are not both 0;
wherein Ab a 、Ab d Each represents an antigen binding domain, wherein at least one of the antigen binding domains is antigen binding domain 1 (ABD 1), antigen binding domain 2 (ABD 2), or antigen binding domain 3 (ABD 3);
wherein X or Y is independently present or absent and comprises an amino acid sequence;
wherein L is b 、L c Each independently comprising one or more linkers; and is also provided with
Wherein DD represents the dimerization domain.
63. The binding agent of any one of claims 48 to 62, wherein said polypeptide chain comprises an antigen binding domain.
64. The binding agent of any one of claims 48 to 62, wherein the polypeptide chain comprises more than one antigen binding domain.
65. The binding agent of any one of claims 48 to 62, wherein the polypeptide chain comprises two or more antigen binding domains.
66. The binding agent of any one of claims 48 to 62, wherein the polypeptide chain comprises three or more antigen binding domains.
67. The binding agent of any one of claims 48 to 62, wherein the polypeptide chain comprises four or more antigen binding domains.
68. The binding agent of any one of claims 48 to 62, wherein the polypeptide chain comprises five or more antigen binding domains.
69. The binding agent of any one of claims 48 to 62, wherein the polypeptide chain comprises six or more antigen binding domains.
70. The binding agent of any one of claims 48 to 62, wherein the polypeptide chain comprises between one and twelve antigen binding domains.
71. The binding agent of any one of claims 62 to 70, wherein when m is 2 or an integer greater than 2, the [ (Ab a )-(L b )]The units are identical.
72. The binding agent of any one of claims 62 to 70, wherein when m is 2 or an integer greater than 2, the [ (Ab a )-(L b )]The units being different。
73. The binding agent of any one of claims 62 to 70, wherein when m is an integer greater than 2, the [ (Ab a )-(L b )]The units comprise the same and different units.
74. The binding agent of any one of claims 62 to 73, wherein when n is 2 or an integer greater than 2, the [ (L) c )-(Ab d )]The units are identical.
75. The binding agent of any one of claims 62 to 73, wherein when n is 2 or an integer greater than 2, the [ (L) c )-(Ab d )]The cells are different.
76. The binding agent of any one of claims 62 to 73, wherein when n is 2 or an integer greater than 2, the [ (L) c )-(Ab d )]The units comprise the same and different units.
77. The binding agent of any one of claims 62 to 70, wherein when m is 2 or an integer greater than 2, each Ab a The same or different.
78. The binding agent of any one of claims 62 to 70 or 77, wherein when n is 2 or an integer greater than 2, each Ab d The same or different.
79. The binding agent of any one of claims 62 to 78, wherein Ab a Representing ABD1.
80. The binding agent of any one of claims 62 to 78, wherein Ab d Representing ABD2.
81. The binding agent of any one of claims 62 to 78, wherein Ab d Representing ABD3.
82. The binding agent of any one of claims 62 to 78, wherein m is 1 and Ab a Representing ABD1.
83. The binding agent of any one of claims 62 to 78, wherein n is 2 and Ab d Represents ABD2.
84. The binding agent of any one of claims 62 to 78, wherein n is 2 and Ab d Represents ABD3.
85. The binding agent of any one of claims 62 to 78, wherein n is 2 and Ab d One of them represents ABD2 and the other Ab d Representing ABD3.
86. The binding agent of any one of claims 62 to 85, wherein m is 2, 3, 4, 5 or an integer greater than 5.
87. The binding agent of any one of claims 62 to 86, wherein n is 3, 4, 5 or an integer greater than 5.
88. The binding agent of any one of claims 62 to 87, wherein L b Is the hinge region of an antibody or antigen binding fragment thereof.
89. The binding agent of any one of claims 62 to 88, wherein L c Is a rigid linker.
90. The binding agent of any one of claims 48 to 89, wherein each of the polypeptide chains independently comprises in N-terminal to C-terminal fashion an amino acid sequence set forth in any one of:
X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 ) -Y (formula II);
X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 ) -Y (formula III);
X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 ) -Y (formula IV);
X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 ) Y (V)
X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-(L c3 )-(Ab d3 ) -Y (formula VI);
X-(Ab a1 )-(L b3 )-(Ab a2 )-(L b2 )-(Ab a3 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 ) -Y (formula VII);
X-(Ab a1 )-(L b3 )-(Ab a2 )-(L b2 )-(Ab a3 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-(L c3 )-(Ab d3 ) Y (formula VIII)
Wherein Ab a1 、Ab a2 、Ab a3 、Ab d1 、Ab d2 、Ab d3 Each representing an antigen binding domain, wherein at least one of the antigen binding domains is antigen binding domain 1 (ABD 1), antigen binding domain 2 (ABD 2), or antigen binding domain 3 (ABD 3)
Wherein L is b1 A hinge region comprising one or more linkers and/or antibodies or antigen binding fragments thereof;
Wherein L is b2 、L b3 、L c1 、L c2 L and L c3 Each independently comprising one or more linkers and;
wherein DD represents the dimerization domain.
91. The binding agent of any one of claims 48 to 90, wherein each of the polypeptide chains independently comprises the amino acid sequence of formula III in N-terminal to C-terminal fashion:
X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 ) -Y (formula III);
wherein Ab a1 、Ab d1 Or Ab d2 Represents antigen binding domain 1 (ABD 1).
92. The binding agent of any one of claims 48 to 90, wherein each of the polypeptide chains independently comprises the amino acid sequence of formula III in N-terminal to C-terminal fashion:
X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 ) -Y (formula III);
wherein Ab a1 、Ab d1 Or Ab d2 Represents antigen binding domain 2 (ABD 2).
93. The binding agent of any one of claims 48 to 90, wherein each of the polypeptide chains independently comprises the amino acid sequence of formula III in N-terminal to C-terminal fashion:
X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 ) -Y (formula III);
wherein Ab a1 、Ab d1 Or Ab d2 Represents antigen binding domain 3 (ABD 3).
94. The binding agent of any one of claims 48 to 90, wherein each of the polypeptide chains independently comprises the amino acid sequence of formula III in N-terminal to C-terminal fashion:
X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 ) -Y (formula III);
wherein Ab a1 、Ab d1 Or Ab d2 One of them represents antigen binding domain 1 (ABD 1), ab a1 、Ab d1 Or Ab d2 One of them represents antigen binding domain 2 (ABD 2), and Ab a1 、Ab d1 Or Ab d2 Represents antigen binding domain 3 (ABD 3).
95. The binding agent of any one of claims 48 to 90, wherein each of the polypeptide chains independently comprises the amino acid sequence of formula III in N-terminal to C-terminal fashion:
X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 ) -Y (formula III);
wherein Ab a1 Representing antigen binding domain 1 (ABD 1), ab d1 Represents antigen binding domain 2 (ABD 2), and Ab d2 Representing antigen binding domain 3 (ABD 3).
96. The binding agent of any one of claims 48 to 90, wherein each of the polypeptide chains independently comprises the amino acid sequence of formula IIIa in N-terminal to C-terminal manner:
X-(ABD1)-(L b1 )-(DD)-(L c1 )-(ABD2)-(L c2 ) - (ABD 3) -Y (formula IIIa).
97. The binding agent of any one of claims 48 to 90, wherein each of the polypeptide chains independently comprises the amino acid sequence of formula IIIb in N-terminal to C-terminal manner:
X-(ABD1)-(L b1 )-(DD)-(L c1 )-(ABD3)-(L c2 ) - (ABD 2) -Y (formula IIIB).
98. The binding agent of any one of claims 36-97, wherein the dimerization domain comprises native human IgG1 CH2 and mutant IgG1 CH3.
99. The binding agent of claim 97, wherein the mutant IgG1 CH3 comprises one or more amino acid substitutions according to EU numbering at positions corresponding to D399, D/E356 and/or K370, or comprises one or more mutations at positions corresponding to D399, E357 and/or K439.
100. The binding agent of claim 99, wherein the mutant IgG1 CH3 domain further comprises one or more amino acid substitutions according to EU numbering at positions corresponding to Y349, T350, L351, P352, S354, R/Q355, T394 and/or P395.
101. The binding agent of any one of claims 99 or 100, wherein the mutant CH3 domain comprises a mutation selected from the group consisting of:
d. mutation D399N, D/E356Q, K370E;
e. mutation D399N, K439E, E357Q;
f. mutation D399Q, D/E356Q, K370E, Y349K and S354K;
g. mutation D399N, D/E356Q, K370E and L351W;
h. mutation D399N, D/E356Q, K370E and S354M;
i. mutation D399N, D/E356Q, K370E and T350I;
j. mutation D399N, D/E356Q, K370E and T350V;
k. mutation D399N, D/E356Q, K370E and P352R;
l. mutation D399N, D/E356Q, K370E and P352E;
m. mutation D399Q, D/E356Q and K370E;
n. mutation D399N, D/E356Q, K370E and L351Y;
o. mutation D399N, D/E356Q, K370E and L351H;
p. mutation D399N, D/E356Q, K370E and R355K;
q. mutation D399N, D/E356Q, K370E and Q355K;
r. mutation D399N, D/E356Q, K370E and S354K;
s. mutation D399N, D/E356Q, K370E and T350L;
t. mutation D399N, D/E356Q, K370E and T394N;
u. mutation D399N, D/E356Q, K370E and P352Y;
v. mutation D399N, D/E356Q, K370E and P352V;
w, mutation D399N, D/E356Q, K370E and P352T;
x, mutation D399N, D/E356Q, K370E and P352L;
y. mutation D399N, D/E356Q, K370E and P352G;
z. mutation D399N, D/E356Q, K370E and P352C;
aa. mutation D399N, D/E356Q, K370E and L351T;
bb. mutation D399N, D/E356Q, K370E and L351A;
cc. mutation D399Q, E357Q, K439E, Y349D and S354D;
dd. mutation D399N, E357Q, K439E and L351R;
ee. mutant In D399N, E357Q, K439E and L351Y;
ff. mutation D399N, E357Q, K439E and T350I;
gg. mutation D399N, E357Q, K439E and T350V;
hh. mutation D399Q, K439E, E357Q;
mutation D399N, K439E, E357Q, S354K;
jj. mutation D399N, K439E, E357Q, S354W;
kk. mutation D399N, K439E, E357Q, Y349R;
ll. mutation D399N, K439E, E357Q, T350 0L;
mm. mutation D399N, K439E, E357Q, R355W;
nn. mutation D399N, K439E, E357Q, Q355W;
oo. mutation D399N, K439E, E357Q, P I;
pp. mutation D399N, K439E, E357Q, P G;
qq. mutation D399N, K439E, E357Q, P E;
rr. mutation D399N, K439E, E357Q, P K;
ss. mutation D399N, K439E, E357Q, P352D; a kind of electronic device with high-pressure air-conditioning system
tt. mutation D399N, K439E, E357Q, L D.
102. The binding agent of any one of claims 62 to 101, wherein each of the linkers is independently at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid residues in length.
103. The binding agent of any one of claims 62 to 102, wherein each of the linkers is independently a flexible linker, a helical linker, or a rigid linker.
104. The binding agent of claim 103, wherein the linker comprises a flexible linker.
105. The binding agent of claim 103, wherein the linker comprises a rigid linker.
106. The binding agent of any one of claims 90 to 105, wherein L c1 、L c2 And/or L c3 Is a rigid linker.
107. The binding agent of any one of claims 62 to 106, wherein the one or more polypeptide chains further comprise a hinge region of an antibody or antigen binding fragment thereof.
108. The binding agent of claim 107, wherein the hinge region is at the N-terminus of the dimerization domain.
109. The binding agent of claim 107 or 108, wherein the hinge region is from IgG1, igG2, igG3, or IgG4.
110. The binding agent of any one of the preceding claims, wherein each antigen binding domain is capable of specifically binding to a different epitope.
111. The binding agent of any one of the preceding claims, wherein each antigen binding domain is capable of specifically binding to a different antigen.
112. The binding agent of any one of the preceding claims, wherein each antigen binding domain is capable of specifically binding to a different protein.
113. The binding agent of any one of claims 50 to 112, wherein
One of the two polypeptide chains comprises a first dimerization domain (DD 1 ) The first dimerization domain comprises a mutant CH3 domain comprising one or more amino acid substitutions at positions corresponding to D399, D/E356 and/or K370 according to EU numbering;
and
the other of the two polypeptide chains comprises a second dimerization domain (DD 2 ) The second dimerization domain comprises a mutant CH3 domain, the mutant CH3 domain comprising one or more amino acid substitutions at positions corresponding to D399, E357 and/or K439 according to EU numbering.
114. The binding agent of claim 113, wherein the first dimerization domain (DD 1 ) Comprises a mutant CH3 domain comprising mutations D399N, D/E356Q and K370E according to EU numbering, and wherein the second dimerization domain (DD 2 ) Comprises a mutant CH3 domain comprising mutations D399N, E357Q and K439E according to EU numbering.
115. The binding agent of claim 113 or 114, wherein the first dimerization domain (DD 1 ) And/or the second dimerization domain (DD 2 ) Comprises a mutant CH3 domain, which mutant CH3 domain further comprises an amino acid substitution at a position corresponding to Y349, T350, L351, P352 and/or S354 according to EU numbering.
116. The binding agent of claim 115, wherein the first dimerization domain (DD 1 ) Comprises a mutant CH3 domain comprising mutations D399Q, D/E356Q, K370E, Y349K and S354K according to EU numbering, and wherein the second dimerization domain (DD 2 ) Comprises a mutant CH3 domain comprising the mutations D399Q, E357Q, K439E, Y349D and S354D according to EU numbering.
117. The binding agent of claim 115, wherein theThe first dimerization domain (DD 1 ) Comprises a mutant CH3 domain comprising mutations D399N, D/E356Q, K E and L351W according to EU numbering, and wherein the second dimerization domain (DD 2 ) Comprises a mutant CH3 domain comprising mutations D399N, E357Q, K439E and L351R according to EU numbering.
118. The binding agent of claim 115, wherein the first dimerization domain (DD 1 ) Comprises a mutant CH3 domain comprising mutations D399N, D/E356Q, K E and S354M according to EU numbering, and wherein the second dimerization domain (DD 2 ) Comprises a mutant CH3 domain comprising mutations D399N, E357Q, K439E and L351Y according to EU numbering.
119. The binding agent of claim 115, wherein the first dimerization domain (DD 1 ) Comprises a mutant CH3 domain comprising mutations D399N, D/E356Q, K E and T350I according to EU numbering, and wherein the second dimerization domain (DD 2 ) Comprises a mutant CH3 domain comprising mutations D399N, E357Q, K439E and T350I according to EU numbering.
120. The binding agent of claim 115, wherein the first dimerization domain (DD 1 ) Comprises a mutant CH3 domain comprising mutations D399N, D/E356Q, K E and T350V according to EU numbering, and wherein the second dimerization domain (DD 2 ) Comprises a mutant CH3 domain comprising mutations D399N, E357Q, K439E and T350V according to EU numbering.
121. The binding agent of claim 115, wherein the first dimerization domain (DD 1 ) Comprises a mutant CH3 domain comprising mutations D399N, D/E356Q, K E and P352R according to EU numbering, and wherein the second dimerization domain (DD 2 ) Comprises a mutant CH3 domain comprising mutations D399N, E357Q, K439E and L351R according to EU numbering.
122The binding agent of claim 115, wherein the first dimerization domain (DD 1 ) Comprises a mutant CH3 domain comprising mutations D399N, D/E356Q, K E and P352E according to EU numbering, and wherein the second dimerization domain (DD 2 ) Comprises a mutant CH3 domain comprising mutations D399N, E357Q, K439E and L351R according to EU numbering.
123. The binding agent of any one of the preceding claims, wherein the binding agent is multispecific.
124. The binding agent of claim 123, wherein the binding agent is bispecific, trispecific, or tetraspecific.
125. The binding agent of any one of the preceding claims, wherein the binding agent is multivalent.
126. The binding agent of any one of claims 48 to 124, wherein the polypeptide chains have the same valency and specificity.
127. The binding agent of any one of claims 48 to 125, wherein the polypeptide chains have different valencies and specificities.
128. The binding agent of any one of claims 48 to 127, wherein each of the polypeptide chains is an antibody heavy chain.
129. The binding agent of any one of claims 48 to 128, wherein the binding agent is a bispecific antibody.
130. The binding agent of claim 129, wherein the bispecific antibody further comprises a first antibody light chain and a second antibody light chain.
131. The binding agent of any one of the preceding claims, wherein one or more antigen binding domains are humanized.
132. The binding agent of any one of claims 62 to 131, wherein X or Y is independently selected from a linker, cytokine, chemokine, tag, masking domain, phage capsid protein (pIII, pVI, pV, pVII or pIX), antigen binding domain, or a combination thereof.
133. The binding agent of any one of the preceding claims, wherein the binding agent is an antibody or antigen binding fragment thereof.
134. The binding agent of any one of the preceding claims, wherein the binding agent is an antibody-like molecule.
135. The binding agent of any one of the preceding claims, wherein the binding agent comprises a protein scaffold.
136. The binding agent of any one of the preceding claims, wherein the binding agent comprises an immune cell modulating agent.
137. A binding agent comprising one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID No. 77.
138. A binding agent comprising one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID No. 78.
139. A binding agent comprising one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID No. 79.
140. A binding agent comprising one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID No. 80.
141. A binding agent comprising one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID No. 81.
142. A binding agent comprising one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID No. 82.
143. A binding agent comprising one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID No. 83.
144. A binding agent comprising one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID No. 85.
145. A binding agent comprising one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID No. 86.
146. A binding agent comprising one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID No. 87.
147. A binding agent comprising one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID No. 88.
148. The binding agent of any one of claims 137-147, wherein the polypeptide chain comprises an amino acid substitution, addition, or deletion outside the complementarity determining region.
149. The binding agent of any one of the preceding claims, wherein the binding agent comprises at least two polypeptide chains capable of assembling to form a dimer, and wherein each polypeptide chain is different.
150. The binding agent of claim 149, wherein the two polypeptide chains are capable of assembling to form a homodimer.
151. The binding agent of claim 149, wherein the two polypeptide chains are capable of assembling to form a heterodimer.
152. A binding agent selected from the group consisting of:
a. A binding agent comprising two polypeptide chains each having the amino acid sequence as set forth in SEQ ID No. 77;
b. a binding agent comprising two polypeptide chains each having an amino acid sequence as set forth in SEQ ID No. 78;
c. a binding agent comprising two polypeptide chains each having the amino acid sequence set forth in SEQ ID No. 79;
d. a binding agent comprising two polypeptide chains each having the amino acid sequence set forth in SEQ ID No. 80;
e. a binding agent comprising two polypeptide chains each having the amino acid sequence set forth in SEQ ID No. 81;
f. a binding agent comprising two polypeptide chains each having the amino acid sequence set forth in SEQ ID No. 82;
g. a binding agent comprising two polypeptide chains each having the amino acid sequence shown in SEQ ID No. 83;
h. a binding agent comprising two polypeptide chains each having the amino acid sequence set forth in SEQ ID No. 85;
i. a binding agent comprising two polypeptide chains each having the amino acid sequence set forth in SEQ ID No. 86;
j. a binding agent comprising two polypeptide chains each having the amino acid sequence set forth in SEQ ID No. 87; or (b)
k. A binding agent comprising two polypeptide chains each having the amino acid sequence set forth in SEQ ID No. 88.
153. The binding agent of any one of the preceding claims, wherein the binding agent is conjugated to a therapeutic moiety.
154. The binding agent of any one of the preceding claims, wherein the binding agent is conjugated to a detectable moiety.
155. The binding agent of any one of the preceding claims, wherein the binding agent is conjugated to a protein that allows for an extended half-life.
156. The binding agent of any one of the preceding claims, wherein the binding agent is linked to a nanoparticle.
157. A composition comprising a binding agent according to any one of the preceding claims.
158. The composition of claim 157, wherein the composition comprises monomers, dimers, and mixtures thereof.
159. The composition of claim 158, wherein more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the composition comprises dimers.
160. The composition of claim 158, wherein more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the composition comprises homodimers.
161. The composition of claim 158, wherein more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the composition comprises heterodimers.
162. A pharmaceutical composition comprising the binding agent of any one of the preceding claims and a pharmaceutically acceptable carrier.
163. A pharmaceutical composition according to claim 162 comprising a binding agent having a purity level of between 80.0% and 99.9%.
164. A nucleic acid or set of nucleic acids encoding the binding agent of any one of the preceding claims.
165. A vector comprising the nucleic acid of claim 164.
166. A cell expressing the binding agent of any one of the preceding claims.
167. A cell comprising the nucleic acid of claim 164 or the vector of claim 165.
168. A kit comprising the binding agent of any one of claims 1 to 156.
169. A kit comprising the nucleic acid of claim 164, or the vector of claim 165, or the cell of claim 166 or 167.
170. A method of treating a disorder or disease comprising administering the binding agent of any one of claims 1-156, the composition of any one of claims 157-161, or the pharmaceutical composition of claim 162 or 163.
171. The method of claim 170, wherein the disorder or disease is cancer.
172. The method of claim 170 or 171, wherein the disorder or disease is a solid tumor.
173. The method according to any one of claims 170-172, wherein the disorder or disease is advanced metastatic solid cancer.
174. The method according to any one of claims 170-172, wherein the disorder or disease is hematological cancer.
175. The method of any one of claims 170-173, wherein the disorder or disease is lung cancer.
176. The method of claim 175, wherein the lung cancer is small cell lung cancer.
177. The method of claim 175, wherein the lung cancer is non-small cell lung cancer.
178. The method of any one of claims 175-177, wherein the lung cancer is metastatic.
179. The method of any one of claims 170-173, wherein the disorder or disease is myeloma.
180. A method of making the binding agent of any one of claims 1-152, the method comprising transforming a cell with one or more vectors, wherein the one or more vectors comprise the nucleic acid or nucleic acid set of claim 163.
181. The method of claim 180, wherein the binding agent is purified from the cells.
182. The method of claim 181, wherein the purity level of the binding agent is between 80.0% and 99.9%.
183. The method of any one of claims 180 to 182, further comprising conjugating or attaching the binding agent to a therapeutic moiety, a detectable moiety, a protein that allows for extended half-life, or to a nanoparticle.
CN202280025997.9A 2021-03-31 2022-03-25 Binding agents targeting tumor and/or immune cells Pending CN117157322A (en)

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US63/168,606 2021-03-31
US63/249,784 2021-09-29
US202263301305P 2022-01-20 2022-01-20
US63/301,305 2022-01-20
PCT/CA2022/050442 WO2022204793A1 (en) 2021-03-31 2022-03-25 Binding agents targeting tumors and/or immune cells

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