Anti-TRAILR2 antigen binding proteins and uses thereof

Abstract

The invention provides a TNF-related apoptosis-inducing ligand receptor 2 (TNF-related apoptosis-inducing ligand receptor 2) and a preparation method of the TNF-related apoptosis-inducing ligand receptor 2 (TNF-related apoptosis-inducing ligand receptor 2 (TNF-related apoptosis-inducing ligand receptor 2). The present invention relates to antigen-binding proteins (e.g., antibodies, such as single domain antibodies) that can bind to an antigen (e.g., TRA5LR2). The present application also provides fusion proteins and conjugates comprising the antigen binding proteins, polynucleotides and recombinant vectors encoding the antigen binding proteins, and host cells and methods for making the antigen binding proteins. The present application further provides pharmaceutical compositions comprising the antigen binding proteins and methods of using the antigen binding proteins in the treatment of diseases or conditions.

Description

[0001] Cross-reference of related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 524,095, filed June 29, 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0003] sequence list

[0004] This application contains a sequence list, which has been submitted electronically in XML format and incorporated herein by reference in its entirety. This XML copy was created on June 12, 2024, named 260525_000049_SL.xml, and has a size of 2,376,169 bytes. Technical Field

[0005] This application relates to antigen-binding proteins (e.g., antibodies, such as single-domain antibodies) that specifically bind to TNF-associated apoptosis-inducing ligand receptor 2 (TRAILR2), their preparation methods, and their uses. Background Technology

[0006] Apoptosis controls cellular homeostasis in normal tissue compartments and is tightly regulated at multiple levels. Cancer cells often evade apoptotic cell death by downregulating pro-apoptotic proteins and / or overexpressing anti-apoptotic proteins, leading to inherent resistance to anticancer therapies. Apoptosis can be induced by TNF-associated apoptosis-inducing ligand receptor 2 (TRAILR2, also known as death receptor 5 or DR5) agonists or recombinant TNF-associated apoptosis-inducing ligand (TRAIL). High TRAILR2 expression has been observed in a variety of indications. Preclinical data have consistently demonstrated that TRAILR2 agonists effectively activate apoptotic pathways in vitro and have significant antitumor efficacy in vivo using xenograft models. However, IgG-based TRAILR2 agonists have been clinically disappointing, with very limited response rates, and poor conversion from preclinical to clinical performance has been hypothesized to be due to suboptimal clustering of receptor agonists and subsequent weak activation of apoptotic pathways. Therefore, there is a need in the art to develop therapeutic molecules that can effectively mimic TRAIL-induced activation, for example, through TRAILR2 polymerization for downstream potent apoptotic signaling. Summary of the Invention

[0007] As mentioned in the background section above, there is an unmet need in the art to develop therapeutic molecules designed for optimal polymerization of TRAILR2 and subsequent induction of potent apoptosis signaling. This application provides compositions and methods for addressing this need and other related needs.

[0008] In one aspect, this article provides an antigen-binding protein that specifically binds to TNF-associated apoptosis-inducing ligand receptor 2 (TRAILR2), comprising a complementarity-determining region 3 (CDR3) containing an amino acid sequence selected from the following: a). (A / V)ASRL(P / V)FNSRSA(I / V)YTDRIYDS (SEQ ID NO: 100); b). AVRRSAWY(S / T)DSIYTVSQYDY (SEQ ID NO: 101); c). NAARSYSR(D / G / N)(G / Y)(E / R)PL(E / K)P(A / D)Y (SEQ ID NO: 102); d). AAASSWSRGG(A / G / I / V)PYGMDY (SEQ ID NO: 103); e). AADS(H / R)FRR(P / Y)(A / T / V)PG(I / Q)QYEY (SEQ ID NO: 104); f). NAA(K / R)SYHRDY(K / S)PL(K / S)(G / P)DY (SEQ ID NO: 105); g). AAAPSFGM(M / R / T)(I / N)PESYVHS (SEQ ID NO: 106); h). AANRGIMSMRLSRYDD (SEQ ID NO: 49); i). T(A / V)GP(A / T)MSYSRGGEF (SEQ ID NO: 107); j). (A / V)ADRGAISRSGAGM(D / N)Y (SEQ ID NO: 108); k). TAGP(A / S)IS(L / Y)SRGGEY (SEQ ID NO: 109); l). A(A / T)NGWGLDP(S / T)TYH(Y / D) (SEQ ID NO: 110); m). SAGWTRRIFQY (SEQ ID NO: 88); n). TAGQSISLSQGGE(H / Y) (SEQ ID NO: 111); o). KAGIRGE(T / V)Y (SEQ ID NO: 112); p). RAYNDGGEY (SEQ ID NO: 2191); q). HS(N / R)WYNL (SEQ ID NO: 2208); and r). (F / M / N)T(A / S)DY, wherein one or more non-alanine residues in the CDR3 sequence are optionally substituted with alanine, and / or one or more alanine residues in the CDR3 sequence are optionally substituted with glycine.

[0009] In some embodiments, CDR3 comprises an amino acid sequence selected from the following: s). (A / G)(A / G)(A / G)(A / S)(A / S)(A / W)(A / S)(A / R)(A / G)(A / G)(A / G / I / V)(A / P)(A / Y)(A / G)(A / M)(A / D)(A / Y); t). (A / T)(A / G / V)(A / G)(A / P)(A / T)(A / M)(A / S)(A / Y)(A / S)(A / R)(A / G)(A / G)(A / E)(A / F) (SEQ ID NO: 2350); u). (A / R)(A / G)(A / Y)(A / N)(A / D)(A / G)(A / G)(A / E)(A / Y); v). (A / H)(A / S)(A / N / R)(A / W)(A / Y)(A / N)(A / L); and w). (A / F / M / N)(A / T)(A / G / S)(A / D)Y.

[0010] In some embodiments, CDR3 comprises an amino acid sequence selected from the following: VASRLPFNSRSAIYTDRIYDS (SEQ ID NO: 3); AVRRSAWYSDSIYTVSQYDY (SEQ ID NO: 8); NAARSYSRDYEPLKPDY (SEQ ID NO: 13); NAARSYSRNYEPLKPDY (SEQ ID NO: 16); NAARSYSRGGEPLKPDY (SEQ ID NO: 20); NAARSYSRGGRPLEPAY (SEQ ID NO: 25); AAASSWSRGGVPYGMDY (SEQ ID NO: 30); AADSHFRRYTPGQQYEY (SEQ ID NO: 35); NAAKSYHRDYSPLSPDY (SEQ ID NO: 40); AAAPSFGMRNPESYVHS (SEQ ID NO: 44); AANRGIMSMRLSRYDD (SEQ ID NO: 49); TAGPTMSYSRGGEF (SEQ ID NO: 30); TAGPTMSYSRGGEF (SEQ ID NO: 49); TAGPTMSYSRGGEF (SEQ ID NO: 30); NAARSYSRDYSPIY ... 54); VADRGAISRSGAGMDY (SEQ ID NO: 64); AADRGAISRSGAGMDY (SEQ ID NO: 69); TAGPAISLSRGGEY (SEQ ID NO: 74); TAGPSISYSRGGEY (SEQ ID NO: 78); 88); TAGQSISLSQGGEY (SEQ ID NO: 92); KAGIRGEVY (SEQ ID NO: 97); RAYNDGGEY (SEQ ID NO: 2191); HSRWYNL (SEQ ID NO: 2197); FTADY (SEQ ID NO: 2203); GAASSWSRGGVPYGMDY (SEQ ID NO: 2298); AGASSWSRGGVPYGMDY (SEQ ID NO: 2299); AAGSSWSRGGVPYGMDY (SEQ ID NO: 2300); AAAASWSRGGVPYGMDY (SEQ ID NO: 2301); AAASAWSRGGVPYGMDY (SEQ ID NO: 2302); AAASSASRGGVPYGMDY (SEQ ID NO: 2303);AAASSWARGGVPYGMDY (SEQ ID NO: 2304);AAASSWSAGGVPYGMDY (SEQ ID NO: 2305);AAASSWSRAGVPYGMDY (SEQ ID NO: 2306);AAASSWSRGAVPYGMDY (SEQ ID NO: 2307) (SEQ ID NO: 2308);AAASSWSRGGVAYGMDY (SEQ ID NO: 2309);AAASSWSRGGVPAGMDY (SEQ ID NO: 2310);AAASSWSRGGVPYAMDY (SEQ ID NO: 2311); 2312);AAASSWSRGGVPYGMAY (SEQ ID NO: 2313);AAASSWSRGGVPYGMDA (SEQ ID NO: 2314);AAGPTMSYSRGGEF (SEQ ID NO: 2315);TGGPTMSYSRGGEF (SEQ ID NO: 2315); 2316);TAAPTMSYSRGGEF (SEQ ID NO: 2317);TAGATMSYSRGGEF (SEQ ID NO: 2318);TAGPAMSYSRGGEF (SEQ ID NO: 2319);TAGPTASYSRGGEF (SEQ ID NO: 2320); NO: 2321);TAGPTMSASRGGEF (SEQ ID NO: 2322);TAGPTMSYARGGEF (SEQ ID NO: 2323);TAGPTMSYSAGGEF (SEQ ID NO: 2324);TAGPTMSYSAGGEF (SEQ ID NO: 2325); NO: 2326);TAGPTMSYSRGGAF (SEQ ID NO: 2327);TAGPTMSYSRGGEA (SEQ ID NO: 2328);AAYNDGGEY(SEQ ID NO: 2329);RGYNDGGEY (SEQ ID NO: 2330);SEQ ID NO: SEQ ID NO: 2331);RAYADGGEY (SEQ ID NO: 2332);RAYNAGGEY (SEQ ID NO: 2333);RAYNDAGEY (SEQ ID NO:2334); RAYNDGAEY (SEQ ID NO: 2335); RAYNDGGAY (SEQ ID NO: 2336); RAYNDGGEA (SEQ ID NO: 2337); ASRWYNL (SEQ ID NO: 2338); HARWYNL (SEQ ID NO: 2339); HSAWYNL (SEQ ID NO: 2340); HSRAYNL (SEQ ID NO: 2341); HSRWANL (SEQ ID NO: 2342); HSRWYAL (SEQ ID NO: 2343); HSRWYNA (SEQ ID NO: 2344); ATADY (SEQ ID NO: 2345); FAADY (SEQ ID NO: 2346); FTGDY (SEQ ID NO: 2347); and FTAAY (SEQ ID NO: 2348).;

[0011] In some embodiments, the antigen-binding protein further comprises CDR1, which contains an amino acid sequence selected from the following: a) GRTFSSNL (SEQ ID NO: 1); b) GGT(F / L)(A / S)N(D / N)G (SEQ ID NO: 113); c) GRTL(D / N / S)(A / D / E)Y(A / G) (SEQ ID NO: 114); d) GRTFS(N / S)YA (SEQ ID NO: 115); e) GRDFSNYV (SEQ ID NO: 33); f) G(L / R)(I / S)FS(D / S)YA (SEQ ID NO: 116); g) GR(A / T)FSTLA (SEQ ID NO: 117); h) GRTFSSDI (SEQ ID NO: 47); i) GRSFGD(D / F / Y)A (SEQ ID NO: 118); j) G(G / R)TLSNYA (SEQ ID NO: 119); k).GRSFGAQGMEG (SEQ ID NO: 72); l).GFFTLDLGAYA (SEQ ID NO: 81); (SEQ ID NO: 2189); p). G(G / S)NFRILS (SEQ ID NO: 2209); and q). G(F / L)(A / T)F(R / S)(R / S)YA (SEQ ID NO: 2212).

[0012] In some embodiments, CDR1 comprises an amino acid sequence selected from the following: GRTFSSNL (SEQ ID NO: 1); GGTLANNG (SEQ ID NO: 6); GRTLDAYG (SEQ ID NO: 11); GRTLSDYA (SEQ ID NO: 23); GRTFSSYA (SEQ ID NO: 28); GRDFSNYV (SEQ ID NO: 33); GRSFSSYA (SEQ ID NO: 38); GRTFSTLA (SEQ ID NO: 43); GRTFSSDI (SEQ ID NO: 47); GRSFGDFA (SEQ ID NO: 52); GGTLSNYA (SEQ ID NO: 62); GRTLSNYA (SEQ ID NO: 67); GRSFGAQGMEG (SEQ ID NO: 72); GFTLDLGAYA (SEQ ID NO: 81); GFTFGALA (SEQ ID NO: 86); GFTLSGYA (SEQ ID NO: 87). NO:95); GSIFGGYN (SEQ ID NO: 2189); GSNFRILS (SEQ ID NO: 2195); and GFTFSRYA (SEQ ID NO: 2201).

[0013] In some embodiments, the antigen-binding protein further comprises CDR2, which contains an amino acid sequence selected from the following: a) VSWNGAST (SEQ ID NO: 2); b) DHR(S / T)GT (SEQ ID NO: 121); c) I(N / S)W(N / S / T)G(T / V)(D / G)T (SEQ ID NO: 122); d) LNW(N / S)G(D / E)ST (SEQ ID NO: 123); e) INWAD(E / T)T (SEQ ID NO: 124); f) INWSGG(S / T)T (SEQ ID NO: 125); g) ISWSDMSA (SEQ ID NO: 48); h) I(N / R)W(A / D / T)G(D / N)T (SEQ ID NO: 126); i) ISQ(S / T)S(D / S)T (SEQ ID NO: 126); 127); j). (I / M)KWTGNT (SEQ ID NO: 128); k). ISN(S / T)GTTT (SEQ ID NO: 129); l). ISNDGEHI (SEQ ID NO: 87); m). ISWNGDIT (SEQ ID NO: 91); n). IT(G / S)(A / S)G(G / S)(N / S)T (SEQ ID NO: 130); o). IFISGN(D / N) (SEQ ID NO: 2207); p). (I / L)T(K / M / S)D(D / G)TT (SEQ ID NO: 2210); and q). ISS(A / G / S)(G / S)G(I / Y)(I / T / V) (SEQ ID NO: 2213).

[0014] In some embodiments, CDR2 comprises an amino acid sequence selected from the following: VSWNGAST (SEQ ID NO: 2); DHRSGT (SEQ ID NO: 7); ISWTGVDT (SEQ ID NO: 12); ISWTGTDT (SEQ ID NO: 19); IWSGVDT (SEQ ID NO: 24); LNWSGEST (SEQ ID NO: 29); INWADET (SEQ ID NO: 34); INWSGGST (SEQ ID NO: 39); ISWSDMSA (SEQ ID NO: 48); IRWTGDT (SEQ ID NO: 53); ISQTSST (SEQ ID NO: 63); ISQSSDT (SEQ ID NO: 68); MKWTGNT (SEQ ID NO: 73); IKWTGNT (SEQ ID NO: 77); ISNTGTTT (SEQ ID NO: 82); ISNDGEHI (SEQ ID NO: 87); ISWNGDIT (SEQ ID NO: 2). NO: 91); ITSAGGST (SEQ ID NO: 96); IFISGNN (SEQ ID NO: 2190); ITSDDTT (SEQ ID NO: 2196); and ISSAGGYI (SEQ ID NO: 2202).

[0015] In some embodiments, the antigen-binding protein provided herein comprises CDR1, wherein CDR1 comprises an amino acid sequence selected from the following: SEQ ID NO: 1, 6, 11, 23, 28, 33, 38, 43, 47, 52, 62, 67, 72, 81, 86, 95, 762 to 1084, 2189, 2195, 2201 and 2252 to 2261; CDR2, wherein CDR2 comprises an amino acid sequence selected from the following: SEQ ID NO: 2, 7, 12, 19, 24, 29, 34, 39, 48, 53, 63, 68, 73, 77, 82, 87, 91, 96, 1085 to 1407, 2190, 2196, 2202 and 2262 to 2271; and / or CDR3, wherein CDR3 comprises an amino acid sequence selected from the following: SEQ ID NO: 3, 8, 13, 16, 20, 25, 30, 35, 40, 44, 49, 54, 64, 69, 74, 78, 83, 88, 92, 97, 1408 to 1730, 2191, 2197, 2203, 2272 to 2277, and 2298 to 2348.

[0016] In some embodiments, the antigen-binding protein comprises: i) a CDR1 containing the amino acid sequence GRTFSSNL (SEQ ID NO: 1), a CDR2 containing the amino acid sequence VSWNGAST (SEQ ID NO: 2), and a CDR3 containing VASRLPFNSRSAIYTDRIYDS (SEQ ID NO: 3); ii) a CDR1 containing the amino acid sequence GGTLANNG (SEQ ID NO: 6), a CDR2 containing the amino acid sequence DHRSGT (SEQ ID NO: 7), and a CDR3 containing the amino acid sequence AVRRSAWYSDSIYTVSQYDY (SEQ ID NO: 8); iii) a CDR1 containing the amino acid sequence GRTLDAYG (SEQ ID NO: 11), a CDR2 containing the amino acid sequence ISWTGVDT (SEQ ID NO: 12), and a CDR3 containing the amino acid sequence NAARSYSRDYEPLKPDY (SEQ ID NO: 13); iv) a CDR3 containing GRTLDAYG (SEQ ID NO: 11). 11) CDR1 containing the amino acid sequence of ISWTGVDT (SEQ ID NO: 12), CDR2 containing the amino acid sequence of NAARSYSRNYEPLKPDY (SEQ ID NO: 16), and CDR3 containing the amino acid sequence of NAARSYSRNYEPLKPDY (SEQ ID NO: 16); v) CDR1 containing the amino acid sequence of GRTLDAYG (SEQ ID NO: 11), CDR2 containing the amino acid sequence of ISWTGTDT (SEQ ID NO: 19), and CDR3 containing the amino acid sequence of NAARSYSRGGEPLKPDY (SEQ ID NO: 20); vi) CDR1 containing the amino acid sequence of GRTLSDYA (SEQ ID NO: 23), CDR2 containing the amino acid sequence of IWSSGVDT (SEQ ID NO: 24), and CDR3 containing the amino acid sequence of NAARSYSRGGRPLEPAY (SEQ ID NO: 25); vii) CDR1 containing the amino acid sequence of GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence of LNWSGEST... CDR2 containing the amino acid sequence of (SEQ ID NO: 29), and CDR3 containing the amino acid sequence of AAASSWSRGGVPYGMDY (SEQ ID NO: 30); viii).CDR1 containing the amino acid sequence GRDFSNYV (SEQ ID NO: 33), CDR2 containing the amino acid sequence INWADET (SEQ ID NO: 34), and CDR3 containing the amino acid sequence AADSHFRRYTPGQQYEY (SEQ ID NO: 35); ix). CDR1 containing the amino acid sequence GRSFSSYA (SEQ ID NO: 38), CDR2 containing the amino acid sequence INNWSGGST (SEQ ID NO: 39), and CDR3 containing the amino acid sequence NAAKSYHRDYSPLSPDY (SEQ ID NO: 40); x). CDR1 containing the amino acid sequence GRTFSTLA (SEQ ID NO: 43), CDR2 containing the amino acid sequence INNWSGGST (SEQ ID NO: 39), and CDR3 containing the amino acid sequence AAAPSFGMRNPESYVHS (SEQ ID NO: 44); xi). CDR1 containing the amino acid sequence GRTFSSDI (SEQ ID NO: 33). CDR1 containing the amino acid sequence of 47), CDR2 containing the amino acid sequence of ISSDMSA (SEQ ID NO: 48), and CDR3 containing the amino acid sequence of AANRGIMSMRLSRYDD (SEQ ID NO: 49); xii). CDR1 containing the amino acid sequence of GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence of IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence of TAGPTMSYSRGGEF (SEQ ID NO: 54); xiii). CDR1 containing the amino acid sequence of GGTLSNYA (SEQ ID NO: 62), CDR2 containing the amino acid sequence of ISQTSST (SEQ ID NO: 63), and CDR3 containing the amino acid sequence of VADRGAISRSGAGMDY (SEQ ID NO: 64); xiv). CDR1 containing the amino acid sequence of GRTLSNYA (SEQ ID NO: 67), CDR2 containing the amino acid sequence of ISQSSDT (SEQ ID NO: 48), and CDR3 containing the amino acid sequence of AANRGIMSMRLSRYDD (SEQ ID NO: 49). CDR2 containing the amino acid sequence of 68), and CDR3 containing the amino acid sequence of AADRGAISSGAGMDY (SEQ ID NO: 69); xv).CDR1 containing the amino acid sequence GRSFGAQGMEG (SEQ ID NO: 72), CDR2 containing the amino acid sequence MKWTGNT (SEQ ID NO: 73), and CDR3 containing the amino acid sequence TAGPAISLSRGGEY (SEQ ID NO: 74); xvi). CDR1 containing the amino acid sequence GRSFGAQGMEG (SEQ ID NO: 72), CDR2 containing the amino acid sequence IKWTGNT (SEQ ID NO: 77), and CDR3 containing the amino acid sequence TAGPSISYSRGGEY (SEQ ID NO: 78); xvii). CDR1 containing the amino acid sequence GFTLDLGAYA (SEQ ID NO: 81), CDR2 containing the amino acid sequence ISNTGTTT (SEQ ID NO: 82), and CDR3 containing the amino acid sequence AANGWGLDPTTYHY (SEQ ID NO: 83); xviii). CDR1 containing the amino acid sequence GFTFGALA (SEQ ID NO: 72). CDR1 containing the amino acid sequence of SEQ ID NO: 86), CDR2 containing the amino acid sequence of ISNDGEHI (SEQ ID NO: 87), and CDR3 containing the amino acid sequence of SAGWTRRIFQY (SEQ ID NO: 88); xix). CDR1 containing the amino acid sequence of GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence of ISWNGDIT (SEQ ID NO: 91), and CDR3 containing the amino acid sequence of TAGQSISLSQGGEY (SEQ ID NO: 92); xx). CDR1 containing the amino acid sequence of GFTLSGYA (SEQ ID NO: 95), CDR2 containing the amino acid sequence of ITSAGGST (SEQ ID NO: 96), and CDR3 containing the amino acid sequence of KAGIRGEVY (SEQ ID NO: 97); xxi). CDR1 containing the amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence of IFISGNN (SEQ ID NO: 2189), and CDR3 containing the amino acid sequence of GSIFGGYN (SEQ ID NO: 2189). CDR2 containing the amino acid sequence of NO: 2190, and CDR3 containing the amino acid sequence of RAYNDGGEY (SEQ ID NO: 2191); xxii). CDR1 containing the amino acid sequence of GSNFRILS (SEQ ID NO: 2195), CDR2 containing the amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and CDR3 containing the amino acid sequence of HSRWYNL (SEQ ID NO: 2197); or xxiii).CDR1 containing the amino acid sequence GFTFSRYA (SEQ ID NO: 2201), CDR2 containing the amino acid sequence ISSAGGYI (SEQ ID NO: 2202), and CDR3 containing the amino acid sequence FTADY (SEQ ID NO: 2203); xxiv). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence GAASSWSRGGVPYGMDY (SEQ ID NO: 2298); xxv). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AGASSWSRGGVPYGMDY (SEQ ID NO: 2299); xxvi). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AGASSWSRGGVPYGMDY (SEQ ID NO: 2299); CDR1 containing the amino acid sequence of (SEQ ID NO: 28), CDR2 containing the amino acid sequence of LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence of AAGSSWSRGGVPYGMDY (SEQ ID NO: 2300); xxvii). CDR1 containing the amino acid sequence of GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence of LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence of AAAASWSRGGVPYGMDY (SEQ ID NO: 2301); xxviii). CDR1 containing the amino acid sequence of GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence of LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence of AAASAWSRGGVPYGMDY (SEQ ID NO: 2302); xxix). CDR1 containing the amino acid sequence of GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence of LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence of AAASAWSRGGVPYGMDY (SEQ ID NO: 2302); CDR1 containing the amino acid sequence of NO: 28, CDR2 containing the amino acid sequence of LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence of AAASSASRGGVPYGMDY (SEQ ID NO: 2303); xxx).CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWARGGVPYGMDY (SEQ ID NO: 2304); xxxi). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSAGGVPYGMDY (SEQ ID NO: 2305); xxxii). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSRAGVPYGMDY (SEQ ID NO: 2304). CDR3 containing the amino acid sequence of GRTFSSYA (SEQ ID NO: 2306); xxxiii) CDR1 containing the amino acid sequence of GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence of LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence of AAASSWSRGAVPYGMDY (SEQ ID NO: 2307); xxxiv) CDR1 containing the amino acid sequence of GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence of LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence of AAASSWSRGGAPYGMDY (SEQ ID NO: 2308); xxxv) CDR1 containing the amino acid sequence of GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence of LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence of AAASSWSRGGVAYGMDY (SEQ ID NO: 2307). CDR3 containing the amino acid sequence of 2309; xxxvi). CDR1 containing the amino acid sequence of GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence of LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence of AAASSWSRGGVPAGMDY (SEQ ID NO: 2310); xxxvii).CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSRGGVPYAMDY (SEQ ID NO: 2311); xxxviii). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSRGGVPYGADY (SEQ ID NO: 2312); xxxix). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSRGGVPYGMAY (SEQ ID NO: 2313); xl). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSRGGVPYGMAY (SEQ ID NO: 2313); xl). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSRGGVPYGMAY (SEQ ID NO: 2313); xl). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSRGGVPYGMAY (SEQ ID NO: 2313). CDR1 containing the amino acid sequence of (SEQ ID NO: 28), CDR2 containing the amino acid sequence of LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence of AAASSWSRGGVPYGMDA (SEQ ID NO: 2314); xli). CDR1 containing the amino acid sequence of GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence of IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence of AAGPTMSYSRGGEF (SEQ ID NO: 2315); xlii). CDR1 containing the amino acid sequence of GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence of IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence of TGGPTMSYSRGGEF (SEQ ID NO: 2316); xliii). CDR1 containing the amino acid sequence of GRSFGDFA (SEQ ID NO: 29), CDR2 containing the amino acid sequence of IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence of TGGPTMSYSRGGEF (SEQ ID NO: 2316); xliii). CDR3 containing the amino acid sequence of GRSFGDFA (SEQ ID NO: 29), CDR3 containing the amino acid sequence of TGGPTMSYSRGGEF (SEQ ID NO: 2316). CDR1 containing the amino acid sequence of 52), CDR2 containing the amino acid sequence of IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence of TAAPTMSYSRGGEF (SEQ ID NO: 2317); xliv).CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGATMSYSRGGEF (SEQ ID NO: 2318); xlv). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPAMSYSRGGEF (SEQ ID NO: 2319); xlvi). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPAMSYSRGGEF (SEQ ID NO: 2320); xlvii). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPAMSYSRGGEF (SEQ ID NO: 2320); CDR1 containing the amino acid sequence of 52), CDR2 containing the amino acid sequence of IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence of TAGPTMAYSRGGEF (SEQ ID NO: 2321); xlviii). CDR1 containing the amino acid sequence of GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence of IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence of TAGPTMAYSRGGEF (SEQ ID NO: 2322); xlix). CDR1 containing the amino acid sequence of GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence of IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence of TAGPTMAYSRGGEF (SEQ ID NO: 2323); l). CDR1 containing the amino acid sequence of GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence of IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence of TAGPTMAYSRGGEF (SEQ ID NO: 2323); l). CDR1 containing the amino acid sequence of GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence of IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence of TAGPTMAYSRGGEF (SEQ ID NO: 2323); CDR1 containing the amino acid sequence of 52), CDR2 containing the amino acid sequence of IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence of TAGPTMSYSAGGEF (SEQ ID NO: 2324); li).CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPTMSYSRAGEF (SEQ ID NO: 2325); lii). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPTMSYSRGAEF (SEQ ID NO: 2326); liii). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPTMSYSRGAEF (SEQ ID NO: 2327); liv). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPTMSYSRGAF (SEQ ID NO: 2327); CDR1 containing the amino acid sequence of 52), CDR2 containing the amino acid sequence of IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence of TAGPTMSYSRGGEA (SEQ ID NO: 2328); lv). CDR1 containing the amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence of IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence of AAYNDGGEY (SEQ ID NO: 2329); lvi). CDR1 containing the amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence of IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence of RGYNDGGEY (SEQ ID NO: 2330); lvii). CDR1 containing the amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence of IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence of RGYNDGGEY (SEQ ID NO: 2330); CDR1 containing the amino acid sequence of 2189, CDR2 containing the amino acid sequence of IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence of RANDGGEY (SEQ ID NO: 2331); lviii).CDR1 containing the amino acid sequence GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence RAYADGGEY (SEQ ID NO: 2332); CDR1 containing the amino acid sequence GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence RAYADGGEY (SEQ ID NO: 2333); CDR1 containing the amino acid sequence GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence RAYADGGEY (SEQ ID NO: 2333); CDR3 containing the amino acid sequence of 2334); lxi). CDR1 containing the amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence of IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence of RAYNDGAEY (SEQ ID NO: 2335); lxii). CDR1 containing the amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence of IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence of RAYNDGGAY (SEQ ID NO: 2336); lxiii). CDR1 containing the amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence of IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence of RAYNDGGEA (SEQ ID NO: 2334). CDR3 containing the amino acid sequence of SEQ ID NO: 2337; lxiv). CDR1 containing the amino acid sequence of GSNFRILS (SEQ ID NO: 2195), CDR2 containing the amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and CDR3 containing the amino acid sequence of ASRWYNL (SEQ ID NO: 2338); lxv).CDR1 containing the amino acid sequence of GSNFRILS (SEQ ID NO: 2195), CDR2 containing the amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and CDR3 containing the amino acid sequence of HARWYNL (SEQ ID NO: 2339); lxvi). CDR1 containing the amino acid sequence of GSNFRILS (SEQ ID NO: 2195), CDR2 containing the amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and CDR3 containing the amino acid sequence of HARWYNL (SEQ ID NO: 2340); lxvii). CDR1 containing the amino acid sequence of GSNFRILS (SEQ ID NO: 2195), CDR2 containing the amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and CDR3 containing the amino acid sequence of HARWYNL (SEQ ID NO: 2341); lxviii). CDR1 containing the amino acid sequence of GSNFRILS (SEQ ID NO: 2195), CDR2 containing the amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and CDR3 containing the amino acid sequence of HARWYNL (SEQ ID NO: 2341); lxviii). CDR1 containing the amino acid sequence of GSNFRILS (SEQ ID NO: 2195), CDR2 containing the amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and CDR3 containing the amino acid sequence of HARWYNL (SEQ ID NO: 2341). CDR1 containing the amino acid sequence of SEQ ID NO: 2195, CDR2 containing the amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and CDR3 containing the amino acid sequence of HSRWANL (SEQ ID NO: 2342); lxix). CDR1 containing the amino acid sequence of GSNFRILS (SEQ ID NO: 2195), CDR2 containing the amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and CDR3 containing the amino acid sequence of HSRWYAL (SEQ ID NO: 2343); lxx). CDR1 containing the amino acid sequence of GSNFRILS (SEQ ID NO: 2195), CDR2 containing the amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and CDR3 containing the amino acid sequence of HSRWYNA (SEQ ID NO: 2344); lxxi). CDR3 containing the amino acid sequence of GFTFSRYA (SEQ ID NO: 2195). CDR1 containing the amino acid sequence of SEQ ID NO: 2201, CDR2 containing the amino acid sequence of ISSAGGYI (SEQ ID NO: 2202), and CDR3 containing the amino acid sequence of ATADY (SEQ ID NO: 2345); lxxii). CDR1 containing the amino acid sequence of GFTFSRYA (SEQ ID NO: 2201), CDR2 containing the amino acid sequence of ISSAGGYI (SEQ ID NO: 2202), and CDR3 containing the amino acid sequence of FAADY (SEQ ID NO: 2346); lxxiii).CDR1 containing the amino acid sequence GFTFSRYA (SEQ ID NO: 2201), CDR2 containing the amino acid sequence ISSAGGYI (SEQ ID NO: 2202), and CDR3 containing the amino acid sequence FTGDY (SEQ ID NO: 2347); or lxxiv). CDR1 containing the amino acid sequence GFTFSRYA (SEQ ID NO: 2201), CDR2 containing the amino acid sequence ISSAGGYI (SEQ ID NO: 2202), and CDR3 containing the amino acid sequence FTAAY (SEQ ID NO: 2348).

[0017] In some embodiments, the antigen-binding protein comprises: i) a CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), a CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and a CDR3 containing the amino acid sequence AAASSWSRGGVPYGMDY (SEQ ID NO: 30); ii) a CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), a CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and a CDR3 containing the amino acid sequence TAGPTMSYSRGGEF (SEQ ID NO: 54); iii) a CDR1 containing the amino acid sequence GSIFGGYN (SEQ ID NO: 2189), a CDR2 containing the amino acid sequence IFISGNN (SEQ ID NO: 2190), and a CDR3 containing the amino acid sequence RAYNDGGEY (SEQ ID NO: 2191); iv) a CDR3 containing GSNFRILS. CDR1 containing the amino acid sequence of (SEQ ID NO: 2195), CDR2 containing the amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and CDR3 containing the amino acid sequence of HSRWYNL (SEQ ID NO: 2197); or v). CDR1 containing the amino acid sequence of GFTFSRYA (SEQ ID NO: 2201), CDR2 containing the amino acid sequence of ISSAGGYI (SEQ ID NO: 2202), and CDR3 containing the amino acid sequence of FTADY (SEQ ID NO: 2203).

[0018] In some implementations, the antigen-binding protein is a single-domain antibody.

[0019] In some implementations, the single-domain antibody is a VHH, VNAR, or VH domain.

[0020] In some implementations, VHH refers to the camel family VHH.

[0021] In some embodiments, VHH comprises an amino acid sequence selected from any one of SEQ ID NO: 4, 9, 14, 17, 21, 26, 31, 36, 41, 45, 50, 55, 65, 70, 75, 79, 84, 89, 93, 98, 146 to 468, 2192, 2198, 2204 and 2214 to 2233, or an amino acid sequence having at least 75% identity with such sequences.

[0022] In some embodiments, VHH comprises an amino acid sequence selected from any one of SEQ ID NO: 4, 9, 14, 17, 21, 26, 31, 36, 41, 45, 50, 55, 65, 70, 75, 79, 84, 89, 93, 98, 2192, 2198, and 2204, or an amino acid sequence having at least 75% identity with such sequences.

[0023] In some embodiments, VHH comprises an amino acid sequence selected from any one of SEQ ID NO: 31, 55, 2192, 2198 and 2204 or an amino acid sequence having at least 75% identity with it.

[0024] In some implementations, VHH is humanized VHH.

[0025] In some embodiments, the humanized VHH comprises an amino acid sequence selected from any one of SEQ ID NO: 5, 10, 15, 18, 22, 27, 32, 37, 42, 46, 51, 56, 66, 71, 76, 80, 85, 90, 94, 99, 469 to 761, 2194, 2200, 2206, 2234 to 2251, and 2354 to 2404, or an amino acid sequence having at least 75% identity with such sequences.

[0026] In some embodiments, the humanized VHH comprises an amino acid sequence selected from any one of SEQ ID NO: 5, 10, 15, 18, 22, 27, 32, 37, 42, 46, 51, 56, 61, 66, 71, 76, 80, 85, 90, 94, 99, 2194, 2200, 2206 and 2354 to 2404 or an amino acid sequence having at least 75% identity with such sequences.

[0027] In some implementations, the humanized VHH comprises an amino acid sequence selected from any of 32, 56, 2200, 2206, 2194, and 2354 to 2404, or an amino acid sequence having at least 75% identity with such sequences.

[0028] In various embodiments, the antigen-binding protein binds to human TRAILR2. In some embodiments, the antigen-binding protein binds at a concentration of less than about 3 × 10⁻⁶. -7 M, for example, about 1×10 -10 Up to 5×10 -8 M of K D It combines with human TRAILR2.

[0029] In various embodiments, the antigen-binding protein binds to the cynomolgus monkey TRAILR2. In some embodiments, the antigen-binding protein binds at a concentration of less than about 3 × 10⁻⁶. -7 M of K D It binds to the cynomolgus monkey TRAILR2. In some embodiments, the antigen-binding protein binds at approximately 1 × 102 -9 Up to 1×10 -7 M of K D It binds to the cynomolgus monkey TRAILR2.

[0030] In various implementation schemes, the antigen-binding protein does not block the binding of TNF-associated apoptosis-inducing ligand (TRAIL) to TRAILR2.

[0031] In various implementation schemes, the antigen-binding protein blocks the binding of TRAIL to TRAILR2.

[0032] In various embodiments, the antigen-binding protein includes one or more modifications that reduce the binding of the antigen-binding protein to pre-existing antibodies present in human blood or serum.

[0033] On the other hand, this document provides a fusion protein that specifically binds to TNF-associated apoptosis-inducing ligand receptor 2 (TRAILR2), comprising one or more of the antigen-binding proteins described herein. In some embodiments, the fusion protein comprises three or more of the antigen-binding proteins. In some embodiments, the fusion protein comprises four of the antigen-binding proteins. In some embodiments, one or more antigen-binding proteins bind to the same epitope on TRAILR2. In some embodiments, one or more antigen-binding proteins bind to different epitopes on TRAILR2. In some embodiments, one or more antigen-binding proteins are one or more single-domain antibodies. In some embodiments, the one or more single-domain antibodies are one or more VHHs.

[0034] In some embodiments, the fusion protein further comprises an immunoglobulin Fc region. The immunoglobulin Fc region may be the Fc region of a human immunoglobulin. The immunoglobulin Fc region may be the Fc region of human IgG1, IgG2, IgG3, or IgG4, or a variant thereof.

[0035] In some implementations, the immunoglobulin Fc region is the Fc region of human IgG1 or a variant thereof. In some implementations, the Fc region of human IgG1 contains one or more mutations selected from the following: Leu234Ala (L234A), Leu234Gly (L234G), Leu234Ser (L234S), Leu234Thr (L234T), Leu234Ala (L234A), Leu235Ala (L235A), Leu235Glu (L235E), Leu235Ser (L235S), Leu235Thr (L235T), Leu235Val (L235V), Leu235Gln (L235Q), Gly236Arg (G236R), Met252Tyr (M252Y), Ser254Thr (S254T), Thr256Glu (T256E), Asp265Asn, according to EU numbers. (D265N), Asp265Ala (D265A), Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala (N297A), Pro329Ala (P329A), Pro239Gly (P329G), Asn325Glu (N325E) and / or Ala327Ser (A327S). In some implementations, the Fc region of human IgG1 contains a set of mutations selected from the following: 1) L234A and L235A; 2) L234A, L235A, and P329A; 3) D265A, N297A, and P329A; 4) L234A, L235A, and G237A; 5) L234G, L235S, and G236R; 6) L234S, L235T, and G236R; 7) L234S, L235V, and G236R; 8) L234T, L235Q, and G236R; 9) L234T, L235T, and G236R; 10) L234A, L235A, and P329G; and 11) M252Y, S254T, and T256E.

[0036] In some embodiments, the immunoglobulin Fc region is the Fc region of human IgG4 or a variant thereof. In some embodiments, the Fc region of human IgG4 contains one or more mutations selected from the following: Ser228Pro (S228P), Leu235Glu (L235E), Leu235Ala (L235A), Phe234Ala (F234A), and / or Pro329Gly (P329G) according to EU numbers. In some embodiments, the Fc region of human IgG4 contains a set of mutations selected from the following: 1) S228P and L235E; 2) S228P and L235A; 3) S228P, F234A, and L235E; 4) S228P, F234A, and L235A; and 5) P329G, S228P, and L235E.

[0037] In some implementations, the fusion protein further includes a portion that binds to serum albumin.

[0038] In some embodiments, the fusion protein comprises the amino acid sequence of any one of SEQ ID NO: 2054 to 2070 or a sequence having at least 75% identity with it.

[0039] In various implementation schemes, the fusion protein acts as an agonist when it binds to TRAILR2.

[0040] On the other hand, this document provides a conjugate comprising the antigen-binding protein or fusion protein described herein, wherein the antigen-binding protein or fusion protein is conjugated to a second portion. In some embodiments, the second portion is selected from detectable markers, drugs, toxins, radionuclides, enzymes, immunomodulators, cytokines, cytotoxic agents, chemotherapeutic agents, diagnostic agents, or combinations thereof.

[0041] In another embodiment, this document provides a polynucleotide molecule encoding an antigen-binding protein or a fusion protein as described herein. In some embodiments, the polynucleotide molecule comprises a nucleotide sequence of any one of SEQ ID NOs: 57 to 61, 131 to 145, 1731 to 2053, 2071 to 2087, 2193, 2199, 2205, and 2278 to 2297, or a nucleotide sequence having at least 70% identity with that sequence. In some embodiments, the polynucleotide molecule comprises a nucleotide sequence of any one of SEQ ID NOs: 57 to 61, 131 to 145, 2071 to 2087, 2193, 2199, and 2205, or a nucleotide sequence having at least 70% identity with that sequence. In some embodiments, the polynucleotide molecule comprises a nucleotide sequence of any one of SEQ ID NOs: 132, 137, 2193, 2199, and 2205, or a nucleotide sequence having at least 70% identity with that sequence.

[0042] On the other hand, this article provides a recombinant vector containing the polynucleotide molecules described herein.

[0043] On the other hand, this article provides a host cell containing either the polynucleotide molecule or the recombinant vector described herein.

[0044] On the other hand, this document provides a kit comprising the antigen-binding protein, fusion protein, conjugate, polynucleotide molecule or recombinant vector described herein, and optionally its instructions and / or packaging.

[0045] On the other hand, this article provides a pharmaceutical composition comprising the antigen-binding protein, fusion protein, conjugate, polynucleotide molecule or recombinant carrier described herein, and pharmaceutically acceptable carrier and / or excipient.

[0046] On the other hand, this article provides a method for preparing an antigen-binding protein or fusion protein that specifically binds to TNF-associated apoptosis-inducing ligand receptor 2 (TRAILR2), the method comprising the steps of: (a) culturing host cells as described herein in a culture medium under conditions suitable for expressing the antigen-binding protein or fusion protein, and (b) isolating the antigen-binding protein or fusion protein from the host cells and / or the culture medium.

[0047] On the other hand, this document provides a method for inducing cell death in cells expressing TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2), the method comprising contacting the cells with an antigen-binding protein, fusion protein, or conjugate described herein. In some embodiments, the contact is performed in vitro. In some embodiments, the contact is performed in vivo. In some embodiments, the method further comprises administering the antigen-binding protein, fusion protein, or conjugate to a subject in need.

[0048] In some implementations, cells expressing TRAILR2 are cancer cells. In some implementations, the cancer is a solid tumor. In some implementations, the cancer is selected from adenoid cystic carcinoma, adrenal tumor, amyloidosis, anal cancer, appendiceal cancer, astrocytoma, ataxia-telangiectasia, Beckwell syndrome, cholangiocarcinoma (cholangiocarcinoma), Birt-Hogg-Dubé syndrome, bladder cancer, bone cancer (osteosarcoma), brainstem glioma, brain tumor, breast cancer, inflammatory breast cancer, metastatic breast cancer, male breast cancer, Carney complex, central nervous system tumors (brain tumors and spinal cord tumors), cervical cancer, childhood cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, desmoidoma, infantile connective tissue proliferative ganglioglioma, childhood tumors, ependymoma, esophageal cancer, Ewing sarcoma, eye cancer, eyelid cancer, familial multiple adenocarcinoma, familial GIST, familial malignant melanoma, familial pancreatic cancer, gallbladder cancer, gastrointestinal stromal tumor (GIST), germ cell tumors, gestational trophoblastic disease, head and neck cancer, hereditary breast and ovarian cancer, hereditary diffuse gastric cancer, hereditary leiomyomatosis and renal cell carcinoma, hereditary mixed polyposis syndrome, hereditary pancreatitis, hereditary papillary renal cell carcinoma, HIV / AIDS-related cancers, juvenile polyposis syndrome, renal cancer, lacrimal adenoma, laryngeal and hypopharyngeal cancer, Rife syndrome, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, Lynch syndrome, mastocytosis, myeloma, melanoma, meningioma, mesothelioma, type 1 multiple endocrine tumor, type 2 multiple endocrine tumor, multiple myeloma, MUTYH (or MYH) related polyposis, myelodysplastic syndrome (MDS), nasal cavity and sinus carcinoma, nasopharyngeal carcinoma, neuroblastoma, gastrointestinal neuroendocrine tumor, pulmonary neuroendocrine tumor, pancreatic neuroendocrine tumor, neuroendocrine tumor, neurofibroma type 1, neurofibroma type 2, nevus-like basal cell tumor syndrome, oral and oropharyngeal carcinoma, osteosarcoma, ovarian, fallopian tube and peritoneal carcinoma, pancreatic cancer, parathyroid carcinoma, penile cancer, Boytz-Yage syndrome, pheochromocytoma and paraganglioma, pituitary adenoma, pleural pulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, Kaposi's sarcoma, soft tissue sarcoma, skin cancer (non-melanoma), small intestine cancer, gastric cancer, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis, uterine cancer, vaginal cancer, Shih-Lin syndrome, vulvar cancer, Werner syndrome, nephroblastoma or xeroderma pigmentosum. In some implementations, the cancer is gastrointestinal cancer, breast cancer, or lung cancer. In other implementations, the gastrointestinal cancer is colorectal cancer, stomach cancer, esophageal cancer, pancreatic cancer, or cholangiocarcinoma.

[0049] In some embodiments, the method further includes contacting the cells with one or more other therapeutic agents.

[0050] On the other hand, this article provides a method for treating or preventing cancer in a subject in need, the method comprising administering to the subject an antigen-binding protein, fusion protein, or conjugate described herein. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is selected from adenoid cystic carcinoma, adrenal tumor, amyloidosis, anal cancer, appendiceal cancer, astrocytoma, ataxia-telangiectasia, Beckwell syndrome, cholangiocarcinoma (cholangiocarcinoma), Birt-Hogg-Dubé syndrome, bladder cancer, bone cancer (osteosarcoma), brainstem glioma, brain tumor, breast cancer, inflammatory breast cancer, metastatic breast cancer, male breast cancer, Carney complex, central nervous system tumors (brain tumors and spinal cord tumors), cervical cancer, childhood cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, desmoidoma, infantile connective tissue proliferative ganglioglioma, childhood tumors, ependymoma, esophageal cancer, Ewing sarcoma, eye cancer, eyelid cancer, familial multiple adenocarcinoma, familial GIST, familial malignant melanoma, familial pancreatic cancer, gallbladder cancer, gastrointestinal stromal tumor (GIST), germ cell tumors, gestational trophoblastic disease, head and neck cancer, hereditary breast and ovarian cancer, hereditary diffuse gastric cancer, hereditary leiomyomatosis and renal cell carcinoma, hereditary mixed polyposis syndrome, hereditary pancreatitis, hereditary papillary renal cell carcinoma, HIV / AIDS-related cancers, juvenile polyposis syndrome, renal cancer, lacrimal adenoma, laryngeal and hypopharyngeal cancer, Rife syndrome, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, Lynch syndrome, mastocytosis, myeloma, melanoma, meningioma, mesothelioma, type 1 multiple endocrine tumor, type 2 multiple endocrine tumor, multiple myeloma, MUTYH (or MYH) related polyposis, myelodysplastic syndrome (MDS), nasal cavity and sinus carcinoma, nasopharyngeal carcinoma, neuroblastoma, gastrointestinal neuroendocrine tumor, pulmonary neuroendocrine tumor, pancreatic neuroendocrine tumor, neuroendocrine tumor, neurofibroma type 1, neurofibroma type 2, nevus-like basal cell tumor syndrome, oral and oropharyngeal carcinoma, osteosarcoma, ovarian, fallopian tube and peritoneal carcinoma, pancreatic cancer, parathyroid carcinoma, penile cancer, Boytz-Yage syndrome, pheochromocytoma and paraganglioma, pituitary adenoma, pleural pulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, Kaposi's sarcoma, soft tissue sarcoma, skin cancer (non-melanoma), small intestine cancer, gastric cancer, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis, uterine cancer, vaginal cancer, Shih-Lin syndrome, vulvar cancer, Werner syndrome, nephroblastoma or xeroderma pigmentosum. In some implementations, the cancer is gastrointestinal cancer, breast cancer, or lung cancer. In other implementations, the gastrointestinal cancer is colorectal cancer, stomach cancer, esophageal cancer, pancreatic cancer, or cholangiocarcinoma.

[0051] In some implementations, the method further includes administering one or more other therapeutic agents.

[0052] In various embodiments using one or more other therapeutic agents, the one or more other therapeutic agents may be selected from chemotherapeutic agents, vascular endothelial growth factor (VEGF) inhibitors, epidermal growth factor receptor (EGFR) inhibitors or apoptosis inducers, immunotherapeutic agents, or combinations thereof. In some implementations, the chemotherapeutic agent is selected from bleomycin, carboplatin, chlorambucil, cisplatin, colchicine, cyclophosphamide, daunorubicin, doxorubicin or doxorubicin liposomes, mitomycin C, actinomycin, diethylstilbestrol, etoposide, 5-fluorouracil, fluxuridine, melphalan, methotrexate, mitomycin, 6-mercaptopurine, teniposide, 6-thioguanine, vincristine and vinblastine, leflunomide, tamoxifen, and interferon-alpha. -2b, glutamate, plicamycin, mercaptopurine, 6-thioguanine, carmustine, BCNU, limousine, CCNU, cytosine arabinose, estramustine, hydroxyurea, procarbazine, busulfan, medroxyprogesterone, estramustine sodium phosphate, vinyl estradiol, estradiol, medroxyprogesterone acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, testosterone lactone, melphalan, chlorambucil, mechlorethamine, thiourea, betamethasone sodium phosphate, dicarbazine, asparagine, mitotane, vincristine sulfate, vinblastine sulfate, FOLFOX (leucovorin, 5-fluorouracil and oxaliplatin) or FOLFIRI (leucovorin, 5-fluorouracil and irinotecan) and combinations thereof.In some embodiments, the VEGF inhibitor is bevacizumab, ramucirumab, regorafenib, or ziv-aflibercept. In some embodiments, the EGFR inhibitor is selected from cetuximab and / or panitumumab. In some embodiments, the apoptosis inducer is selected from B-cell lymphoma 2 (BCL2) inhibitors, BCL super-large (BCL-XL) inhibitors, or inhibitors of apoptosis proteins (IAP) inhibitors, or combinations thereof. In some embodiments, the immunotherapy agent is an anti-CTLA4 agent, an anti-PD1 agent, an anti-PD-L1 agent, an anti-LAG3 agent, or an anti-TIM3 agent.

[0053] In various implementation schemes, the subjects are mammals, such as humans. Attached Figure Description

[0054] Figure 1 Exemplary general screening strategies for immune library construction and screening strategies for discovering TRAILR2 binders are described.

[0055] Figure 2 This illustrates the sample selection process for next-generation sequencing (NGS) during phage display. Sequencing was performed on three initial libraries, 12 TRAILR2 samples from the first round of panning, and 18 TRAILR2 samples from the second round of panning, with 20 million, 2 million, and 2 million reads respectively. Comparing the V-body enrichment of the initial libraries with that of the first and second rounds of panning enabled the identification of potential V-body candidates.

[0056] Figure 3 A schematic diagram of an exemplary NGS workflow is shown. After phage display, the VHH region of the phage elution is amplified by polymerase chain reaction (PCR), fused with unique and sample-specific barcodes, and NGS is performed using Genewiz's Illumina NovaSeq platform. The raw data is demultiplexed and then processed through the Pipebio NGS analysis workflow. Forward and reverse sequence pairs are merged via overlapping regions, and VHHs are labeled including CDRs. V-body sequences are clustered based on CDR3 identity, allowing for detailed analysis of V-body enrichment, sequence diversity, CDR3 length distribution, and cluster abundance during phage display. Based on these analyses, up to 500 candidates are selected for Twist DNA synthesis and further characterization.

[0057] Figures 4A to 4BThe study showed that the anti-TRAILR2 V monomer binds to the cells of an engineered cell line that overexpresses TRAILR2.

[0058] Figures 5A to 5B The anti-TRAILR2 V antibody monomer binds to cells expressing endogenous TRAILR2.

[0059] Figure 6 A schematic diagram of an exemplary experimental setup for determining the binding affinity of a V-body to its respective target via surface plasmon resonance (SPR) is shown. The diagram discloses “HHHHHH” as SEQ ID NO:2407.

[0060] Figures 7A to 7G Surface plasmon resonance (SPR) sensor maps depicting the binding of VHH to TNFR2 in humans, cynomolgus monkeys, and mice are presented. These include fitted binding curves and calculated dissociation constants (KL). D The first figure shows the binding affinity of a single V-body to human TRAILR2. Each plot represents a global analysis of the binding of human TRAILR2 (extracellular domain) to a single V-body captured at discrete points, reporting K... D Values. Global fitting of the line representing the increase in TRAILR2 concentration shows that the binding kinetics are adequately described by a simple Langmuir mode. Points with non-ideal properties are boxed (e.g., insufficient binding / dissociation (on-rate / off-rate) information, inactive binding, or almost no binding). Binding affinity was measured using 11 different antigen concentrations (2-fold serial dilution, starting at 1 µM) under physiological conditions (50 mM HEPES pH 7.4, 150 mM NaCl, 0.1% (w / v) BSA, 0.05% (v / v) Tween20, 25°C). The second figure shows the binding affinity of a single V-body to the extracellular domain of cynomolgus monkey TRAILR2. The third figure shows the binding affinity of a single V-body to the extracellular domain of mouse TRAILR2. Two competing molecules were used as Figure 7G The reference material is shown in the figure. The figure discloses "HHHHHH" as SEQ ID NO: 2407.

[0061] Figure 8 This diagram illustrates an exemplary experimental setup for determining competition between ligands and TRAIL.

[0062] Figures 9A to 9ISurface plasmon resonance (SPR) sensor maps used to determine TRAIL blocking are depicted. Each tile represents an overlay of sensor maps captured at discrete points on a single V-body. Figures 9A to 9G In the diagram, the solid line sensor plot shows TRAIL competition: additional binding of TRAIL following association with human TRAILR2 (extracellular domain), indicating unoccupied epitopes (non-overlapping epitopes), or the absence of TRAIL binding, indicating epitope blocking (overlapping epitopes); while the dashed line sensor plot shows the buffer control: association and dissociation of human TRAILR2 in the absence of TRAIL. Figures 9H to 9I In the figure, ligand and buffer control sensor diagrams are shown as indicated. Human TRAILR2 (extracellular domain) (500 nM) was injected under physiological conditions (50 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% (w / v) BSA, 0.05% (v / v) Tween 20, 25°C), followed by human TRAIL (1 µM).

[0063] Figure 10 To depict the potency of TRAILR2 V agonists with different valences in inducing necrosis in cell lines expressing endogenous TRAILR2 compared to a tetravalent competitor molecule (competitor #1 in the SPR characterization data).

[0064] Figure 11 To depict the time course of Annexin V induction by TRAILR2 V agonists of different valences in cell lines expressing endogenous TRAILR2.

[0065] Figures 12A to 12B Depicting different tetravalent forms reveals comparable dynamics and efficiencies.

[0066] Figure 13An exemplary design of a multivalent anti-TRAILR2 V-body construct is shown. Different V-body forms for testing TRAILR2 signal induction are depicted. The anti-TRAILR2 V-body is shown as an elliptical shape, with the linkers shown as lines, where the glycine-serine (GS) linker is shown as a curve, the rigid linker as a straight line, and the Fc domain as a dimer bar. The first set of constructs contains an anti-TRAILR2 V-body linked via a flexible GS linker. The second set of constructs with the same anti-TRAILR2 valence contains a mixture of flexible and rigid linkers. The rigid proline-rich linker included in some constructs mimics the approximately 90 to 100 Å distance that a conventional antibody (e.g., a monoclonal antibody, mAb) can span, which is approximately equal to the size of the death-induced signal transduction complex (DISC) complex. V-bodies fused with the Fc domain of a silencing antibody are also included. The rigidity of the Fc domain and the distance it spans indicate its suitability for TRAILR2 signal induction. A set of Fc-mounted V-bodies is also designed, including the fusion of the V-bodies with the Fc structural domain at the N and C ends with different valences. This figure discloses "PAPAP" as SEQ ID NO: 2408.

[0067] Figure 14 High-throughput kinetic analysis of ODY-N2280Hu1 and ODY-28B1Hu1 is depicted. The binding affinity of human TRAILR2 is shown in the above figure. Each plot represents a global analysis of the binding of human TRAILR2 (extracellular domain) to a single V-body captured at discrete points, reporting K... D Values. Global fitting of the line representing the increase in TRAILR2 concentration showed that the binding kinetics were adequately described by a simple Langmuir model. Binding affinity was measured under physiological conditions (50 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% (w / v) BSA, 0.05% (v / v) Tween20, 25°C) using 10 different antigen concentrations (2-fold serial dilutions, starting at 200 nM (human TRAILR2), 300 nM (cynomolgus monkey TRAILR2), or 1 µM (mouse TRAILR2)). The binding affinity for cynomolgus monkey TRAILR2 is shown in the middle plot. The binding affinity for mouse TRAILR2 is shown in the lower plot. This figure discloses “HHHHHH” as SEQ ID NO: 2407.

[0068] Figure 15 High-throughput kinetic analysis of ODY-N1039Hu1 and ODY-N1047Hu1 is depicted. The binding affinity of human TRAILR2 is shown in the above figure. Each plot represents a global analysis of the binding of human TRAILR2 (extracellular domain) to single V-body structures captured at discrete points, reporting K...D Values. Global fitting of the line representing the increase in TRAILR2 concentration showed that the binding kinetics were adequately described by a simple Langmuir model. Binding affinity was measured under physiological conditions (50 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% (w / v) BSA, 0.05% (v / v) Tween20, 25°C) using 10 different antigen concentrations (2-fold serial dilutions, starting at 62.5 nM (human TRAILR2), 125 nM (cynomolgus monkey TRAILR2), or 1 µM (mouse TRAILR2)). The binding affinity of cynomolgus monkey TRAILR2 is shown in the middle plot. The binding affinity of mouse TRAILR2 is shown in the lower plot. This figure discloses “HHHHHH” as SEQ ID NO: 2407.

[0069] Figure 16 Off-target binding analyses of ODY-30A9Hu1, ODY-N1047Hu1, ODY-28B1Hu1, ODY-N2280Hu1, and ODY-N1039Hu1 are shown. The binding affinity of V bodies to human TRAILR2 compared to homologous proteins TRAILR1, TRAILR3, and TRAILR4 is evaluated. Each plot represents a global analysis of receptor binding to a single V body captured at discrete points, reporting K... D Values. A global fit to the line representing the increase in receptor concentration shows that the binding kinetics are adequately described by a simple Langmuir model. Points with non-ideal characteristics are shown in boxes (e.g., insufficient binding / dissociation information, inactive binding, or almost no binding). Binding affinity was measured under physiological conditions (50 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% (w / v) BSA, 0.05% (v / v) Tween20, 25°C) using 12 different antigen concentrations (2-fold serial dilutions, TRAILR1, TRAILR3, and TRAILR4 starting at 500 nM, and TRAILR2 starting at 31.3 nM).

[0070] Figure 17 Constructs mapping the V-body domains were displayed. Exchange constructs were generated to identify the TRAILR2 domains targeted by different V-bodies. Individual cysteine-rich domains (CRDs) or stem regions were exchanged from human protein sequences for cameloid protein sequences to generate hybrid TRAILR2 extracellular receptor domains (ECDs).

[0071] Figures 18A to 18BThe following are examples of TRAILR2 binding V-types: ODY-28B1Hu1, ODY-N829Hu1, ODY-N2280Hu1, ODY-23D12Hu1, ODY-24F10Hu1, and ODY-25C08Hu1. Figure 18A ), and ODY-N1039Hu1 and ODY-N1047Hu1 ( Figure 18B Domain mapping of TRAILR2. Determining the binding of V-bodies to the human / camelidae exchanged extracellular receptor domain (ECD) protein. Each tile represents a global analysis of TRAILR2 binding to a single V-body captured at a discrete point. These lines represent the rising concentration of the injected receptor. Points where no binding events were observed compared to the WT TRAILR2 ECD sensing map are shown in boxes.

[0072] Figures 19A to 19B The diagram shows epitope binning of TRAILR2 with V bodies. Simultaneous binding of two V bodies to TRAILR2 was determined to test for overlapping epitopes. The V bodies were coupled to an SPR chip, TRAILR2 was saturated, and binding of the injected other V body was tested. An example of the experimental setup used for competitive binding and epitope binning analysis is depicted in... Figure 19A The results of the binning analysis are shown in a matrix representation (in the middle). Figure 19B In the diagram, dotted squares indicate that two V-body cells cannot bind simultaneously, suggesting overlapping epitopes on TRAILR2. Striped squares indicate that the tested V-body cells may bind simultaneously. Therefore, these V-body cells target different epitopes on TRAILR2.

[0073] Figures 20A to 20G Kinetic analysis of the CDR3 alanine (Ala) scanning V-body mutant is shown. This analysis targets the Ala scanning mutant ODY-25C08Hu1 ( Figures 20A to 20B ), ODY-28B1Hu1 ( Figures 20C to 20D ), ODY-1039Hu1 ( Figure 20E ), ODY-1047Hu1 ( Figure 20F ) and ODY-2280Hu1 ( Figure 20G This study determined the binding affinity of the CDR3 alanine (Ala) scanning mutant V variant to human TRAILR2. Each plot represents a global analysis of the binding of human or cynomolgus monkey TRAILR2 to a single V variant captured at discrete points, reporting K... DValues. A global fit to the line representing the increase in receptor concentration shows that the binding kinetics are adequately described by a simple Langmuir model. Points with non-ideal characteristics are shown in boxes (e.g., insufficient binding / dissociation information, inactive binding, or almost no binding). Binding affinity was measured under physiological conditions (50 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% (w / v) BSA, 0.05% (v / v) Tween20, 25°C) using seven different antigen concentrations (2-fold serial dilutions, human and cynomolgus monkey TAILR2 starting at 15.6 nM).

[0074] Figures 21A to 21E The scan of CDR3 alanine (Ala) in the V-body mutant ODY-25C08Hu1 shows... Figure 21A ), ODY-28B1Hu1 ( Figure 21B ), ODY-1039Hu1 ( Figure 21C ), ODY-1047Hu1 ( Figure 21D ) and ODY-2280Hu1 ( Figure 21E Fluorescence-activated cell sorting (FACS) analysis was performed on the cells containing the nucleotides. All data are expressed as a percentage of mean fluorescence intensity (MFI) relative to parental V. The figure discloses, in order of appearance, SEQ ID NOs: 2328, 2327, 2326, 2325, 2324, 2323, 2322, 2321, 2320, 2319, 2318, 2317, 2316, 2315, 2314, 2313, 2312, 2311, 2310, 2309, 2308, 2307, 2306, 2305, 2304, and 2305. 03, 2302, 2301, 2300, 2299, 2298, 2344, 2343, 2342, 2341, 2340, 2339, 2338, 2348, 2347, 2346, 2345, 2337, 2336, 2335, 2334, 2333, 2332, 2331, 2330, and 2329. Invention Details

[0076] definition

[0077] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For the purposes of interpreting this specification, the following terminology will be used, and where appropriate, terms used in the singular will also include the plural, and vice versa. All patents, applications, publications, and other publications are incorporated herein by reference in their entirety. In the event of any conflict between any description of terms set forth herein and any reference incorporated herein by reference, the description of terms set forth herein shall prevail.

[0078] As used herein, when referring to a specific numerical value, the term “about” means that the value may differ from the stated value by no more than 5%. For example, as used herein, the expression “about 100” includes 95 and 105 and all values ​​in between (e.g., 96, 97, 98, 99, etc.).

[0079] The term "antigen" encompasses any agent (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleotide, portion thereof, or combination thereof) that can specifically bind to a particular humoral or cellular immune product (such as antibody molecules or T-cell receptors). In various embodiments of this disclosure, the antigen described herein is TRAILR2, including human, cynomolgus monkey, and / or mouse TRAILR2.

[0080] The term "epitope" refers to an antigenic determinant on the surface of an antigen to which an antibody molecule binds. A single antigen may have more than one epitope. Therefore, different antibodies can bind to different regions of the antigen and may have different biological effects (e.g., agonist or antagonist). Epitopes can be conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are generated from adjacent amino acid residues in a polypeptide chain. In some cases, epitopes may include non-peptide portions of the antigen, such as sugars, phosphoryl groups, or sulfonyl groups.

[0081] The term "antigen-binding protein" in its broadest sense refers to a protein that specifically binds to an antigen (e.g., TRAILR2). In some embodiments, the antigen-binding protein is an antibody or an antigen-binding fragment of an antibody, such as human antibodies, humanized antibodies; camelid antibodies; chimeric antibodies; recombinant antibodies; heavy chain antibodies; single-domain antibodies (e.g., VHH); single-chain antibodies (e.g., single-chain fragment variable (scFv)); double-chain antibodies; triple-chain antibodies; quadruple-chain antibodies; Fab fragments; F(ab')2 fragments; IgD antibodies; IgE antibodies; IgM antibodies; IgG1 antibodies; IgG2 antibodies; IgG3 antibodies; or IgG4 antibodies; and fragments thereof. The term "antigen-binding protein" also encompasses, for example, alternative protein backbones or artificial backbones having a transplanted CDR or CDR derivative. Such backbones include, but are not limited to, antibody-derived backbones containing mutations introduced, for example, to stabilize the three-dimensional structure of the antigen-binding protein, and fully synthetic backbones containing, for example, biocompatible polymers. In addition, peptide antibody mimics and antibody mimics based on fibronectin components (such as fibronectin type III domain (FN3)) can be used as the backbone.

[0082] The terms “TNF-associated apoptosis-inducing ligand receptor 2” or “TRAILR2” or similar terms are used interchangeably herein and may refer to any isotype, variant, and / or species homolog of TRAILR2 from any source, such as mammals, including primates (e.g., humans and monkeys) and rodents (e.g., rats and mice). The term covers naturally occurring variants of TRAILR2, such as, but not limited to, allelic variants and splice variants. In addition to any form of TRAILR2 that can be produced by processing (such as intracellular processing), the term also covers “full-length” or unprocessed TRAILR2. In some embodiments, TRAILR2 is human TRAILR2. In some embodiments, TRAILR2 is cynomolgus monkey (“cyno”) TRAILR2.

[0083] The terms “antibody” and “immunoglobulin” or “Ig” are used interchangeably herein and are used in the broadest sense to encompass, for example, single monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length or intact monoclonal antibodies), antibody compositions having multi-epitope or single-epitope specificity, polyclonal antibodies, monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), single-domain antibodies (e.g., VHH), single-chain antibodies, intracellular antibodies, anti-idiotype (anti-Id) antibodies, and antigen-binding fragments of antibodies, as described below. Antibodies can be human, humanized, camelified, recombinant-generated, chimeric, synthetic, affinity-dematurated and / or affinity-maturated antibodies, and antibodies derived from other species (e.g., mice, camels, llamas, rabbits, etc.). In certain embodiments, the specific target antigens that can be bound by the antibodies provided herein include TRAILR2 peptides, TRAILR2 fragments, or TRAILR2 epitopes. "Antigen-binding fragment" generally refers to a portion of the antibody heavy chain and / or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment is derived. Non-limiting examples of antigen-binding fragments include single-domain antibodies (e.g., VHH), single-chain Fv (scFv), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), Fd fragments, Fv fragments, double-chain antibodies, triple-chain antibodies, quadruple-chain antibodies, and microantibodies, or chemically modified derivatives thereof. Specifically, the antibodies described herein include immunoglobulin molecules and molecules containing the immunoactive portion of immunoglobulin molecules, such as one or more complementarity-determining regions (CDRs) of antibodies binding to TRAILR2. Such antibody fragments can be found in, for example, the following descriptions: Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989); Myers (ed.), Molec. Biology and Biotechnology: A Comprehensive Desk Reference, New York: VCH Publisher, Inc.; Huston et al., Cell Biophysics, 22:189-224 (1993); Plückthun and Skerra, Meth. Enzymol., 178:497-515 (1989); and Day, ED, Advanced Immunochemistry 2nd Edition, Wiley-Liss, Inc., New York, NY (1990).The antibodies described herein may be any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b).

[0084] As used herein, the term "single-domain antibody" or "sdAb" refers to an antibody or antibody fragment containing a single antibody variable domain capable of binding to a specific antigen independently without requiring another antibody variable domain. The complementarity-determining region (CDR) of a single-domain antibody is part of the single-antibody variable domain. Examples of single-domain antibodies include, but are not limited to, heavy-chain antibodies, naturally occurring antibodies lacking light chains, single-domain antibodies derived from conventional four-chain antibodies, engineered antibodies, variable domains derived from the aforementioned antibodies, and single-domain backbones other than those derived from antibody backbones. Single-domain antibodies can be derived from any species, including but not limited to mice, humans, camels, llamas, sharks, goats, rabbits, and / or cattle. In some embodiments, the single-domain antibodies used herein are naturally occurring single-domain antibodies, referred to as light-chain-free heavy-chain antibodies. For clarity, variable domains derived from naturally occurring light-chain-free heavy-chain antibodies are referred to herein as VHHs to distinguish them from the conventional VHs of four-chain immunoglobulins. Such VHH molecules can be derived from antibodies produced in camel species, such as camels, llamas, dromedaries, alpacas, and guanacos. Other species besides camels can also produce naturally occurring heavy-chain antibodies that do not contain light chains, which are also within the scope of this invention. For example, cartilaginous fish (such as sharks) can produce immunoglobulin-like structures called VNARs. In some embodiments, single-domain antibodies can be obtained from camel VH domains. In some embodiments, single-domain antibodies can be obtained from camelified human VHs. See Saerens et al., Current Opinion in Pharmacology, 2008, 8:600-608, the disclosure of which is incorporated herein by reference as a review of single-domain antibodies.

[0085] The term "specific binding" as used in this article refers to the formation of a complex between an antigen-binding protein and a target antigen that is relatively stable under physiological conditions. Specific binding is characterized by its dissociation constant (K). D It is approximately 1×10 -6 M or lower (e.g., less than 10) -6 M, less than 5×10 -7 M, less than 10 -7 M, less than 5×10 -8 M, less than 10 -8 M, less than 5×10 -9M, less than 10 -9 M, or less than 10 -10 Methods for determining the binding affinity of an antigen-binding protein (e.g., an antibody or antibody fragment) to a target antigen are well known in the art and include, for example, surface plasmon resonance (e.g., BIACORE® assay), biolayer interferometry, ligand binding assays (e.g., enzyme-linked immunosorbent assay (ELISA)), equilibrium dialysis, fluorescence activated cell sorting (FACS), or flow cytometry-based binding assays. Specific binding to a specific target antigen from a particular species does not preclude the antigen-binding protein from specifically binding to similar targets from different species. For example, specific binding to human TRAILR2 does not preclude the antigen-binding protein from specifically binding to TRAILR2 from cynomolgus monkeys (“cyno”) or mice.

[0086] When used in the context of antigen-binding proteins (e.g., antibodies, such as single-domain antibodies), peptides, polynucleotides, and carriers, the term "isolated" refers to antigen-binding proteins (e.g., antibodies, such as single-domain antibodies), peptides, polynucleotides, and carriers that are at least partially free of other biomolecules from the cells or cell cultures in which they are produced. Such biomolecules include nucleic acids, proteins, other antibody or antigen-binding fragments, lipids, carbohydrates, or other substances such as cell debris and growth media. Isolated antigen-binding proteins may further be at least partially free of expression system components, such as biomolecules from the host cell or its growth medium. Generally, the term "isolated" is not intended to mean that such biomolecules are completely absent (e.g., may retain small or insignificant amounts of impurities) or free of water, buffers or salts, or components of pharmaceutical formulations that include antigen-binding proteins (e.g., antibodies, such as single-domain antibodies).

[0087] As used herein, the term "operably linked" can refer to a functional relationship between two or more regions of a polypeptide chain, wherein the two or more regions are linked to produce a functional polypeptide.

[0088] As used herein, the terms “variant,” “derived from,” “derived from,” or “derived from” in the context of a protein or polypeptide (e.g., an antigen-binding protein or its domain) mean: (a) a polypeptide having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the polypeptide from which the variant or derivative is derived; (b) a polypeptide encoded by a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the nucleotide sequence encoding the polypeptide from which the variant or derivative is derived; or (c) containing the numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, relative to the polypeptide from which the variant or derivative is derived. (d) A polypeptide with mutations (i.e., additions, deletions, and / or substitutions) of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids; (e) A polypeptide encoded by a nucleic acid that can hybridize with the coding nucleic acid of a polypeptide derived from a variant or derivative under high, medium, or typical stringency hybridization conditions; (f) A polypeptide encoded by a nucleotide sequence that can hybridize with a fragment of a polypeptide derived from a variant or derivative under high, medium, or typical stringency hybridization conditions (having at least 20, 30, 40, 50, 75, 100, 125, or 150 consecutive amino acids); or (f) A fragment of a polypeptide derived from a variant or derivative. The term also covers fusion proteins or polypeptides comprising polypeptides derived from variants or derivatives.

[0089] When referring to nucleic acids or fragments thereof, the terms "substantial identity" or "substantially identical" mean that, when optimally aligned with another nucleic acid (or its complementary strand) for appropriate nucleotide insertions or deletions, nucleotide sequence identity is present in at least about 95%, and preferably at least about 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any well-known algorithm for sequence identity (such as FASTA, BLAST, or Gap), as discussed below. Nucleic acid molecules that are substantially identical to a reference nucleic acid molecule may, in certain circumstances, encode polypeptides having the same or substantially similar amino acid sequences as the polypeptide encoded by the reference nucleic acid molecule.

[0090] When applied to peptides, the term "substantially similar" or "substantially similar" means that two peptide sequences share at least 95% sequence identity, and preferably at least 98% or 99% sequence identity, when best aligned using default gap weights, such as through procedures like GAP or BESTFIT. Preferably, the difference in the position of the inconsistent residues is a conserved amino acid substitution. A "conserved amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced by another amino acid residue with a side chain (R group) having similar chemical properties (e.g., charge or hydrophobicity). Generally, conserved amino acid substitutions do not substantially alter the functional properties of the protein. In cases where the difference between two or more amino acid sequences is a conserved substitution, the percentage of sequence identity or similarity can be adjusted upwards to correct for the nature of the conserved substitution. The manner in which this adjustment is made is well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference. Examples of groups of amino acids with side chains having similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change with a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445, which is incorporated herein by reference. A “moderately conservative” permutation is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0091] Sequence analysis software is commonly used to measure the sequence similarity of peptides, also known as sequence identity. Protein analysis software uses similarity measures assigned to various substitutions, deletions, and other modifications (including conserved amino acid substitutions) to match similar sequences. For example, GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related peptides (such as homologous peptides from different species) or between wild-type proteins and their mutant proteins. See, for example, GCG version 6.1. FASTA can also be used, with default or recommended parameters, with the program in GCG version 6.1 to compare peptide sequences. FASTA (e.g., FASTA2 and FASTA3) provides alignment of the best overlapping regions between the query and retrieved sequences and a percentage of sequence identity (see Pearson (2000) above). When comparing sequences disclosed herein with databases containing large amounts of sequences from different organisms, another preferred algorithm is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, which are each incorporated herein by reference.

[0092] The terms “enhancement,” “promotion,” “increase,” “amplification,” or “modification” refer to the ability of the composition covered herein to produce, initiate, or induce a greater physiological response (i.e., downstream effects) compared to the response induced by the mediator or control molecule / composition. Measurable physiological responses may include increased immune cell expansion, activation, effector function, persistence, and / or increased tumor cell killing capacity, particularly as apparent from the understanding in the art and in the description herein. In some embodiments, the amount of “increase” or “enhancement” may be a “statistically significant” amount and may include an increase of 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, or more (e.g., 500-fold, 1000-fold) (including all integers and decimals between these and greater than 1, e.g., 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, etc.) in the response produced by the mediator or control composition.

[0093] The terms “reduction” or “lowering” or “mitigation” or “reduction” generally refer to a smaller physiological response (i.e., downstream effect) produced, initiated, or induced by the composition covered herein compared to the response induced by the mediator or control molecule / composition. In some embodiments, the amount of “reduction” or “reduction” may be a “statistically significant” amount and may include a reduction of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, or more times (e.g., 500, 1000 times) (including all integers and decimals between the two and greater than 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the response produced by the mediator or control composition (reference response).

[0094] The term “treatment” or “management” for a state, symptom, or condition includes: (1) preventing, delaying, or reducing the incidence and / or likelihood of the occurrence and development of at least one clinical or subclinical symptom of a state, symptom, or condition in a subject who may have or be susceptible to the state, symptom, or condition but has not yet experienced or exhibited clinical or subclinical symptoms of the state, symptom, or condition; or (2) suppressing the state, symptom, or condition, i.e., preventing, reducing, or delaying the development of the disease or its recurrence or at least one of its clinical or subclinical symptoms; or (3) alleviating the disease, i.e. causing the resolution of the state, symptom, or condition or at least one of its clinical or subclinical symptoms. The benefit to the subject treated is statistically significant or at least perceptible to the patient or physician.

[0095] The term "effective dose" or "therapeutic effective dose" refers to the amount and / or concentration of an active ingredient (e.g., anti-TRAILR2 antigen-binding protein) that, when administered to a patient alone (i.e., as a monotherapy) or in combination with another therapeutic agent, significantly reduces disease progression, for example, by improving or eliminating symptoms and / or causes of the disease. An effective dose can be an amount that alleviates, reduces, or relieves at least one symptom or biological response or effect associated with the disease or condition, prevents the progression of the disease or condition, or improves the patient's physiological function. Therapeutic effective doses of compositions containing active agents can vary depending on factors such as disease state, individual age, sex, and weight, and the ability of the active agent to elicit the desired response in the individual. A therapeutic effective dose is also an amount in which the beneficial therapeutic effect outweighs any toxic or harmful effects of the active agent. A therapeutic effective dose can be delivered by a single or multiple administrations. A therapeutic effective dose is the amount that effectively achieves the desired therapeutic and / or preventative outcome at the necessary dose and within the necessary time.

[0096] The terms “individual,” “subject,” and “patient” are used interchangeably herein and refer to an animal; for example, a mammal. The terms include human and veterinary subjects. In some embodiments, methods of treating mammals are provided, including but not limited to humans, rodents, apes, felines, canines, equines, bovines, suidae, sheep, goats, mammalian laboratory animals, mammalian livestock, mammalian locusts, and mammalian pets. Subjects may be male or female and may be of any suitable age, including infants, young children, adolescents, adults, and elderly subjects. In some embodiments, subjects may be subjects who require treatment for a disease or ailment. In a particular embodiment, the subject is a human.

[0097] Anti-TRAILR2 antigen-binding protein

[0098] This disclosure provides antigen-binding proteins (e.g., antibodies, such as single-domain antibodies) that bind to TNF-associated apoptosis-inducing ligand receptor 2 (TRAILR2, also known as death receptor 5 or DR5).

[0099] TRAIL receptor 2 (TRAILR2) is a single-cell type 1 membrane protein with a molecular weight of approximately 42 kDa, belonging to the TNFR superfamily. TRAILR2 contains an extracellular domain with three cysteine-rich domains (CRDs) and an intracellular death domain involved in apoptosis signaling. The cysteine-rich domains contain a total of seven disulfide bonds that stabilize the protein's elongated structure. CRD1, also known as the pre-ligand assembly domain (PLAD), has been found to participate in the formation of low-affinity (>1 µM) TRAILR2 dimers in the absence of TRAIL. The TRAILR2 death domain is a bundle of six α-helices that participates in the binding of intracellular chaperones and the formation of the death-inducing signaling complex (DISC) to promote apoptosis signaling. Furthermore, O-glycosylation of TRAILR2 at two positions within CRD1 and CRD2 has been found to alter its apoptosis-inducing ability but not its affinity for TRAIL. In addition, it has been reported that fucosylation of O-glycans is crucial for the apoptosis-inducing ability of TRAILR2.

[0100] The TRAIL ligand itself consists of β-sandwich folds and homotrimerizes in solution. The trimer is non-covalent, and TRAIL dissociates at low concentrations, leading to rapid degradation of the monomer units. The TRAIL trimer is stabilized by zinc ions coordinated to three free cysteine ​​residues (one per monomer) at the trimerization interface. The TRAIL ligand exists in both soluble (sTRAIL) and membrane-anchored (mTRAIL) forms.

[0101] The extracellular domain of TRAILR2 has been structurally characterized as a trimeric complex in the presence of TRAIL ligand. Furthermore, NMR structures of the transmembrane domain trimer and hexamer have been reported, but structural data on the intracellular death domain of TRAILR2 are unavailable. However, homologous structures of the death domain and other signaling components provide structural insights into the signaling complex.

[0102] In the absence of the trimeric TRAIL ligand, TRAILR2 has been reported to exist in monomeric or dimer states. The dimer state is mediated via low-affinity (>1 µM) interactions of the PLAD domain. The dimer has been reported to employ two distinct conformations: an active parallel conformation, where the TRAIL binding site is accessible, or an inactive antiparallel conformation, where the TRAIL binding site is masked. In the presence of soluble or membrane-bound trimeric TRAIL, TRAILR2 can bind to the TRAIL trimer with high affinity (<2 nM) and assemble on the membrane into a hexameric TRAILR23TRAIL3 complex containing three TRAILR2 molecules bound to the trimeric TRAIL. The TRAIL binding site on TRAILR2 is located at the concave surface formed by CRD1 and CRD2. Furthermore, in addition to homologous complexes formed with TRAILR2 bound to trimeric TRAIL, heterologous complexes containing TRAILR1 and TRAILR4 have been reported, exhibiting lower overall signal transduction efficacy upon activation. As for other receptors in the TNFR superfamily, it has been reported that the dimers and trimers of TRAILR2 may not be randomly distributed across the entire membrane, but may be arranged in a higher-order hexagonal pattern.

[0103] The actual distribution of TRAILR2 oligomers in any given normal or cancerous cell remains largely unknown. It has been confirmed that in the absence of TNF ligands, TNFR1 exists primarily in monomeric and dimer states, while in the presence of TNF, most receptors assemble into trimers or higher-order oligomers of trimers, with only a small number of monomers observed and no dimers. Whether these results translate to TRAILR2 remains to be seen.

[0104] Consistent with these observations, TRAILR2 oligomerization has been consistently reported as potentially essential for efficient signal transduction. The precise mechanisms of signal transduction complex assembly and protein stoichiometry are not fully understood, but it is hypothesized that two TRAILR2 trimers bound to TRAIL need to be very close to achieve efficient apoptosis signaling. More specifically, it has been shown that the membrane-bound TRAIL trimer binds to three TRAILR2 receptor molecules assembled into a symmetrical trimer complex, with each TRAILR2 molecule interacting with two TRAIL monomers at the center of the complex. This trimerization has been proposed to induce a rearrangement of the transmembrane helix, causing a conformational change of the TRAILR2 death domain to an open conformation. In its open conformation, TRAILR2-DD can recruit an adaptor protein called the Fas-associated death domain (FADD) via a tight helix-helix interaction similar to Fas-FADD. Once this interaction is established, procaspase-8 can bind to the currently exposed FADD death-effector domain (DED). A single death-induced signaling complex (DISC) was found to be unable to induce downstream signaling because caspaseogen-8 requires homodimerization to exhibit autocatalytic activity and subsequent activation. Therefore, a second TRAILR2 complex, which binds to all intracellular components, may need to be very close to achieve caspaseogen-8 dimerization. DISC complex dimerization is mediated solely via membrane-bound TRAIL (mTRAIL), not soluble TRAIL. The anchoring of mTRAIL in the lipid bilayer restricts protein diffusion into two dimensions, which would effectively increase the co-localization of mTRAIL molecules and improve the efficiency of agonist-induced TRAILR2 clustering compared to the efficiency promoted by the soluble form.

[0105] Structural models of the entire signal transduction complex suggest that a distance >90 Å is required between the two TRAILR23TRAIL3 complexes to accommodate the intracellular DISC complex. This distance is closely correlated with the distance traversed by monoclonal antibodies (mAbs) that have been shown to induce TRAILR2 clustering and signal transduction.

[0106] In some implementations, human TRAILR2 may be encoded by the TNF receptor superfamily member 10b (TNFRSF10B) gene (NCBI Gene ID 8795) and have the following amino acid sequence: MEQRGQNAPAASGARKRHGPGPREARGARPGPRVPKTLVLVVAAVLLLVSAESALITQQDLAPQQRAAPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKESGTKHSGEVPAVEETVTSSPGTPASPCSL SGIIIGVTVAAVVLIVAVFVCKSLLWKKVLPYLKGICSGGGGDPERVDRSSQRPGAEDNVLNEIVSILQPTQVPEQEMEVQEPAEPTGVNMLSPGESEHLLEPAEAERSQRRRL LVPANEGDPTETLRQCFDDFADLVPFDSWEPLMRKLGLMDNEIKVAKAEAAGHRDTLYTMLIKWVNKTGRDASVHTLLDALETLGERLAKQKIEDHLLSSGKFMYLEGNADSAMS (UniProtKB accession number O14763) (SEQ ID NO: 2149).

[0107] In some implementations, cynomolgus monkey TRAILR2 can be encoded by the TNF receptor superfamily member 10b (TNFRSF10B) gene (NCBI Gene ID 102133727) and has the following amino acid sequence: MGQLRQSAPAASGARKGRGPGPREARGARPGLRVLKTLVLVVAAAAVLLSVSADCAPITRQSLDPQRRAAPQQKRSSPTEGLCPPGHHISEDSRECISCKYGQDYSTHWNDFLFCLRCTKCDSGEVEVNSCTTTRNTVCQCEEGTFREEDSPEICRKCRTGCPRGMVKVKDCTPWSDIECVHKESGTKHTGEVPAVEKTVTTSPGTPASPC SLSGIIIGVIVLVVIVVVAVIVWKTSLWKKVLPYLKGVCSGGGGDPERVDSSSHSPQRPGAEDNALNEIVSIVQPSQVPEQEMEVQEPAEQTDVNTLSPGESEHLLEPAKAEGPQRR GQLVPVNENDPTETLRQCFDDFAAIVPFDAWEPLVRQLGLTNNEIKVAKAEAASSRDTLYVMLIKWVNKTGRAASVNTLLDALETLEERLAKQKIQDRLLSSGKFMYLEDNADSATS (UniProtKB accession number A0A2K5TXK0|A0A2K5TXK0) (SEQ ID NO: 2150).

[0108] In some implementations, mouse TRAILR2 can be generated by the TNF receptor superfamily member 10b (TNFRSF10B) gene (NCBI Gene ID). 21933) encodes and has the following amino acid sequence: MEPPGPSTPTASAAARADHYTPGLRPLPKRRLLYSFALLLAVLQAVFVPVTANPAHNRPAGLQRPEESPSRGPCLAGQYLSEGNCKPCREGIDYTSHSNHSLDSCILCTVCKEDKVVETRCNITTNTVCRCKPGTFEDKDSPEICQSCSNCTDGEEELTSCTPRENRKCVSKTAWASWHKL GLWIGLLVPVVLLIGALLVWKTGAWRQWLLCIKRGCERDPESANSVHSSLLDRQTSSTTNDSNHNTEPGKTQKTGKKLLVPVNGNDSADDLKFIFEYCSDIVPFDSWNRLMRQLGLTDNQIQMVKAETLVTREALYQMLLKWRHQTGRSASINHLLDALEAVEERDAMEKIEDYAVKSGRFTYQNAAAQPETGPGGSQCV (UniProtKB registration number Q9QZM4) (SEQ ID NO: 2151).

[0109] In various embodiments, the antigen-binding protein of this disclosure has agonist activity upon binding to TRAILR2. While not wishing to be bound by theory, agonistic TRAILR2 conjugates can promote or increase TRAILR2 activation and / or enhance TRAILR2-mediated one or more signal transduction pathways. Agonistic TRAILR2 conjugates can promote or increase TRAILR2 activation by binding to TRAILR2, for example, to induce TRAILR2 polymerization, thereby conferring biological activity to the receptor. For example, agonistic TRAILR2 conjugates can nucleate the trimerization of TRAILR2 and further promote the close proximity of the TRAILR2 trimer associated with the TRAILR2 agonist conjugate to one or more other TRAILR2 trimers associated with the TRAILR2 agonist conjugate, in a manner similar to the interaction of TRAILR2 with its homologous ligand TRAIL, thereby inducing TRAILR2-mediated signal transduction. In some embodiments, the agonistic TRAILR2 conjugates described herein can bind, for example, three TRAILR2 molecules, which can assemble into a trimeric complex (i.e., a TRAILR2 trimer).

[0110] In some embodiments, the agonistic TRAILR2 binding protein of this disclosure may enable TRAILR2, as described herein, to bind to intracellular binding chaperones (e.g., adaptor proteins, such as (but not limited to) Fas-associated death domains (FADD)) and form death-inducing signaling complexes (DISC) to promote apoptosis signaling.

[0111] In some embodiments, the antigen-binding protein of this disclosure binds to human TRAILR2. In some embodiments, the antigen-binding protein of this disclosure (e.g., an antibody, such as a single-domain antibody) may be less than about 1 × 10⁻⁶. -6 M, for example, less than about 5 × 10 -7 M, less than approximately 3 × 10 -7 M, less than approximately 1×10 -7 M, less than approximately 8 × 10 -8 M, less than approximately 5 × 10 -8 M, less than approximately 3 × 10 -8 M, less than approximately 1×10 -8 M, less than approximately 9 × 10 -9 M, less than approximately 8 × 10 -9 M, less than approximately 7 × 10 -9 M, less than approximately 6 × 10 -9 M, less than approximately 5 × 10 -9 M, less than approximately 4 × 10 -9 M, less than approximately 3 × 10 -9 M, less than approximately 2 × 10 -9 M, or less than approximately 1×10 -9 M, or approximately 1×10 -10 Up to 1×10 -9 M, 1×10 -10 Up to 5×10 -9 M, approximately 1×10 -10 Up to 1×10 -8 M, approximately 1×10 -10 Up to 5×10 -8 M, approximately 1×10 -9 Up to 5×10 -9 M, approximately 5×10 -9 Up to 1×10 -8 M, approximately 1×10 -9 Up to 1×10 -8 M, approximately 1×10 -9 Up to 5×10 -8 M, approximately 1×10 -9 Up to 1×10 -7 M, or approximately 1×10 -8 Up to 1×10 -7 M of KD It combines with human TRAILR2.

[0112] In some embodiments, the antigen-binding protein of this disclosure (e.g., an antibody, such as a single-domain antibody) may be smaller than about 3 × 10⁻⁶. -7 M of K D Binds to human TRAILR2. In some embodiments, the antigen-binding protein of this disclosure (e.g., an antibody, such as a single-domain antibody) may be smaller than about 1 × 10⁻⁶. -10 Up to 5×10 -8 M of K D It combines with human TRAILR2.

[0113] In some embodiments, the antigen-binding protein of this disclosure binds to cynomolgus monkey (“cyno”) TRAILR2. In some embodiments, the antigen-binding protein of this disclosure (e.g., an antibody, such as a single-domain antibody) may be less than about 1 × 10⁻⁶. -6 M, for example, less than about 5 × 10 -7 M, less than approximately 3 × 10 -7 M, less than approximately 1×10 -7 M, less than approximately 8 × 10 -8 M, less than approximately 5 × 10 -8 M, less than approximately 3 × 10 -8 M, less than approximately 1×10 -8 M, less than approximately 8 × 10 -9 M, less than approximately 5 × 10 -9 M, less than approximately 3 × 10 -9 M, or less than approximately 1×10 -9 M, or approximately 1×10 -10 Up to 1×10 -9 M, 1×10 -10 Up to 5×10 -9 M, approximately 1×10 -10 Up to 1×10 -8 M, approximately 1×10 -10 Up to 5×10 -8 M, approximately 1×10 -9 Up to 1×10 -8 M, approximately 1×10 -9 Up to 5×10 -8 M, approximately 1×10 -9 Up to 1×10 -7 M, approximately 1×10 -9 Up to 2×10 -7 M, approximately 1×10 -9 Up to 5×10 -7 M, approximately 1×10 -8 Up to 1×10-7 M, approximately 1×10 -8 Up to 2×10 -7 M, approximately 1×10 -8 Up to 5×10 -7 M, or approximately 1×10 -8 Up to 1×10 -6 M of K D It binds to the cynomolgus monkey TRAILR2.

[0114] In some embodiments, the antigen-binding protein of this disclosure (e.g., an antibody, such as a single-domain antibody) may be smaller than about 3 × 10⁻⁶. -7 M of K D It binds to TRAILR2 in cynomolgus monkeys. In some embodiments, the antigen-binding protein of this disclosure (e.g., an antibody, such as a single-domain antibody) can be approximately 1 × 10⁻⁶. -9 Up to 1×10 -7 M of K D It binds to the cynomolgus monkey TRAILR2.

[0115] In some embodiments, the antigen-binding protein of this disclosure binds to mouse TRAILR2. In some embodiments, the antigen-binding protein of this disclosure may be less than about 1 × 10⁻⁶. -6 M, for example, less than about 5 × 10 -7 M, less than approximately 3 × 10 -7 M, less than approximately 1×10 -7 M, less than approximately 8 × 10 -8 M, less than approximately 5 × 10 -8 M, less than approximately 3 × 10 -8 M, less than approximately 1×10 -8 M, less than approximately 8 × 10 -9 M, less than approximately 5 × 10 -9 M, less than approximately 3 × 10 -9 M, or less than approximately 1×10 -9 M, or approximately 1×10 -10 Up to 1×10 -9 M, 1×10 -10 Up to 5×10 -9 M, approximately 1×10 -10 Up to 1×10 -8 M, approximately 1×10 -10 Up to 5×10 -8 M, approximately 1×10 -9 Up to 1×10 -8 M, approximately 1×10 -9 Up to 5×10 -8 M, approximately 1×10 -9 Up to 1×10-7 M, approximately 1×10 -9 Up to 2×10 -7 M, approximately 1×10 -9 Up to 5×10 -7 M, approximately 1×10 -8 Up to 1×10 -7 M, approximately 1×10 -8 Up to 2×10 -7 M, approximately 1×10 -8 Up to 5×10 -7 M, or approximately 1×10 -8 Up to 1×10 -6 M of K D It binds to mouse TRAILR2. In some embodiments, the antigen-binding protein of this disclosure does not bind to mouse TRAILR2.

[0116] In some embodiments, the antigen-binding protein of this disclosure binds to rat TRAILR2. In some embodiments, the antigen-binding protein of this disclosure may be less than about 1 × 10⁻⁶. -6 M, for example, less than about 5 × 10 -7 M, less than approximately 3 × 10 -7 M, less than approximately 1×10 -7 M, less than approximately 8 × 10 -8 M, less than approximately 5 × 10 -8 M, less than approximately 3 × 10 -8 M, less than approximately 1×10 -8 M, less than approximately 8 × 10 -9 M, less than approximately 5 × 10 -9 M, less than approximately 3 × 10 -9 M, or less than approximately 1×10 -9 M, or approximately 1×10 -10 Up to 1×10 -9 M, 1×10 -10 Up to 5×10 -9 M, approximately 1×10 -10 Up to 1×10 -8 M, approximately 1×10 -10 Up to 5×10 -8 M, approximately 1×10 -9 Up to 1×10 -8 M, approximately 1×10 -9 Up to 5×10 -8 M, approximately 1×10 -9 Up to 1×10 -7 M, approximately 1×10 -9 Up to 2×10 -7 M, approximately 1×10 -9Up to 5×10 -7 M, approximately 1×10 -8 Up to 1×10 -7 M, approximately 1×10 -8 Up to 2×10 -7 M, approximately 1×10 -8 Up to 5×10 -7 M, or approximately 1×10 -8 Up to 1×10 -6 M of K D It binds to rat TRAILR2. In some embodiments, the antigen-binding protein of this disclosure does not bind to rat TRAILR2.

[0117] In some implementations, the anti-TRAILR2 antigen-binding protein described herein does not block the binding of TNF-associated apoptosis-inducing ligand (TRAIL) to TRAILR2.

[0118] In some embodiments, the anti-TRAILR2 antigen-binding protein described herein blocks the binding of TNF-associated apoptosis-inducing ligand (TRAIL) to TRAILR2. In some embodiments, the anti-TRAILR2 antigen-binding protein described herein can block the binding of TNF-associated apoptosis-inducing ligand (TRAIL) to TRAILR2 by about 10% or more. In some embodiments, the binding of TNF-associated apoptosis-inducing ligand (TRAIL) to TRAILR2 can be blocked by the anti-TRAILR2 antigen-binding protein described herein by about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, more than 60%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, more than about 70%, about 70% to about 80%, about 70% to about 90%, more than about 80%, about 80% to about 90%, more than 90%, about 90% to about 95%, about 90% to about 98%, more than 95%, about 95% to about 98%, more than about 98%, or more than about 99%. The binding of TNF-associated apoptosis-inducing ligand (TRAIL) to TRAILR2 can be blocked by approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or even 100%. In some embodiments, the binding of TNF-associated apoptosis-inducing ligand (TRAIL) to TRAILR2 can be blocked by approximately 1%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, or more by the anti-TRAILR2 antigen-binding protein described herein.

[0119] In some embodiments, the anti-TRAILR2 antigen-binding protein of this disclosure can specifically bind to TRAILR2 without exhibiting specific binding to another receptor in the tumor necrosis factor receptor (TNFR) superfamily.

[0120] In some embodiments, the anti-TRAILR2 antigen-binding protein of this disclosure does not specifically bind to TRAILR1, TRAILR3, and / or TRAILR4. In some embodiments, the anti-TRAILR2 antigen-binding protein of this disclosure does not specifically bind to TRAILR1. In some embodiments, the anti-TRAILR2 antigen-binding protein of this disclosure does not specifically bind to TRAILR3. In some embodiments, the anti-TRAILR2 antigen-binding protein of this disclosure does not specifically bind to TRAILR4. In some embodiments, the anti-TRAILR2 antigen-binding protein of this disclosure does not specifically bind to TRAILR1, TRAILR3, and TRAILR4.

[0121] In some embodiments, the anti-TRAILR2 antigen-binding protein of this disclosure binds to the cysteine-rich domain (CRD) 1 (CRD1) and / or CRD2 of TRAILR2. In some embodiments, the anti-TRAILR2 antigen-binding protein of this disclosure binds to CRD1 of TRAILR2. In some embodiments, the anti-TRAILR2 antigen-binding protein of this disclosure binds to CRD2 of TRAILR2. In some embodiments, the anti-TRAILR2 antigen-binding protein of this disclosure binds to both CRD1 and CRD2 of TRAILR2.

[0122] In some embodiments, the anti-TRAILR2 antigen-binding protein of this disclosure binds to CRD2 and / or CRD3 of TRAILR2. In some embodiments, the anti-TRAILR2 antigen-binding protein of this disclosure binds to CRD2 of TRAILR2. In some embodiments, the anti-TRAILR2 antigen-binding protein of this disclosure binds to CRD3 of TRAILR2. In some embodiments, the anti-TRAILR2 antigen-binding protein of this disclosure binds to both CRD2 and CRD3 of TRAILR2.

[0123] The binding affinity of molecular interactions between two molecules can be measured using various techniques, such as surface plasmon resonance (SPR), biolayer interferometry (BLI), enzyme-linked immunosorbent assay (ELISA), equilibrium dialysis, fluorescence activated cell sorting (FACS), flow cytometry, or isothermal titration calorimetry (ITC). SPR is a biosensor technique that allows the analysis of real-time, biospecific interactions by detecting changes in protein concentration within a biosensor matrix, where one molecule is immobilized on a biosensor chip and the other molecule passes through the immobilized molecule under flow conditions (see, for example, Ober et al. 2001, Intern. Immunology 13: 1551-1559). SPR can be performed, for example, using a BIACORE® system or a Carterra LSA system. Another biosensor technique that can be used to determine the affinity of biomolecular interactions is biolayer interferometry (BLI) (see, for example, Abdiche et al. 2008, Anal. Biochem. 377: 209-217). Biolayer interferometry (BLI) is a label-free optical technique for analyzing the interference pattern of light reflected from two surfaces: an internal reference layer (reference beam) and a protein layer immobilized on the biosensor tip (signal beam). Changes in the number of molecules binding to the biosensor tip result in changes in the interference pattern, reported as wavelength changes (nm), a direct measure of the number of molecules bound to the biosensor tip surface. Because interactions can be measured in real time, binding and dissociation rates, as well as affinity, can be determined. BLI can be performed, for example, using an Octet® system. Alternatively, affinity can be measured in kinetic exclusion assays (KinExA) (see, for example, Drake et al. 2004, Anal. Biochem., 328:35-43), a solution-based method for measuring the true equilibrium binding affinity and kinetics of unmodified molecules. An equilibrium solution of an antibody / antigen complex is passed through a column containing beads pre-coated with antigen (or antibody), causing free antibody (or antigen) to bind to the coated molecule. The detection of the captured antibodies (or antigens) is accomplished by using fluorescently labeled proteins to bind to the antibodies (or antigens).

[0124] The antigen-binding proteins disclosed herein may include antibodies or antigen-binding fragments of antibodies, such as human antibodies, humanized antibodies; camel antibodies; chimeric antibodies; recombinant antibodies; heavy chain antibodies; single-domain antibodies (e.g., VHH); single-chain antibodies (e.g., single-chain variable fragment (scFv)); double-chain antibodies; triple-chain antibodies; quadruple-chain antibodies; Fab fragments; F(ab')2 fragments; IgD antibodies; IgE antibodies; IgM antibodies; IgG1 antibodies; IgG2 antibodies; IgG3 antibodies; or IgG4 antibodies, and fragments thereof.

[0125] In some embodiments, the antigen-binding protein that binds to TRAILR2 is a single-domain antibody (also referred to as "sdAb"). The single-domain antibodies of this disclosure can be derived from many sources, including but not limited to, VHH, VNAR, or VH domains (naturally occurring or engineered VH domains). VHH can be generated solely from camel heavy chain antibodies and their libraries (e.g., synthetic libraries). VNAR can be generated solely from cartilaginous fish heavy chain antibodies and their libraries (e.g., synthetic libraries). Various methods have been implemented to generate monomeric sdAbs from conventional heterodimeric VH and VK domains, including interface engineering and selection of specific strain families. In some embodiments, the sdAbs described in this invention are human or humanized.

[0126] In some embodiments, the single-domain antibody described herein is a VHH fragment (also known as a nanobody). The VHH fragment is also referred to herein as a "V-body". In some embodiments, the VHH is a camel VHH, a humanized VHH, or a camel-derived VH. In some embodiments, the single-domain antibody described herein is a VH domain. In some embodiments, the single-domain antibody described herein is a naturally occurring VH domain or an engineered VH domain.

[0127] The variable domains of the antigen-binding proteins (e.g., antibodies, such as single-domain antibodies) disclosed herein comprise at least three complementarity-determining regions (CDRs) that define their binding specificity. Preferably, in the variable domain, the CDRs are distributed between framework regions (FRs). The variable domain typically contains framework regions separated by 4 and 3 CDRs, resulting in the following typical antibody variable domain structures: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDRs and / or FRs of the single-domain antibodies of the present invention may be fragments or derivatives of naturally occurring antibody variable domains or may be synthetic.

[0128] The sequence identifiers corresponding to the exemplary anti-TRAILR2 VHH antibodies provided herein are listed in Table 1-1. Table 1-1 illustrates the sequence identifiers of the amino acid sequences of the complementarity-determining regions (CDR1, CDR2, and CDR3), the amino acid and DNA sequences of the full-length camelid VHH antibody, and the amino acid sequences of the corresponding humanized VHH antibody. Further exemplary anti-TRAILR2 VHH antibodies and their corresponding humanized VHH antibody amino acid sequences are provided in Table 1-2.

[0129] Table 1-1. Sequence identifiers of exemplary anti-TRAILR2 VHH antibodies

[0130] Table 1-2. Sequence identifiers of other exemplary VHH antibodies and humanized VHH antibodies

[0131] In some embodiments, the anti-TRAILR2 antigen-binding protein (e.g., an antibody, such as a single-domain antibody) described herein may further include a complementarity-determining region 1 (CDR1) comprising an amino acid sequence selected from the following (amino acids listed in parentheses represent possible amino acids at specific positions):

[0132] a).GRTFSSNL (SEQ ID NO: 1);

[0133] b). GGT(F / L)(A / S)N(D / N)G (SEQ ID NO: 113);

[0134] c).GRTL(D / N / S)(A / D / E)Y(A / G) (SEQ ID NO: 114);

[0135] d). GRTFS(N / S)YA (SEQ ID NO: 115);

[0136] e). GRDFSNYV (SEQ ID NO: 33);

[0137] f). G(L / R)(I / S)FS(D / S)YA (SEQ ID NO: 116);

[0138] g). GR(A / T)FSTLA (SEQ ID NO: 117);

[0139] h). GRTFSSDI (SEQ ID NO: 47);

[0140] i). GRSFGD(D / F / Y)A (SEQ ID NO: 118);

[0141] j). G(G / R)TLSNYA (SEQ ID NO: 119);

[0142] k). GRSFGAQGMEG (SEQ ID NO: 72);

[0143] l). GFTLDLGAYA (SEQ ID NO: 81);

[0144] m). GFTFGALA (SEQ ID NO: 86);

[0145] n). GFTLS(G / S)YA (SEQ ID NO: 120);

[0146] o). GSIFGGYN (SEQ ID NO: 2189);

[0147] p). G(G / S)NFRILS (SEQ ID NO: 2209); and

[0148] q). G(F / L)(A / T)F(R / S)(R / S)YA (SEQ ID NO: 2212).

[0149] In some embodiments, the anti-TRAILR2 antigen-binding protein of this disclosure (e.g., an antibody, such as a single-domain antibody) comprises a complementarity-determining region 1 (CDR1) containing an amino acid sequence selected from the following: GRTFSSNL (SEQ ID NO: 1); GGTLANNG (SEQ ID NO: 6); GRTLDAYG (SEQ ID NO: 11); GRTLSDYA (SEQ ID NO: 23); GRTFSSYA (SEQ ID NO: 28); GRDFSNYV (SEQ ID NO: 33); GRSFSSYA (SEQ ID NO: 38); GRTFSTLA (SEQ ID NO: 43); GRTFSSDI (SEQ ID NO: 47); GRSFGDFA (SEQ ID NO: 52); GGTLSNYA (SEQ ID NO: 62); GRTLSNYA (SEQ ID NO: 67); GRSFGAQGMEG (SEQ ID NO: 72); GFTLDLGAYA (SEQ ID NO: 52); GGTLSNYA (SEQ ID NO: 62); GRTLSNYA (SEQ ID NO: 67); GRSFGAQGMEG (SEQ ID NO: 72); GFTLDLGAYA (SEQ ID NO: 6 ... 81); GFTFGALA (SEQ ID NO: 86); GFTSGYA (SEQ ID NO: 95); GSIFGGYN (SEQ ID NO: 2189); GSNFRILS (SEQ ID NO: 2195); and GFTFSRYA (SEQ ID NO: 2201).

[0150] In some embodiments, the anti-TRAILR2 antigen-binding protein (e.g., an antibody, such as a single-domain antibody) described herein may further include a complementarity-determining region 2 (CDR2) comprising an amino acid sequence selected from the following (the amino acids listed in parentheses represent possible amino acids at specific positions):

[0151] a).VSWNGAST (SEQ ID NO: 2);

[0152] b). DHR(S / T)GT (SEQ ID NO: 121);

[0153] c). I(N / S)W(N / S / T)G(T / V)(D / G)T (SEQ ID NO: 122);

[0154] d). LNW(N / S)G(D / E)ST (SEQ ID NO: 123);

[0155] e). INWAD(E / T)T (SEQ ID NO: 124);

[0156] f). INWSGG(S / T)T (SEQ ID NO: 125);

[0157] g). ISWSDMSA (SEQ ID NO: 48);

[0158] h). I(N / R)W(A / D / T)G(D / N)T(SEQ ID NO: 126);

[0159] i). ISQ(S / T)S(D / S)T (SEQ ID NO: 127);

[0160] j). (I / M)KWTGNT (SEQ ID NO: 128);

[0161] k). ISN(S / T)GTTT (SEQ ID NO: 129);

[0162] l). ISNDGEHI (SEQ ID NO: 87);

[0163] m). ISWNGDIT (SEQ ID NO: 91);

[0164] n). IT(G / S)(A / S)G(G / S)(N / S)T (SEQ ID NO: 130);

[0165] o). IFISGN(D / N) (SEQ ID NO: 2207);

[0166] p). (I / L)T(K / M / S)D(D / G)TT (SEQ ID NO: 2210); and

[0167] q). ISS(A / G / S)(G / S)G(I / Y)(I / T / V) (SEQ ID NO: 2213).

[0168] In some embodiments, the anti-TRAILR2 antigen-binding protein of this disclosure (e.g., an antibody, such as a single-domain antibody) comprises a complementarity-determining region 2 (CDR2) containing an amino acid sequence selected from the following: VSWNGAST (SEQ ID NO: 2); DHRSGT (SEQ ID NO: 7); ISWTGVDT (SEQ ID NO: 12); ISWTGTDT (SEQ ID NO: 19); IWSGVDT (SEQ ID NO: 24); LNWSGEST (SEQ ID NO: 29); INWADET (SEQ ID NO: 34); INWSGGST (SEQ ID NO: 39); ISWSDMSA (SEQ ID NO: 48); IRWTGDT (SEQ ID NO: 53); ISQTSST (SEQ ID NO: 63); ISQSSDT (SEQ ID NO: 68); MKWTGNT (SEQ ID NO: 73); IKWTGNT (SEQ ID NO: 77); ISNTGTTT (SEQ ID NO: 78). ID NO: 82); ISNDGEHI (SEQ ID NO: 87); ISWNGDIT (SEQ ID NO: 91); ITSAGGST (SEQ ID NO: 96); IFISGNN (SEQ ID NO: 2190); ITSDDTT (SEQ ID NO: 2196); and ISSAGGYI (SEQ ID NO: 2202).

[0169] In some embodiments, the anti-TRAILR2 antigen-binding protein of this disclosure (e.g., an antibody, such as a single-domain antibody) includes a complementarity-determining region 3 (CDR3) comprising an amino acid sequence selected from the following (the amino acids listed in parentheses represent possible amino acids at specific positions):

[0170] a). (A / V)ASRL(P / V)FNSRSA(I / V)YTDRIYDS (SEQ ID NO: 100);

[0171] b). AVRRSAWY(S / T)DSIYTVSQYDY (SEQ ID NO: 101);

[0172] c).NAARSYSR(D / G / N)(G / Y)(E / R)PL(E / K)P(A / D)Y (SEQ ID NO: 102);

[0173] d). AAASSWSRGG(A / G / I / V)PYGMDY (SEQ ID NO: 103);

[0174] e). AADS(H / R)FRR(P / Y)(A / T / V)PG(I / Q)QYEY (SEQ ID NO: 104);

[0175] f). NAA(K / R)SYHRDY(K / S)PL(K / S)(G / P)DY (SEQ ID NO: 105);

[0176] g). AAAPSFGM(M / R / T)(I / N)PESYVHS (SEQ ID NO: 106);

[0177] h). AANRGIMSMRLSRYDD (SEQ ID NO: 49);

[0178] i). T(A / V)GP(A / T)MSYSRGGEF (SEQ ID NO: 107);

[0179] j). (A / V)ADRGAISRSGAGM(D / N)Y (SEQ ID NO: 108);

[0180] k). TAGP(A / S)IS(L / Y)SRGGEY (SEQ ID NO: 109);

[0181] l). A(A / T)NGWGLDP(S / T)TYH(Y / D) (SEQ ID NO: 110);

[0182] m). SAGWTRRIFQY (SEQ ID NO: 88);

[0183] n). TAGQSISLSQGGE(H / Y) (SEQ ID NO: 111);

[0184] o). KAGIRGE(T / V)Y (SEQ ID NO: 112);

[0185] p). RAYNDGGEY (SEQ ID NO: 2191);

[0186] q). HS(N / R)WYNL (SEQ ID NO: 2208); and

[0187] r). (F / M / N)T(A / S)DY,

[0188] One or more non-alanine residues in the CDR3 sequence are optionally replaced with alanine, and / or one or more alanine residues in the CDR3 sequence are optionally replaced with glycine.

[0189] In some embodiments, one non-alanine residue in the CDR3 sequence is replaced with an alanine residue. In some embodiments, two non-alanine residues in the CDR3 sequence are replaced with an alanine residue. In some embodiments, three non-alanine residues in the CDR3 sequence are replaced with an alanine residue. In some embodiments, four non-alanine residues in the CDR3 sequence are replaced with an alanine residue. In some embodiments, five or more non-alanine residues in the CDR3 sequence are replaced with an alanine residue.

[0190] In some embodiments, one alanine residue in the CDR3 sequence is substituted with glycine. In some embodiments, two alanine residues in the CDR3 sequence are substituted with glycine. In some embodiments, three alanine residues in the CDR3 sequence are substituted with glycine. In some embodiments, four alanine residues in the CDR3 sequence are substituted with glycine. In some embodiments, five or more alanine residues in the CDR3 sequence are substituted with glycine.

[0191] In some embodiments, any of the aforementioned non-alanine residues in the CDR3 sequence may be combined with alanine substitutions with glycine substitutions of any of the aforementioned alanine residues in the CDR3 sequence.

[0192] In some implementations, CDR3 comprises an amino acid sequence selected from the following:

[0193] s). (A / G)(A / G)(A / G)(A / S)(A / S)(A / W)(A / S)(A / R)(A / G)(A / G)(A / G / I / V)(A / P)(A / Y)(A / G)(A / M)(A / D)(A / Y);

[0194] t). (A / T)(A / G / V)(A / G)(A / P)(A / T)(A / M)(A / S)(A / Y)(A / S)(A / R)(A / G)(A / G)(A / E) (A / F);

[0195] u). (A / R)(A / G)(A / Y)(A / N)(A / D)(A / G)(A / G)(A / E)(A / Y);

[0196] v). (A / H)(A / S)(A / N / R)(A / W)(A / Y)(A / N)(A / L); and

[0197] w). (A / F / M / N)(A / T)(A / G / S)(A / D)Y。

[0198] In some embodiments, the anti-TRAILR2 antigen-binding protein of this disclosure (e.g., an antibody, such as a single-domain antibody) includes a complementarity-determining region 3 (CDR3) comprising an amino acid sequence selected from the following: VASRLPFNSRSAIYTDRIYDS (SEQ ID NO: 3); AVRRSAWYSDSIYTVSQYDY (SEQ ID NO: 8); NAARSYSRDYEPLKPDY (SEQ ID NO: 13); NAARSYSRNYEPLKPDY (SEQ ID NO: 16); NAARSYSRGGEPLKPDY (SEQ ID NO: 20); NAARSYSRGGRPLEPAY (SEQ ID NO: 25); AAASSWSRGGVPYGMDY (SEQ ID NO: 30); AADSHFRRYTPGQQYEY (SEQ ID NO: 35); NAAKSYHRDYSPLSPDY (SEQ ID NO: 40); AAAPSFGMRNPESYVHS (SEQ ID NO: 30); NAARSYSRDYSPLSPDY (SEQ ID NO: 40); NAARSYSRDYSPLSPDY (SEQ ID NO: 3 ... 44); AANRGIMSMRLSRYDD (SEQ ID NO: 49); TAGPTMSYSRGGEF (SEQ ID NO: 54); VADRGAISRSGAGMDY (SEQ ID NO: 64); AADRGAISRSGAGMDY (SEQ ID NO: 69); TAGPAISLSRGGEY (SEQ ID NO: 74); TAGPSISYSRGGEY (SEQ ID NO: 78); AANGWGLLDPTTYHY (SEQ ID NO: 83); SAGWTRRIFQY (SEQ ID NO: 88); TAGQSISLSQGGEY (SEQ ID NO: 92); KAGIRGEVY (SEQ ID NO: 97); RAYNDGGEY (SEQ ID NO: 2191); HSRWYNL (SEQ ID NO: 2197); FTADY (SEQ ID NO: 2203); GAASSWSRGGVPYGMDY (SEQ ID NO: 2298); AGASSWSRGGVPYGMDY (SEQ ID NO: 2299); AAGSSWSRGGVPYGMDY (SEQ ID NO: 2300); AAAASWSRGGVPYGMDY (SEQ ID NO: 2301); AAASAWSRGGVPYGMDY (SEQ ID NO: 2302);AAASSASRGGVPYGMDY (SEQ ID NO: 2303);AAASSWARGGVPYGMDY (SEQ ID NO: 2304);AAASSWSAGGVPYGMDY (SEQ ID NO: 2305);AAASSWSRAGVPYGMDY (SEQ ID NO: 2306) (SEQ ID NO: 2307);AAASSWSRGGAPYGMDY (SEQ ID NO: 2308);AAASSWSRGGVAYGMDY (SEQ ID NO: 2309);AAASSWSRGGVPAGMDY (SEQ ID NO: 2310); 2311);AAASSWSRGGVPYGADY (SEQ ID NO: 2312);AAASSWSRGGVPYGMAY (SEQ ID NO: 2313);AAASSWSRGGVPYGMDA (SEQ ID NO: 2314);AAGPTMSYSRGGEF (SEQ ID NO: NO: NO 2315);TGGPTMSYSRGGEF (SEQ ID NO: 2316);TAAPTMSYSRGGEF (SEQ ID NO: 2317);TAGATMSYSRGGEF (SEQ ID NO: 2318);TAGPAMSYSRGGEF (SEQ ID NO: 2319) ID NO: 2320);TAGPTMAYSRGGEF (SEQ ID NO: 2321);TAGPTMSASRGGEF (SEQ ID NO: 2322);TAGPTMSYARGGEF (SEQ ID NO: 2323);TAGPTMSYSAGGEF (SEQ ID NO: 2324) NO: 2325);TAGPTMSYSRGAEF (SEQ ID NO: 2326);TAGPTMSYSRGGAF (SEQ ID NO: 2327);TAGPTMSYSRGGEA (SEQ ID NO: 2328);AAYNDGGEY(SEQ ID NO: 2329) 2330);RAANDGGEY (SEQ ID NO: 2331);RAYADGGEY (SEQ ID NO: 2332); RAYNAGGEY (SEQ ID NO: 2333); RAYNDAGEY (SEQ ID NO: 2334); RAYNDGAEY (SEQ ID NO: 2335); RAYNDGGAY (SEQ ID NO: 2336); RAYNDGGEA (SEQ ID NO: 2337); ASRWYNL (SEQ ID NO: 2338); HARWYNL (SEQ ID NO: 2339); HSAWYNL (SEQ ID NO: 2340); HSRAYNL (SEQ ID NO: 2341); (SEQ ID NO: 2345); FAADY (SEQ ID NO: 2346); FTGDY (SEQ ID NO: 2347); and FTAAY (SEQ ID NO: 2348). ;

[0199] In some embodiments, the anti-TRAILR2 antigen-binding protein (e.g., an antibody, such as a single-domain antibody) disclosed herein comprises:

[0200] i) CDR1 containing the amino acid sequence GRTFSSNL (SEQ ID NO: 1), CDR2 containing the amino acid sequence VSWNGAST (SEQ ID NO: 2), and CDR3 containing the amino acid sequence (A / V)ASRL(P / V)FNSRSA(I / V)YTDRIYDS (SEQ ID NO: 100);

[0201] ii) CDR1 containing the amino acid sequence GGT(F / L)(A / S)N(D / N)G (SEQ ID NO: 113), CDR2 containing the amino acid sequence DHR(S / T)GT (SEQ ID NO: 121), and CDR3 containing the amino acid sequence AVRRSAWY(S / T)DSIYTVSQYDY (SEQ ID NO: 101);

[0202] iii) CDR1 containing the amino acid sequence GRTL(D / N / S)(A / D / E)Y(A / G) (SEQ ID NO: 114), CDR2 containing the amino acid sequence I(N / S)W(N / S / T)G(T / V)(D / G)T (SEQ ID NO: 122), and CDR3 containing the amino acid sequence NAARSYSR(D / G / N)(G / Y)(E / R)PL(E / K)P(A / D)Y (SEQ ID NO: 102);

[0203] iv). CDR1 containing the amino acid sequence GRTFS(N / S)YA (SEQ ID NO: 115), CDR2 containing the amino acid sequence LNW(N / S)G(D / E)ST (SEQ ID NO: 123), and CDR3 containing the amino acid sequence AAASSWSRGG(A / G / I / V)PYGMDY (SEQ ID NO: 103);

[0204] v). CDR1 containing the amino acid sequence GRDFSNYV (SEQ ID NO: 33), CDR2 containing the amino acid sequence INWAD(E / T)T (SEQ ID NO: 124), and CDR3 containing the amino acid sequence AADS(H / R)FRR(P / Y)(A / T / V)PG(I / Q)QYEY (SEQ ID NO: 104);

[0205] vi). CDR1 containing the amino acid sequence G(L / R)(I / S)FS(D / S)YA (SEQ ID NO: 116), CDR2 containing the amino acid sequence INNWSGG(S / T)T (SEQ ID NO: 125), and CDR3 containing the amino acid sequence NAA(K / R)SYHRDY(K / S)PL(K / S)(G / P)DY (SEQ ID NO: 105);

[0206] vii). CDR1 containing the amino acid sequence GR(A / T)FSTLA (SEQ ID NO: 117), CDR2 containing the amino acid sequence INNSGG(S / T)T (SEQ ID NO: 125), and CDR3 containing the amino acid sequence AAAPSFGM(M / R / T)(I / N)PESYVHS (SEQ ID NO: 106);

[0207] viii). CDR1 containing the amino acid sequence GRTFSSDI (SEQ ID NO: 47), CDR2 containing the amino acid sequence ISWSDMSA (SEQ ID NO: 48), and CDR3 containing the amino acid sequence AANRGIMSMRLSRYDD (SEQ ID NO: 49);

[0208] ix). CDR1 containing the amino acid sequence GRSFGD(D / F / Y)A (SEQ ID NO: 118), CDR2 containing the amino acid sequence I(N / R)W(A / D / T)G(D / N)T (SEQ ID NO: 126), and CDR3 containing the amino acid sequence T(A / V)GP(A / T)MSYSRGGEF (SEQ ID NO: 107);

[0209] x). CDR1 containing the amino acid sequence G(G / R)TLSNYA (SEQ ID NO: 119), CDR2 containing the amino acid sequence ISQ(S / T)S(D / S)T (SEQ ID NO: 127), and CDR3 containing the amino acid sequence (A / V)ADRGAISRSGAGM(D / N)Y (SEQ ID NO: 108);

[0210] xi). CDR1 containing the amino acid sequence GRSFGAQGMEG (SEQ ID NO: 72), CDR2 containing the amino acid sequence (I / M)KWTGNT (SEQ ID NO: 128), and CDR3 containing the amino acid sequence TAGP(A / S)IS(L / Y)SRGGEY (SEQ ID NO: 109);

[0211] xii). CDR1 containing the amino acid sequence GFTLDLGAYA (SEQ ID NO: 81), CDR2 containing the amino acid sequence ISN(S / T)GTTT (SEQ ID NO: 129), and CDR3 containing the amino acid sequence A(A / T)NGWGLDP(S / T)TYH(Y / D) (SEQ ID NO: 110);

[0212] xiii). CDR1 containing the amino acid sequence GFTFGALA (SEQ ID NO: 86), CDR2 containing the amino acid sequence ISNDGEHI (SEQ ID NO: 87), and CDR3 containing the amino acid sequence SAGWTRRIFQY (SEQ ID NO: 88);

[0213] xiv). CDR1 containing the amino acid sequence GRTFS(N / S)YA (SEQ ID NO: 115), CDR2 containing the amino acid sequence ISWNGDIT (SEQ ID NO: 91), and CDR3 containing the amino acid sequence TAGQSISLSQGGE(H / Y) (SEQ ID NO: 111);

[0214] xv). CDR1 containing the amino acid sequence GFTLS(G / S)YA (SEQ ID NO: 120), CDR2 containing the amino acid sequence IT(G / S)(A / S)G(G / S)(N / S)T (SEQ ID NO: 130), and CDR3 containing the amino acid sequence KAGIRGE(T / V)Y (SEQ ID NO: 112);

[0215] xvi). CDR1 containing the amino acid sequence GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence IFISN(D / N) (SEQ ID NO: 2207), and CDR3 containing the amino acid sequence RAYNDGGEY (SEQ ID NO: 2191);

[0216] xvii). CDR1 containing the amino acid sequence G(G / S)NFRILS (SEQ ID NO: 2209), CDR2 containing the amino acid sequence (I / L)T(K / M / S)D(D / G)TT (SEQ ID NO: 2210), and CDR3 containing the amino acid sequence HS(N / R)WYNL (SEQ ID NO: 2208);

[0217] xviii). CDR1 containing the amino acid sequence G(F / L)(A / T)F(R / S)(R / S)YA (SEQ ID NO: 2212), CDR2 containing the amino acid sequence ISS(A / G / S)(G / S)G(I / Y)(I / T / V) (SEQ ID NO: 2213), and CDR3 containing the amino acid sequence (F / M / N)T(A / S)DY;

[0218] xix). CDR1 containing the amino acid sequence GRTFS(N / S)YA (SEQ ID NO: 115), CDR2 containing the amino acid sequence LNW(N / S)G(D / E)ST (SEQ ID NO: 123), and CDR3 containing the amino acid sequence (A / G)(A / G)(A / G)(A / S)(A / S)(A / W)(A / S)(A / R)(A / G)(A / G)(A / G)(A / G / I / V)(A / P)(A / Y)(A / G)(A / M)(A / D)(A / Y);

[0219] xx). CDR1 containing the amino acid sequence GRSFGD(D / F / Y)A (SEQ ID NO: 118), CDR2 containing the amino acid sequence I(N / R)W(A / D / T)G(D / N)T (SEQ ID NO: 126), and CDR3 containing the amino acid sequence (A / T)(A / G / V)(A / G)(A / P)(A / T)(A / M)(A / S)(A / Y)(A / S)(A / R)(A / G)(A / G)(A / E)(A / F);

[0220] xxi). CDR1 containing the amino acid sequence GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence IFISN(D / N) (SEQ ID NO: 2207), and CDR3 containing the amino acid sequence (A / R)(A / G)(A / Y)(A / N)(A / D)(A / G)(A / G)(A / E)(A / Y);

[0221] xxii). CDR1 containing the amino acid sequence G(G / S)NFRILS (SEQ ID NO: 2209), CDR2 containing the amino acid sequence (I / L)T(K / M / S)D(D / G)TT (SEQ ID NO: 2210), and CDR3 containing the amino acid sequence (A / H)(A / S)(A / N / R)(A / W)(A / Y)(A / N)(A / L); or

[0222] xxiii). CDR1 containing the amino acid sequence G(F / L)(A / T)F(R / S)(R / S)YA (SEQ ID NO: 2212), CDR2 containing the amino acid sequence ISS(A / G / S)(G / S)G(I / Y)(I / T / V) (SEQ ID NO: 2213), and CDR3 containing the amino acid sequence (A / F / M / N)(A / T)(A / G / S)(A / D)Y.

[0223] This article provides an anti-TRAILR2 antigen-binding protein (e.g., an antibody, such as a single-domain antibody) comprising CDR1 (CDR1), wherein the CDR1 comprises an amino acid sequence selected from any of the CDR1 amino acid sequences listed in Tables 1-1 or 6, or a similar sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with it.

[0224] In some embodiments, the anti-TRAILR2 antigen-binding protein (e.g., an antibody, such as a single-domain antibody) comprises CDR1, which comprises an amino acid sequence selected from SEQ ID NO: 1, 6, 11, 23, 28, 33, 38, 43, 47, 52, 62, 67, 72, 81, 86, 95, 762 to 1084, 2189, 2195, 2201 and 2252 to 2261 or a similar sequence having at least 70%, at least 80%, at least 90% or at least 95% sequence identity with it.

[0225] This article provides an anti-TRAILR2 antigen-binding protein (e.g., an antibody, such as a single-domain antibody) comprising CDR2 (CDR2), said CDR2 comprising an amino acid sequence selected from any of the CDR2 amino acid sequences listed in Tables 1-1 or 6, or a similar sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with it.

[0226] In some embodiments, the anti-TRAILR2 antigen-binding protein (e.g., an antibody, such as a single-domain antibody) comprises CDR2, said CDR2 comprising an amino acid sequence selected from SEQ ID NO: 2, 7, 12, 19, 24, 29, 34, 39, 48, 53, 63, 68, 73, 77, 82, 87, 91, 96, 1085 to 1407, 2190, 2196, 2202 and 2262 to 2271 or a similar sequence having at least 70%, at least 80%, at least 90% or at least 95% sequence identity with it.

[0227] This article provides an anti-TRAILR2 antigen-binding protein (e.g., an antibody, such as a single-domain antibody) comprising CDR3 (CDR3), said CDR3 comprising an amino acid sequence selected from any of the CDR3 amino acid sequences listed in Tables 1-1 or 6, or a similar sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with it.

[0228] In some embodiments, the anti-TRAILR2 antigen-binding protein (e.g., an antibody, such as a single-domain antibody) comprises CDR3, which comprises an amino acid sequence selected from SEQ ID NO: 3, 8, 13, 16, 20, 25, 30, 35, 40, 44, 49, 54, 64, 69, 74, 78, 83, 88, 92, 97, 1408 to 1730, 2191, 2197, 2203, 2272 to 2277, and 2298 to 2348, or a similar sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with it.

[0229] This document provides an anti-TRAILR2 antigen-binding protein (e.g., an antibody, such as a single-domain antibody) comprising a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within any of the exemplary anti-TRAILR2 VHH antibodies listed in Tables 1-1, 1-2, or 6. In some embodiments, the anti-TRAILR2 antigen-binding protein (e.g., an antibody, such as a single-domain antibody) of this disclosure comprises:

[0230] i) CDR1 containing the amino acid sequence GRTFSSNL (SEQ ID NO: 1), CDR2 containing the amino acid sequence VSWNGAST (SEQ ID NO: 2), and CDR3 containing the amino acid sequence VASRPPFNSRSAIYTDRIYDS (SEQ ID NO: 3);

[0231] ii) CDR1 containing the amino acid sequence GGTLANNG (SEQ ID NO: 6), CDR2 containing the amino acid sequence DHRSGT (SEQ ID NO: 7), and CDR3 containing the amino acid sequence AVRRSAWYSDSIYTVSQYDY (SEQ ID NO: 8);

[0232] iii) CDR1 containing the amino acid sequence GRTLDAYG (SEQ ID NO: 11), CDR2 containing the amino acid sequence ISWTGVDT (SEQ ID NO: 12), and CDR3 containing the amino acid sequence NAARSYSRDYEPLKPDY (SEQ ID NO: 13);

[0233] iv) CDR1 containing the amino acid sequence GRTLDAYG (SEQ ID NO: 11), CDR2 containing the amino acid sequence ISWTGVDT (SEQ ID NO: 12), and CDR3 containing the amino acid sequence NAARSYSRNYEPLKPDY (SEQ ID NO: 16);

[0234] v) CDR1 containing the amino acid sequence GRTLDAYG (SEQ ID NO: 11), CDR2 containing the amino acid sequence ISWTGTDT (SEQ ID NO: 19), and CDR3 containing the amino acid sequence NAARSYSRGGEPLKPDY (SEQ ID NO: 20);

[0235] vi) CDR1 containing the amino acid sequence GRTLSDYA (SEQ ID NO: 23), CDR2 containing the amino acid sequence ISWSGVDT (SEQ ID NO: 24), and CDR3 containing the amino acid sequence NAARSYSRGGRPLEPAY (SEQ ID NO: 25);

[0236] vii) CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSRGGVPYGMDY (SEQ ID NO: 30);

[0237] viii) CDR1 containing the amino acid sequence GRDFSNYV (SEQ ID NO: 33), CDR2 containing the amino acid sequence INWADET (SEQ ID NO: 34), and CDR3 containing the amino acid sequence AADSHFRRYTPGQQYEY (SEQ ID NO: 35);

[0238] ix) CDR1 containing the amino acid sequence GRSFSSYA (SEQ ID NO: 38), CDR2 containing the amino acid sequence INNWSGGST (SEQ ID NO: 39), and CDR3 containing the amino acid sequence NAAKSYHRDYSPLSPDY (SEQ ID NO: 40);

[0239] x) CDR1 containing the amino acid sequence GRTFSTLA (SEQ ID NO: 43), CDR2 containing the amino acid sequence INNWSGGST (SEQ ID NO: 39), and CDR3 containing the amino acid sequence AAAPSFGMRNPESYVHS (SEQ ID NO: 44);

[0240] xi) CDR1 containing the amino acid sequence GRTFSSDI (SEQ ID NO: 47), CDR2 containing the amino acid sequence ISWSDMSA (SEQ ID NO: 48), and CDR3 containing the amino acid sequence AANRGIMSMRLSRYDD (SEQ ID NO: 49);

[0241] xii) CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPTMSYSRGGEF (SEQ ID NO: 54);

[0242] xiii) CDR1 containing the amino acid sequence GGTLSNYA (SEQ ID NO: 62), CDR2 containing the amino acid sequence ISQTSST (SEQ ID NO: 63), and CDR3 containing the amino acid sequence VADRGAISRSGAGMDY (SEQ ID NO: 64);

[0243] xiv) CDR1 containing the amino acid sequence GRTLSNYA (SEQ ID NO: 67), CDR2 containing the amino acid sequence ISQSSDT (SEQ ID NO: 68), and CDR3 containing the amino acid sequence AADRGAISRSGAGMDY (SEQ ID NO: 69);

[0244] xv) CDR1 containing the amino acid sequence GRSFGAQGMEG (SEQ ID NO: 72), CDR2 containing the amino acid sequence MKWTGNT (SEQ ID NO: 73), and CDR3 containing the amino acid sequence TAGPAISLSRGGEY (SEQ ID NO: 74);

[0245] xvi) CDR1 containing the amino acid sequence GRSFGAQGMEG (SEQ ID NO: 72), CDR2 containing the amino acid sequence IKWTGNT (SEQ ID NO: 77), and CDR3 containing the amino acid sequence TAGPSISYSRGGEY (SEQ ID NO: 78);

[0246] xvii) CDR1 containing the amino acid sequence GFTLDLGAYA (SEQ ID NO: 81), CDR2 containing the amino acid sequence ISNTGTTT (SEQ ID NO: 82), and CDR3 containing the amino acid sequence AANGWGLDPTTYHY (SEQ ID NO: 83);

[0247] xviii) CDR1 containing the amino acid sequence GFTFGALA (SEQ ID NO: 86), CDR2 containing the amino acid sequence ISNDGEHI (SEQ ID NO: 87), and CDR3 containing the amino acid sequence SAGWTRRIFQY (SEQ ID NO: 88);

[0248] xix) CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence ISWNGDIT (SEQ ID NO: 91), and CDR3 containing the amino acid sequence TAGQSISLSQGGEY (SEQ ID NO: 92);

[0249] CDR1 containing the amino acid sequence GFTLSGYA (SEQ ID NO: 95), CDR2 containing the amino acid sequence ITSAGGST (SEQ ID NO: 96), and CDR3 containing the amino acid sequence KAGIRGEVY (SEQ ID NO: 97);

[0250] xxi). CDR1 containing the amino acid sequence GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence RAYNDGGEY (SEQ ID NO: 2191);

[0251] xxii). CDR1 containing the amino acid sequence GSNFRILS (SEQ ID NO: 2195), CDR2 containing the amino acid sequence ITSDDTT (SEQ ID NO: 2196), and CDR3 containing the amino acid sequence HSRWYNL (SEQ ID NO: 2197);

[0252] xxiii). CDR1 containing the amino acid sequence GFTFSRYA (SEQ ID NO: 2201), CDR2 containing the amino acid sequence ISSAGGYI (SEQ ID NO: 2202), and CDR3 containing the amino acid sequence FTADY (SEQ ID NO: 2203);

[0253] xxiv). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence GAASSWSRGGVPYGMDY (SEQ ID NO: 2298);

[0254] xxv). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AGASSWSRGGVPYGMDY (SEQ ID NO: 2299);

[0255] xxvi). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAGSSWSRGGVPYGMDY (SEQ ID NO: 2300);

[0256] xxvii). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAAASWSRGGVPYGMDY (SEQ ID NO: 2301);

[0257] xxviii). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASAWSRGGVPYGMDY (SEQ ID NO: 2302);

[0258] xxix). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSASRGGVPYGMDY (SEQ ID NO: 2303);

[0259] xxx). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWARGGVPYGMDY (SEQ ID NO: 2304);

[0260] xxxi). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSAGGVPYGMDY (SEQ ID NO: 2305);

[0261] xxxii). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSRAGVPYGMDY (SEQ ID NO: 2306);

[0262] xxxiii). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSRGAVPYGMDY (SEQ ID NO: 2307);

[0263] xxxiv). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSRGGAPYGMDY (SEQ ID NO: 2308);

[0264] xxxv). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSRGGVAYGMDY (SEQ ID NO: 2309);

[0265] xxxvi). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSRGGVPAGMDY (SEQ ID NO: 2310);

[0266] xxxvii). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSRGGVPYAMDY (SEQ ID NO: 2311);

[0267] xxxviii). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSRGGVPYGADY (SEQ ID NO: 2312);

[0268] xxxix). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSRGGVPYGMAY (SEQ ID NO: 2313);

[0269] xl). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSRGGVPYGMDA (SEQ ID NO: 2314);

[0270] xli). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence AAGPTMSYSRGGEF (SEQ ID NO: 2315);

[0271] xlii). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TGGPTMSYSRGGEF (SEQ ID NO: 2316);

[0272] xliii). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAAPTMSYSRGGEF (SEQ ID NO: 2317);

[0273] xliv). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGATMSYSRGGEF (SEQ ID NO: 2318);

[0274] xlv). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPAMSYSRGGEF (SEQ ID NO: 2319);

[0275] xlvi). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPTASYSRGGEF (SEQ ID NO: 2320);

[0276] xlvii). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPTMAYSRGGEF (SEQ ID NO: 2321);

[0277] xlviii). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPTMSASRGGEF (SEQ ID NO: 2322);

[0278] xlix). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPTMSYARGGEF (SEQ ID NO: 2323);

[0279] l) CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPTMSYSAGGEF (SEQ ID NO: 2324);

[0280] li). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPTMSYSRAGEF (SEQ ID NO: 2325);

[0281] lii). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPTMSYSRGAEF (SEQ ID NO: 2326);

[0282] liii). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPTMSYSRGGAF (SEQ ID NO: 2327);

[0283] CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPTMSYSRGGEA (SEQ ID NO: 2328);

[0284] lv). CDR1 containing the amino acid sequence GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence AAYNDGGEY (SEQ ID NO: 2329);

[0285] lvi). CDR1 containing the amino acid sequence GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence RGYNDGGEY (SEQ ID NO: 2330);

[0286] lvii). CDR1 containing the amino acid sequence GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence RAANDGGEY (SEQ ID NO: 2331);

[0287] lviii). CDR1 containing the amino acid sequence GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence RAYADGGEY (SEQ ID NO: 2332);

[0288] CDR1 containing the amino acid sequence GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence RAYNAGGEY (SEQ ID NO: 2333);

[0289] lx). CDR1 containing the amino acid sequence GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence RAYNDAGEY (SEQ ID NO: 2334);

[0290] lxi). CDR1 containing the amino acid sequence GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence RAYNDGAEY (SEQ ID NO: 2335);

[0291] lxii). CDR1 containing the amino acid sequence GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence RAYNDGGAY (SEQ ID NO: 2336);

[0292] lxiii). CDR1 containing the amino acid sequence GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence RAYNDGGEA (SEQ ID NO: 2337);

[0293] (lxiv). CDR1 containing the amino acid sequence GSNFRILS (SEQ ID NO: 2195), CDR2 containing the amino acid sequence ITSDDTT (SEQ ID NO: 2196), and CDR3 containing the amino acid sequence ASRWYNL (SEQ ID NO: 2338);

[0294] lxv). CDR1 containing the amino acid sequence GSNFRILS (SEQ ID NO: 2195), CDR2 containing the amino acid sequence ITSDDTT (SEQ ID NO: 2196), and CDR3 containing the amino acid sequence HARWYNL (SEQ ID NO: 2339);

[0295] lxvi). CDR1 containing the amino acid sequence GSNFRILS (SEQ ID NO: 2195), CDR2 containing the amino acid sequence ITSDDTT (SEQ ID NO: 2196), and CDR3 containing the amino acid sequence HSAWYNL (SEQ ID NO: 2340);

[0296] lxvii). CDR1 containing the amino acid sequence GSNFRILS (SEQ ID NO: 2195), CDR2 containing the amino acid sequence ITSDDTT (SEQ ID NO: 2196), and CDR3 containing the amino acid sequence HSRAYNL (SEQ ID NO: 2341);

[0297] lxviii). CDR1 containing the amino acid sequence GSNFRILS (SEQ ID NO: 2195), CDR2 containing the amino acid sequence ITSDDTT (SEQ ID NO: 2196), and CDR3 containing the amino acid sequence HSRWANL (SEQ ID NO: 2342);

[0298] lxix). CDR1 containing the amino acid sequence GSNFRILS (SEQ ID NO: 2195), CDR2 containing the amino acid sequence ITSDDTT (SEQ ID NO: 2196), and CDR3 containing the amino acid sequence HSRWYAL (SEQ ID NO: 2343);

[0299] lxx). CDR1 containing the amino acid sequence GSNFRILS (SEQ ID NO: 2195), CDR2 containing the amino acid sequence ITSDDTT (SEQ ID NO: 2196), and CDR3 containing the amino acid sequence HSRWYNA (SEQ ID NO: 2344);

[0300] lxxi). CDR1 containing the amino acid sequence GFTFSRYA (SEQ ID NO: 2201), CDR2 containing the amino acid sequence ISSAGGYI (SEQ ID NO: 2202), and CDR3 containing the amino acid sequence ATADY (SEQ ID NO: 2345);

[0301] lxxii). CDR1 containing the amino acid sequence GFTFSRYA (SEQ ID NO: 2201), CDR2 containing the amino acid sequence ISSAGGYI (SEQ ID NO: 2202), and CDR3 containing the amino acid sequence FAADY (SEQ ID NO: 2346);

[0302] lxxiii). CDR1 containing the amino acid sequence GFTFSRYA (SEQ ID NO: 2201), CDR2 containing the amino acid sequence ISSAGGYI (SEQ ID NO: 2202), and CDR3 containing the amino acid sequence FTGDY (SEQ ID NO: 2347); or

[0303] lxxiv). CDR1 containing the amino acid sequence GFTFSRYA (SEQ ID NO: 2201), CDR2 containing the amino acid sequence ISSAGGYI (SEQ ID NO: 2202), and CDR3 containing the amino acid sequence FTAAY (SEQ ID NO: 2348).

[0304] In some embodiments, the anti-TRAILR2 antigen-binding protein (e.g., an antibody, such as a single-domain antibody) of this disclosure comprises:

[0305] i) CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSRGGVPYGMDY (SEQ ID NO: 30);

[0306] ii) CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPTMSYSRGGEF (SEQ ID NO: 54);

[0307] iii) CDR1 containing the amino acid sequence GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence RAYNDGGEY (SEQ ID NO: 2191);

[0308] iv) CDR1 containing the amino acid sequence GSNFRILS (SEQ ID NO: 2195), CDR2 containing the amino acid sequence ITSDDTT (SEQ ID NO: 2196), and CDR3 containing the amino acid sequence HSRWYNL (SEQ ID NO: 2197); or

[0309] v). CDR1 containing the amino acid sequence GFTFSRYA (SEQ ID NO: 2201), CDR2 containing the amino acid sequence ISSAGGYI (SEQ ID NO: 2202), and CDR3 containing the amino acid sequence FTADY (SEQ ID NO: 2203).

[0310] One or more non-alanine residues in the CDR3 sequence are optionally replaced with alanine, and / or one or more alanine residues in the CDR3 sequence are optionally replaced with glycine.

[0311] In related embodiments, this document provides an anti-TRAILR2 antigen-binding protein (e.g., an antibody, such as a single-domain antibody) comprising a set of three CDRs (i.e., CDR1-CDR2-CDR3) within the VHH amino acid sequence as defined by any of the exemplary anti-TRAILR2 VHH antibodies listed in Tables 1-1, 1-2, or 6. For example, this document provides an antibody or antigen-binding fragment thereof comprising the CDR1-CDR2-CDR3 amino acid sequence set within the VHH amino acid sequence selected from SEQ ID NO: 4, 9, 14, 17, 21, 26, 31, 36, 41, 45, 50, 55, 65, 70, 75, 79, 84, 89, 93, 98, 146 to 468, 2192, 2198, 2204, 2214 to 2233, and 2354 to 2404.

[0312] In some embodiments, the anti-TRAILR2 antigen-binding protein (e.g., an antibody, such as a single-domain antibody) disclosed herein may include:

[0313] a) A variable structural domain comprising CDR1, CDR2 and CDR3 contained within such a VHH, wherein the VHH comprises SEQ ID NO: 4;

[0314] b) A variable structural domain comprising CDR1, CDR2 and CDR3 contained within such a VHH, wherein the VHH comprises SEQ ID NO: 9;

[0315] c) A variable structural domain comprising CDR1, CDR2 and CDR3 contained within such a VHH, wherein the VHH comprises SEQ ID NO: 14;

[0316] d) A variable structural domain comprising CDR1, CDR2 and CDR3 contained within such a VHH, wherein the VHH comprises SEQ ID NO: 17;

[0317] e) A variable structural domain comprising CDR1, CDR2 and CDR3 contained within such a VHH, wherein the VHH comprises SEQ ID NO: 21;

[0318] f) A variable structural domain comprising CDR1, CDR2 and CDR3 contained within such a VHH, wherein the VHH comprises SEQ ID NO: 26;

[0319] g) A variable structural domain comprising CDR1, CDR2 and CDR3 contained within such a VHH, wherein the VHH contains SEQ ID NO: 31;

[0320] h) A variable structural domain comprising CDR1, CDR2 and CDR3 contained within such a VHH, wherein the VHH comprises SEQ ID NO: 36;

[0321] i) A variable structural domain comprising CDR1, CDR2 and CDR3 contained within such a VHH, wherein the VHH comprises SEQ ID NO: 41;

[0322] j) A variable structural domain comprising CDR1, CDR2 and CDR3 contained within such a VHH, wherein the VHH comprises SEQ ID NO: 45;

[0323] k) includes a variable structural domain containing CDR1, CDR2 and CDR3 within such a VHH, wherein the VHH contains SEQ ID NO: 50;

[0324] l) A variable structural domain comprising CDR1, CDR2 and CDR3 contained within such a VHH, wherein the VHH comprises SEQ ID NO: 55;

[0325] m) contains variable structural domains of CDR1, CDR2 and CDR3 contained within such a VHH, wherein the VHH contains SEQ ID NO: 65;

[0326] n) includes a variable structural domain containing CDR1, CDR2 and CDR3 within such a VHH, wherein the VHH contains SEQ ID NO: 70;

[0327] o) A variable structural domain containing CDR1, CDR2 and CDR3 contained in such a VHH, wherein the VHH contains SEQ ID NO: 75;

[0328] p) includes a variable structural domain containing CDR1, CDR2 and CDR3 within such a VHH, wherein the VHH contains SEQ ID NO: 79;

[0329] q) A variable structural domain containing CDR1, CDR2 and CDR3 contained in such a VHH, wherein the VHH contains SEQ ID NO: 84;

[0330] r) includes a variable structural domain containing CDR1, CDR2 and CDR3 within such a VHH, wherein the VHH contains SEQ ID NO: 89;

[0331] s) includes variable structural domains containing CDR1, CDR2 and CDR3 within such a VHH, wherein the VHH contains SEQ ID NO: 93;

[0332] t) includes variable structural domains containing CDR1, CDR2 and CDR3 within such a VHH, wherein the VHH contains SEQ ID NO: 98;

[0333] u) includes a variable structural domain containing CDR1, CDR2 and CDR3 within such a VHH, wherein the VHH contains SEQ ID NO: 2192;

[0334] v) A variable structural domain comprising CDR1, CDR2, and CDR3 contained within a VHH, wherein the VHH contains SEQ ID NO: 2198; or

[0335] w) includes a variable structural domain containing CDR1, CDR2 and CDR3 within such a VHH, wherein the VHH contains SEQ ID NO: 2204.

[0336] In the embodiments provided herein, the anti-TRAILR2 antigen-binding protein (e.g., antibody, such as a single-domain antibody) of this disclosure may include a VHH amino acid sequence selected from SEQ ID NO: 4, 9, 14, 17, 21, 26, 31, 36, 41, 45, 50, 55, 65, 70, 75, 79, 84, 89, 93, 98, 146 to 468, 2192, 2198, 2204 and 2214 to 2233 or a similar sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with it.

[0337] In some embodiments, the anti-TRAILR2 antigen-binding protein of this disclosure (e.g., an antibody, such as a single-domain antibody) may include a VHH amino acid sequence selected from SEQ ID NO: 4, 9, 14, 17, 21, 26, 31, 36, 41, 45, 50, 55, 65, 70, 75, 79, 84, 89, 93, 98, 146 to 468, 2192, 2198, 2204 and 2214 to 2233, or a sequence having at least 75% identity with such sequences.

[0338] In the embodiments provided herein, the anti-TRAILR2 antigen-binding protein (e.g., antibody, such as a single-domain antibody) of this disclosure may include a VHH amino acid sequence selected from SEQ ID NO: 4, 9, 14, 17, 21, 26, 31, 36, 41, 45, 50, 55, 65, 70, 75, 79, 84, 89, 93, 98, 2192, 2198, and 2204, or a similar sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it.

[0339] In some embodiments, the anti-TRAILR2 antigen-binding protein of this disclosure (e.g., an antibody, such as a single-domain antibody) may include a VHH amino acid sequence selected from 4, 9, 14, 17, 21, 26, 31, 36, 41, 45, 50, 55, 65, 70, 75, 79, 84, 89, 93, 98, 2192, 2198, and 2204, or a sequence having at least 75% identity with such sequences.

[0340] In some embodiments, the anti-TRAILR2 antigen-binding protein of this disclosure (e.g., an antibody, such as a single-domain antibody) may include a VHH amino acid sequence selected from any of SEQ ID NO: 31, 55, 2192, 2198 and 2204 or an amino acid sequence having at least 75% identity with it.

[0341] In some embodiments of any of the above antigen-binding proteins, VHH may be humanized.

[0342] In the embodiments provided herein, the anti-TRAILR2 antigen-binding protein (e.g., antibody, such as a single-domain antibody) of this disclosure may include a humanized VHH amino acid sequence selected from SEQ ID NO: 5, 10, 15, 18, 22, 27, 32, 37, 42, 46, 51, 56, 66, 71, 76, 80, 85, 90, 94, 99, 469-761, 2194, 2200, 2206, 2234 to 2251 and 2354 to 2404 or a similar sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it.

[0343] In some embodiments, the anti-TRAILR2 antigen-binding protein of this disclosure (e.g., an antibody, such as a single-domain antibody) may include a humanized VHH amino acid sequence selected from SEQ ID NO: 5, 10, 15, 18, 22, 27, 32, 37, 42, 46, 51, 56, 66, 71, 76, 80, 85, 90, 94, 99, 469 to 761, 2194, 2200, 2206, 2234 to 2251, and 2354 to 2404, or a sequence having at least 75% identity with it.

[0344] In the embodiments provided herein, the anti-TRAILR2 antigen-binding protein (e.g., antibody, such as a single-domain antibody) of this disclosure may include a humanized VHH amino acid sequence selected from SEQ ID NO: 5, 10, 15, 18, 22, 27, 32, 37, 42, 46, 51, 56, 66, 71, 76, 80, 85, 90, 94, 99, 2194, 2200, 2206 and 2354 to 2404 or a similar sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with it.

[0345] In some embodiments, the anti-TRAILR2 antigen-binding protein of this disclosure (e.g., an antibody, such as a single-domain antibody) may include a humanized VHH amino acid sequence selected from SEQ ID NO: 5, 10, 15, 18, 22, 27, 32, 37, 42, 46, 51, 56, 66, 71, 76, 80, 85, 90, 94, 99, 2194, 2200, 2206 and 2354 to 2404 or a sequence having at least 75% identity with it.

[0346] In some embodiments, the anti-TRAILR2 antigen-binding protein of this disclosure (e.g., an antibody, such as a single-domain antibody) may include a humanized VHH amino acid sequence selected from any of SEQ ID NO: 32, 56, 2200, 2206 and 2194 or an amino acid sequence having at least 75% identity with it.

[0347] In some embodiments, the anti-TRAILR2 antigen-binding protein of this disclosure (e.g., an antibody, such as a single-domain antibody) may include a humanized VHH amino acid sequence selected from any of SEQ ID NO: 2354 to 2404 or an amino acid sequence having at least 75% identity with it.

[0348] In some embodiments, this disclosure also provides an anti-TRAILR2 antigen-binding protein (e.g., an antibody, such as a single-domain antibody) that competes with any of the exemplary anti-TRAILR2 VHH antibodies listed in Tables 1-1, 1-2, or 6 for binding to TRAILR2.

[0349] In some embodiments, this disclosure also provides an anti-TRAILR2 antigen-binding protein (e.g., an antibody, such as a single-domain antibody) that binds to the same epitope on TRAILR2 with any of the exemplary anti-TRAILR2 VHH antibodies listed in Tables 1-1, 1-2, or 6.

[0350] Single domain antibodies

[0351] Single-domain antibodies (e.g., VHH) can be obtained by immunizing dromedary camels, camels, llamas, alpacas, or sharks with the desired antigen and subsequently isolating the mRNA encoding heavy chain antibodies. The antigen can be from a natural source or purified during recombinant manufacturing. Immunization and / or screening of immunoglobulin sequences can be performed using peptide fragments of such antigens. Gene libraries containing millions of clones of single-domain antibodies can be generated by reverse transcription and polymerase chain reaction (PCR). Screening techniques such as phage display, yeast display, and ribosome display help identify clones that bind to the antigen. Methods for generating heavy chain antibody fragments are described, for example, in WO 94 / 04678; Hamers-Casterman et al. 1993; Muyldermans et al. 2001; and Arbabi Ghahroudi, M. et al. (1997). FEBS Letters 414 (3): 521-526, the entire contents of each of which are incorporated herein by reference.

[0352] Different approaches can utilize gene libraries from animals that have not previously been immunized. Such initial libraries typically contain only antibodies with low affinity for the desired antigen, making it necessary to apply affinity maturation as an additional step through random mutagenesis. See, for example, Saerens, D. et al. (2008). “Single-domain antibodies as building blocks for novel therapeutics”. Current Opinion in Pharmacology 8 (5): 600-608.

[0353] Affinity maturation strategies can be categorized into targeted / rational methods or non-targeted / randomized methods. Targeted methods require information about the VHH of interest, such as affinity maturation hotspots or structural information about the VHH:antigen complex, while non-targeted methods do not require prior information. Targeted methods applicable to VHH affinity maturation include site-directed in vitro mutagenesis and in-silico / computational methods. Common non-targeted methods for VHH affinity maturation include randomized in vitro mutagenesis, CDR exchange, and autonomous hypermutant yeast surface display, the latter two being novel, emerging, and very time-efficient techniques. Most of these strategies follow a common approach: after applying a randomization strategy to generate a mutant library, the resulting library can be screened using standard display techniques such as yeast, phage, or ribosome display to select the optimal binding compound. The choice of display system is typically guided by the size of the library to be displayed, with yeast display capable of handling ~10T 7 -10 9 Library size, phage display ~10 8 -10 10 Ribosome demonstration ~10 12 -10 13 (Chan and Groves, 2021). Notably, during affinity maturation, the number of highly interacting residues (such as aromatic amino acids) typically increases in the CDR region. The selected affinity-matured clones can be further evaluated through exploitability assessments to test for undesirable properties, such as off-target nonspecific binding or VHH instability.

[0354] For targeted in vitro mutagenesis, a selected set of residues within the CDR of VHH can be mutated (Tiller et al., 2017; Yau et al., 2005). Hotspot residues for mutation can be identified using alanine scanning or preselected by identifying the sites to be mutated using structural data of the antigen:VHH complex. These sites can then be saturated mutagenesis to replace specific sites with all possible amino acids, or to perform specific amino acid substitutions, generating several smaller libraries. After mutagenesis, the conjugates can be displayed to select the best maturation candidate. Typically, several rounds of targeted mutagenesis are performed with individual sublibraries to obtain a combination of single mutations that synergistically increase binding affinity.

[0355] Computer-aided / computer simulation methods are commonly used to guide targeted in vitro mutagenesis. Using a target:VHH complex or docking homology model, mutation hotspots can be identified, followed by in vitro mutagenesis (Bert Schepens et al., 2021; Cheng et al., 2019; Inoue et al., 2013; Mahajan et al., 2018). Additionally, computer simulation methods can search virtual libraries in a considerably short time (approximately 10...). 40 All designed variants of the drug (among its members) are used to identify a number of promising candidates that are experimentally feasible to test. Such techniques can be especially valuable if structural data on drug-target interactions are available.

[0356] Non-targeted / random affinity maturation strategies applicable to VHH affinity maturation include random in vitro mutagenesis, CDR shuffling / exchange, and in vivo affinity maturation via yeast display. For random in vitro mutagenesis, the entire VHH or only the CDR sequence is randomly mutated (Chen et al., 2021; Ye et al., 2021; Zupancic et al., 2021). The most commonly used technique is error-prone PCR, which uses DNA polymerases lacking proofreading activity and PCR conditions that further increase the polymerase error rate. This technique can be applied without further structural knowledge or information about the importance of residues that contribute to antigen:VHH interactions. The resulting mutant library can then be displayed to select the best maturation candidates. This technique can also be combined with NGS sequencing of the displayed eluents to deeply read all obtained candidates, enabling the identification of low-abundance but still promising clones (Chen et al., 2021).

[0357] In some implementations, CDR shuffling or exchange is applied to VHH affinity maturation, as described in Zupancic et al., 2021. For CDR exchange, enriched libraries can be used as input material for PCR reactions to individually amplify the CDRs of VHHs. Overlap PCR can then be used to mix and reassemble the PCR products to generate the entire plasmid for further multiple rounds of display to select the optimal maturation binder. One limitation of this approach is that it can only be used with VHHs containing the same framework as in the case of synthetic libraries.

[0358] In some implementations, in vivo affinity maturation via yeast display is applied to VHH affinity maturation, as described in Wellner et al., 2021. This method is based on Autonomous Hypermutant Yeast Surface Display (AHEAD), which mimics somatic hypermutation during VHH selection using engineered yeast strains. Yeast's error-prone orthogonal DNA replication system can generate new variants by randomly introducing mutations during plasmid replication. Yeast surface display can then be used to display and select these new variants to identify the optimal binder. This allows for the generation of high-affinity clones in a very short time (approximately 2 weeks), significantly faster than classical affinity maturation procedures. This method can be applied using synthetic or immunolibrium libraries, using unenriched libraries, or subpopulations of preselected clones.

[0359] When a medium-affinity conjugate is required, as is the case with the anti-TRAILR2 V conjugate, and the affinity of the identified candidates needs to be reduced, very similar techniques can be applied. For example, the same targeted or non-targeted methods described for affinity maturation can be used to introduce mutations designed to reduce affinity. Selection can then be adapted accordingly. If a large library is generated that needs to be screened via display technology, the selection strategy can be adapted to enrich for isoaffinity conjugates while excluding high-affinity candidates. For example, this could be pre-screening with a low antigen concentration in phage display to remove all high-affinity candidates, followed by selection with a high antigen concentration to obtain medium-affinity VHHs. For library sizes of up to 1000 candidates, kinetic dissociation rate characterization can be used to obtain real-time information on the kinetic behavior of the candidates.

[0360] Once the most potent clone has been identified, its DNA sequence can be optimized, for example, to improve its stability against enzymes. Another goal is humanization to prevent immune responses in human organisms to antibodies. Humanization can be achieved based on the homology between camel VHH fragments and human VH fragments, which is described in further detail below. Finally, optimized single-domain antibodies can be translated and expressed in suitable organisms such as *Escherichia coli* or *Saccharomyces cerevisiae*.

[0361] Single-domain antibodies can also be derived from conventional antibodies. In some embodiments, single-domain antibodies can be made from conventional murine or human IgG with four chains. The process is similar, involving a gene library from an immunized or initial donor and a display technique for identifying the most specific antigen. However, the binding region of conventional IgG consists of two domains (VH and VL), which tend to dimerize or aggregate due to their lipophilicity. Monomerization can be achieved by replacing lipophilic amino acids with hydrophilic amino acids. (See, for example, Borrebaeck, CAK; Ohlin, M. (2002). “Antibody evolution beyond Nature”. Nature Biotechnology 20 (12): 1189-90). If affinity can be retained after monomerization, single-domain antibodies can also be produced in E. coli, Saccharomyces cerevisiae, or other suitable organisms.

[0362] "Humanized antibody" refers to a chimeric, genetically engineered antibody in which an amino acid sequence (typically a CDR) from an antibody (donor antibody) (e.g., a camelid antibody) is grafted onto a human antibody (recipient antibody). Therefore, a humanized antibody typically contains a CDR from the donor antibody and a variable region framework and a constant region (if present) from the human antibody. Thus, a "humanized VHH" contains a CDR corresponding to a naturally occurring VHH domain (e.g., a camelid VHH) but which has been "humanized." A humanized VHH can be prepared by replacing one or more amino acid residues in the amino acid sequence (especially in the framework sequence) of a naturally occurring VHH sequence with one or more amino acid residues appearing at corresponding positions in the VH domain of a conventional 4-strand human antibody. Such a humanized VHH can be obtained in any suitable manner known to those skilled in the art, and is therefore not strictly limited to the methods described herein.

[0363] Humanization of VHH can be achieved using surface resurfacing or CDR transplantation. Surface resurfacing strategies have been described, for example, in Conrath et al., 2005 J Mol Biol; Kazemi-Lomedasht et al., 2018; Vincke et al., 2009 J Biol Chem, and CDR transplantation strategies have been described, for example, in ben Abderrazek et al., 2011; van Faassen et al., 2020 FASEB; Li et al., 2018; Vaneycken et al., 2010; Vincke et al., 2009 J Biol Chem; and Yu et al., 2017, the entire contents of each of which are incorporated herein by reference.

[0364] To humanize camel VHHs using a surface remodeling approach, a human phylogenetic reference was identified that most closely resembled the selected VHH's camel phylogenetic sequence. Most isolated camel VHHs in the literature belong to the subfamily IGHV3 of the camel family (Nguyen et al., 2000, EMBO J), with DP-47 / VH3-23 of the IGHV3 family commonly used as a human reference. The scaffold of the camel VHH was then compared to the human reference sequence. Surface-exposed residues were replaced with their human counterparts, presumably because their contribution to protein stability was assumed to be relatively low. However, masked residues remained camel-derived, as they might contribute to the overall stability of the VHH. Humanization of scaffold regions 1, 3, and 4 generally did not affect the physicochemical properties of the VHH, while general humanization of scaffold 2 would significantly increase local hydrophobicity. Residues H37, H44, H45, and H47 (Chothia number) in framework 2, the so-called tetrad residues or marker residues, are quite hydrophobic in human VH (VGLW) because they are partially masked and participate in VH / VL pairing. However, in camel VHH, these residues are partially charged (FERG), which significantly increases VHH solubility and inhibits camel VL pairing (Soler et al., 2021; Biomolecules; Conrath et al., 2005; J Mol Biol). Furthermore, residues H37 and H47 are known to interact with the CDR-H3 loop in many VHHs, stabilizing their conformation and thus contributing to antigen-binding affinity. Additionally, a large number of VHHs utilize framework 2 residues H44, H45, and H47 for antigen binding (Zavrtanik et al., 2018; J Mol Biol). Therefore, complete humanization of such residues typically leads to reduced solubility or aggregation of VHH, as well as reduced or complete loss of binding affinity to the target antigen (van Faassen et al., 2020; Vincke et al., 2009). Thus, when VHH is humanized, all or at least some of these marker residues in framework 2 still originate from camels.

[0365] Another method that can be used to humanize VHHs is CDR transplantation. The CDR of the selected VHH can be transplanted onto a partially or fully humanized generic VHH framework (Saerens et al., 2009 J Biol Chem; Soler et al., 2021; Vincke et al., 2009 J Biol Chem). CDR transplantation has been successful in some cases but has failed in several others, where the VHH often loses its potential to bind to the desired antigen and / or becomes structurally unstable and highly prone to aggregation (van Faassen et al., 2020, FASEB). This is mainly attributed to the interaction of CDR3 with specific residues in framework 2, which are important for CDR3 conformation, general VHH stability, and overall hydrophobicity, and are impaired by this method. Sometimes, camel reversion mutations are introduced into the framework to compensate for such effects (van Faassen et al., 2020, FASEB).

[0366] An alternative strategy to mitigate the need for humanization of selected VHH sequences is to use fully or partially humanized synthetic VHH libraries instead of camel immune libraries for VHH discovery (Moutel et al. 2016, eLife; McMahon, 2018, NSMB; Zimmermann et al. 2018, eLife). For the reasons discussed above, in many such libraries, the marker residues remain of camel origin.

[0367] Other suitable humanization substitutions are described in WO 09 / 138519 and WO 08 / 020079, and Tables A-3 to A-8 from WO 08 / 020079 (which are lists showing possible humanization substitutions), each of which is incorporated herein by reference in its entirety. Non-limiting examples of such humanization substitutions include Q108L and A14P. Such humanization substitutions may also be suitably combined with one or more other mutations described herein, such as one or more mutations that reduce binding to pre-existing antibodies.

[0368] In some implementations, the humanized VHH sequence retains residues associated with protein A binding. In some implementations, engineering activities during humanization can be used to engineer the protein A binding properties to those of a VHH that previously did not interact with protein A (Graille et al., 2000, PNAS).

[0369] Similar to “humanized antibodies,” “camelized antibodies” refer to antibodies having an amino acid sequence (typically a CDR) from a donor antibody (e.g., a human antibody) and a variable region framework and constant region (if present) from a camel-dwelling antibody. Thus, a “camelized VH” contains an amino acid sequence corresponding to the naturally occurring VH domain, but which has been “camelized.” Camelized VHs can be prepared by replacing one or more amino acid residues in the naturally occurring VH domain of a conventional 4-chain antibody with one or more amino acid residues appearing at corresponding positions in the VHH domain of a heavy-chain antibody. This can be done, for example, as described in WO 2008 / 020079. Such “camelization” substitutions are typically inserted at amino acid positions forming and / or present at the VH-VL interface, and / or inserted at so-called camel-dwelling marker residues, such as F37, E44, R45, and F47 (see, for example, WO 94 / 04678 and Davies and Riechmann (1994 and 1996)). In one embodiment, the VH sequence used as the starting material or starting point for generating or designing camel-like VHs is a VH sequence derived from mammals or a VH sequence from a human antibody. However, such camel-like VHs can be obtained in any suitable manner known to those skilled in the art, and are therefore not strictly limited to peptides obtained using peptides containing naturally occurring VH domains as starting materials.

[0370] The amino acid residues of single-domain antibodies can be numbered according to the general numbering of the VH domain given by Kabat et al. (“Sequence of proteins of immune interest”, US Public Health Services, NIH Bethesda, Md., Publication 91), applicable to Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240 (1-2): 185-195; see, for example, that publication). Figure 2 The VHH domain of camels described in [reference needed]. The total number of amino acid residues in each of the CDRs can vary and may not correspond to the total number of amino acid residues indicated by the Kabat number. For example, the actual sequence may occupy one or more positions according to the Kabat number, or the actual sequence may contain more amino acid residues than allowed by the Kabat number. Therefore, the Kabat number may or may not correspond to the actual number of amino acid residues in the actual sequence. The total number of amino acid residues in the VH domain and the VHH domain is typically in the range of 110 to 120, and usually in the range of 112 to 115. However, smaller and longer sequences may also be suitable for the purposes described herein.

[0371] The determination of the CDR region in a single-domain antibody can be accomplished using different methods, including those described below: Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745 (“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, January 2003; 27(1):55-77 (“IMGT” numbering scheme); Honegger A and Plückthun A, “Yet another numbering scheme for immunoglobulin variabledomains: an automatic modeling and analysis tool”, J Mol Biol, June 8, 2001; 309(3):657-70, (“Aho” numbering scheme); and Martin et al., “Modeling antibody hypervariable loops: a combined algorithm”, PNAS, 1989, 86(23):9268-9272, (“AbM” numbering scheme). Each reference cited in this paper is incorporated herein by reference in its entirety.

[0372] The boundaries of a given CDR or framework (FR) can vary depending on the scheme used for identification. For example, the Kabat scheme is based on structure alignment, while the Chothia scheme is based on structural information. Both the Kabat and Chothia schemes number antibodies based on the length of the most common antibody region sequence, where some antibodies exhibit insertions and deletions with insert letters (e.g., "30a"). The two schemes place certain insertions and deletions ("insertions and / or deletions (indels)") in different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar to the Chothia numbering scheme in several ways. The AbM scheme is a compromise between the Kabat and Chothia definitions, based on the scheme used by Oxford Molecular's AbM antibody modeling software.

[0373] In some implementations, CDRs may be defined according to any of the Kabat numbering scheme, the Chothia numbering scheme, a combination of Kabat and Chothia, the AbM numbering scheme, and / or the Contact numbering scheme. A VHH typically contains three CDRs, referred to as CDR1, CDR2, and CDR3. Tables 1-3 below list exemplary positional boundaries of CDR-H1, CDR-H2, and CDR-H3 identified according to the Kabat, Chothia, AbM, and Contact schemes, respectively. For CDR-H1, residue numbering is listed using both the Kabat and Chothia numbering schemes. FRs lie between multiple CDRs; for example, FR-H1 precedes CDR-H1, FR-H2 lies between CDR-H1 and CDR-H2, FR-H3 lies between CDR-H2 and CDR-H3, and so on. It should be noted that because the Kabat numbering scheme shown makes the insertions located at H35A and H35B, the end of the Chothia CDR-H1 ring varies between H32 and H34 when numbered using the Kabat numbering convention shown, depending on the length of the ring.

[0374] Table 1-3. CDR definitions according to various numbering schemes.

[0375]

[0376] 1 Kabat et al. (1991), “Sequences of Proteins of Immunological Interest”, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD;

[0377] 2 Al-Lazikani et al., (1997) JMB 273, 927-948.

[0378] Therefore, unless otherwise stated, a “CDR” or “complementarity-determining region” or a single specific CDR (e.g., CDR-H1, CDR-H2, CDR-H3) of a given antibody or its regions (such as its variable regions) should be understood to encompass (or specific) CDRs defined by any of the above schemes. For example, when a specific CDR (e.g., CDR-H3) is stated to contain the amino acid sequence of the corresponding CDR in a given VHH amino acid sequence, it should be understood that such a CDR has the sequence of the corresponding CDR (e.g., CDR-H3) within the VHH, as defined by any of the above schemes. In some embodiments, a specific CDR sequence is specified. Exemplary CDR sequences of the antibodies provided are described using various numbering schemes (see, for example, Tables 1-3), but it should be understood that the antibodies provided may include CDRs described according to any of the other numbering schemes described above or other numbering schemes known to those skilled in the art.

[0379] In the single-domain antibody sequences disclosed herein, the framework sequence can be any suitable framework sequence. For example, the framework sequence can be a framework sequence derived from the heavy chain variable domain (e.g., a VH sequence or a VHH sequence). In some embodiments, the framework sequence is a framework sequence derived from a VHH sequence (wherein the framework sequence may optionally be partially or fully humanized) or a conventional VH sequence (wherein the framework sequence may optionally be partially or fully camelized).

[0380] This disclosure also covers antigen-binding fragments (or combinations of fragments) of any of the single-domain antibodies described herein, such as fragments containing one or more CDR sequences, suitably side-joined with one or more framework sequences, and / or linked via one or more framework sequences.

[0381] However, it should be noted that this disclosure is not limited to the source of single-domain antibodies (or the nucleotide sequences used to express them), nor to the manner in which single-domain antibodies or nucleotide sequences are generated or obtained. Therefore, the antigen-binding protein of this disclosure may comprise naturally occurring sequences (from suitable species), recombinant sequences, or synthetic or semi-synthetic sequences. Similarly, the nucleotide sequence encoding the antigen-binding protein of this disclosure may comprise naturally occurring nucleotide sequences, recombinant sequences, or synthetic or semi-synthetic sequences (e.g., sequences prepared by PCR or isolated from a library).

[0382] Compared to the exemplary antibody sequences provided herein, the anti-TRAILR2 antigen-binding proteins (e.g., antibodies, such as single-domain antibodies) of this disclosure may contain one or more amino acid substitutions, insertions, and / or deletions in the framework region and / or CDR region of the heavy chain variable domain. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences obtained from, for example, public antibody sequence databases. The antigen-binding molecules of this disclosure may contain an antigen-binding domain derived from any of the exemplary amino acid sequences disclosed herein, wherein one or more amino acids in one or more framework regions and / or CDR regions are mutated to corresponding residues of a germline sequence that derives the antibody, or mutated to corresponding residues of another germline sequence, or mutated to conserved amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). Using the heavy chain and light chain variable region sequences disclosed herein as starting materials, those skilled in the art can readily generate numerous antibody and antigen-binding fragments containing one or more single germline mutations or combinations thereof. In some embodiments, all framework and / or CDR residues within the VHH domain are mutated back to residues found in the original germline sequence from which the antigen-binding domain was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, such as mutant residues found only in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or mutant residues found only in CDR1, CDR2, or CDR3. In other embodiments, one or more framework and / or CDR residues are mutated to corresponding residues in a different germline sequence (i.e., a germline sequence different from the one from which the antigen-binding domain was originally derived).

[0383] Furthermore, the antigen-binding domain may contain any combination of two or more germline mutations within the framework region and / or CDR region, such that certain individual residues are mutated to corresponding residues of a specific germline sequence, while certain other residues different from the original germline sequence remain unchanged or are mutated to corresponding residues of a different germline sequence. Once obtained, one or more desired properties of the antigen-binding domain containing one or more germline mutations can be readily tested, such as improved binding specificity, increased binding affinity, improved or enhanced biological properties (e.g., agonist effect), reduced immunogenicity, etc. This disclosure covers antigen-binding proteins comprising one or more antigen-binding domains obtained in this generally manner.

[0384] This document provides anti-TRAILR2 antigen-binding proteins comprising variants of any of the VHH and / or CDR amino acid sequences disclosed herein, having one or more amino acid substitutions. For example, this disclosure includes anti-TRAILR2 antigen-binding proteins having VHH and / or CDR amino acid sequences having, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, 3 or fewer, 2 or 1 amino acid substitutions relative to any of the VHH and / or CDR amino acid sequences set forth in Tables 1-1 and 1-2 herein. Amino acid substitutions may be introduced into the antigen-binding protein of interest, and the resulting variants may be screened for desired activities, such as maintaining / modifying antigen binding, reducing immunogenicity, or reducing ADCC or CDC.

[0385] Amino acids can be grouped according to common side chain properties: (1) hydrophobic: leucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. In some embodiments, amino acid substitution is a conservative substitution, which means exchanging an amino acid for another amino acid of the same class. In some embodiments, amino acid substitution may also include non-conservative substitution, which means exchanging an amino acid for an amino acid of a different class. Other exemplary amino acid substitutions are shown in Tables 1-4.

[0386] Table 1-4. Exemplary amino acid substitutions

[0387]

[0388] In some embodiments, the single-domain antibody of this disclosure (e.g., VHH) may contain one or more mutations to reduce the oxidation level of oxidatively unstable residues (such as Met(M)). In some embodiments, it may be necessary to address the oxidation tendency of Met(M) by mutation of the Met(M) residue. In some embodiments, the single-domain antibody of this disclosure (e.g., VHH) may contain one or more mutations (e.g., substitution mutations) of the Met residue to reduce oxidation. As a non-limiting example, any of the single-domain antibodies described herein may be substituted with, for example, Ile(I), Ala(A), or Leu(L) to reduce oxidation.

[0389] In some embodiments, the single-domain antibody (e.g., VHH) of this disclosure comprises one or more modifications that reduce the binding of the single-domain antibody (e.g., VHH) to pre-existing antibodies found in human blood or serum. In some embodiments, the single-domain antibody (e.g., VHH) of this disclosure is modified by a mutation at amino acid position 11 (e.g., Leu11Glu (L11E), Leu11Lys (L11K), or Leu11Val (L11V)). In one embodiment, the single-domain antibody (e.g., VHH) of this disclosure may comprise valine (V) at amino acid position 11 and leucine (L) at amino acid position 89 (according to Kabat designation). As another example, the single-domain antibody (e.g., VHH) of this disclosure may include an extension of 1 to 5 (naturally occurring) amino acids, such as a monoalanine (A) extension, at the C-terminus of the single-domain antibody (e.g., VHH). The C-terminus of VHH is typically VTVSS (SEQ ID NO: 2166). In one embodiment, the single-domain antibody (e.g., VHH) of this disclosure comprises lysine (K) or glutamine (Q) at position 110 (according to the Kabat number). In another embodiment, the single-domain antibody (e.g., VHH) of this disclosure comprises lysine (K) or glutamine (Q) at position 112 (according to the Kabat number). Therefore, the C-terminus of the single-domain antibody (e.g., VHH) may be any of VKVSS (SEQ ID NO: 2167), VQVSS (SEQ ID NO: 2168), VTVKS (SEQ ID NO: 2169), VTVQS (SEQ ID NO: 2170), VKVKS (SEQ ID NO: 2171), VKVQS (SEQ ID NO: 2172), VQVKS (SEQ ID NO: 2173), or VQVQS (SEQ ID NO: 2174). In another embodiment, the single-domain antibody of this disclosure (e.g., VHH) comprises valine (V) at amino acid position 11 (according to Kabat number) and leucine (L) at amino acid position 89, optionally lysine (K) or glutamine (Q) at position 110 (according to Kabat number), and an extension of 1 to 5 (naturally occurring) amino acids, such as a single alanine (A) extension at the C-terminus of the single-domain antibody (e.g., VHH) (thereby such that the C-terminus of the single-domain antibody (e.g., VHH) has, for example, the sequence VTVSSA (SEQ ID NO: 2175), VKVSSA (SEQ ID NO: 2176) or VQVSSA (SEQ ID NO: 2177)).In other embodiments, the single-domain antibody of this disclosure (e.g., VHH) is modified by changing the carboxyl-terminal region, for example, by changing it to a terminal sequence having the sequence GQGTLVTVKPGG (SEQ ID NO: 2178) or GQGTLVTVEPGG (SEQ ID NO: 2179) or a modification thereof. Other modifications that reduce binding to pre-existing antibodies in human serum can be found in, for example, WO2012 / 175741, WO2015 / 173325, WO2016 / 150845, WO2011 / 003622, WO2013 / 024059, US 11,426,468, and US 10,526,397, which are incorporated herein by reference in their entirety.

[0390] In one embodiment, the single-domain antibody of this disclosure (e.g., VHH) contains the amino acid sequence VAGG (SEQ ID NO: 2405) or VPAG (SEQ ID NO: 2406) at the carboxyl terminus starting at position 111 according to Chothia. In another embodiment, the single-domain antibody of this disclosure (e.g., VHH) contains the amino acid sequence VAGG (SEQ ID NO: 2405) at the carboxyl terminus starting at position 111 according to Chothia. In yet another embodiment, the single-domain antibody of this disclosure (e.g., VHH) contains the amino acid sequence VPAG (SEQ ID NO: 2406) at the carboxyl terminus starting at position 111 according to Chothia.

[0391] In some embodiments, the single-domain antibody (e.g., VHH) of this disclosure comprises a subset selected from SEQ ID NO: The amino acid sequences of any one of the following: 4, 5, 9, 10, 14, 15, 17, 18, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 45, 46, 50, 51, 55, 56, 65, 66, 70, 71, 75, 76, 79, 80, 84, 85, 89, 90, 93, 94, 98, 99, 146 to 761, 2192, 2194, 2198, 2200, 2204, 2206, and 2214 to 2251, or sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the above sequences, wherein the amino acid sequence beginning at the carboxyl terminus at position 111 according to Chothia contains VAGG (SEQ ID NO: 2405) or VPAG (SEQ ID NO: 2406).

[0392] In some embodiments, the single-domain antibody of this disclosure (e.g., VHH) comprises an amino acid sequence selected from any one of SEQ ID NO: 4, 9, 14, 17, 21, 26, 31, 36, 41, 45, 50, 55, 65, 70, 75, 79, 84, 89, 93, 98, 2192, 2198, and 2204, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with it, wherein the amino acid sequence beginning at the carboxyl terminus at position 111 according to Chothia comprises VAGG (SEQ ID NO: 2405) or VPAG (SEQ ID NO: 2406).

[0393] In some embodiments, the single-domain antibody of this disclosure (e.g., VHH) comprises an amino acid sequence selected from any one of SEQ ID NO: 31, 55, 2192, 2198 and 2204 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity with it, wherein the amino acid sequence beginning at the carboxyl terminus at position 111 according to Chothia comprises VAGG (SEQ ID NO: 2405) or VPAG (SEQ ID NO: 2406).

[0394] In some embodiments, the single-domain antibody of this disclosure (e.g., VHH) comprises an amino acid sequence selected from any of SEQ ID NO: 5, 10, 15, 18, 22, 27, 32, 37, 42, 46, 51, 56, 66, 71, 76, 80, 85, 90, 94, 99, 2194, 2200, 2206, and 2354 to 2404, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with such sequence, wherein the amino acid sequence beginning at the carboxyl terminus at position 111 according to Chothia comprises VAGG (SEQ ID NO: 2405) or VPAG (SEQ ID NO: 2406).

[0395] In some embodiments, the single-domain antibody of this disclosure (e.g., VHH) comprises an amino acid sequence selected from any one of SEQ ID NO: 32, 56, 2200, 2206 and 2194 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity with it, wherein the amino acid sequence beginning at the carboxyl terminus at position 111 according to Chothia comprises VAGG (SEQ ID NO: 2405) or VPAG (SEQ ID NO: 2406).

[0396] In some embodiments, the single-domain antibodies of this disclosure (e.g., VHH) are modified to enhance binding to staphylococcal protein A (SpA) or streptococcal protein G (SpG). The binding of SpA and SpG to the antibody or antibody fragment is applicable to the manufacturing process of the antibody or antibody fragment. High-affinity interactions between the IgG Fc region and SpA and SpG have been extensively developed and have become the gold standard for monoclonal antibody purification (Björck and Kronvall, 1984). Other Fc-free antibody fragments, such as VHH and Fab, do not have the ability to bind SpA or SpG via their Fc regions. However, these Fc-free antibody fragments have been shown to have sequence-dependent interactions with SpA (Graille et al., 2000; Henry et al., 2016). This feature avoids the potential use of affinity tags fused to drug candidates in affinity chromatography, which have the disadvantage of being considered sequence liability because they can affect protein immunogenicity as well as protein structure and stability, and can impair functionality. The interaction between single-domain antibodies (e.g., VHH) and SpA depends on alternative binding modes, with an affinity of 1 to 5 µM, comparable to 0.2 to 3 µM as measured for VH-SpA interaction (To et al., JBC, 2005; Henry et al., Plos One, 2016).

[0397] In some embodiments, the single-domain antibody of this disclosure (e.g., VHH) has or is modified to have a SpA binding motif. For example, the VHH-SpA interface has been mapped to thirteen residues clustered within a framework on the back side of the V body, away from the CDR (Graille et al., 2000; Henry et al., 2016). In the absence of the VHH-SpA costructure, the superposition of the SpA-Fab crystal structure with VHH allows visualization of the binding mode. Based on structural and functional analysis, the thirteen residues at the VHH-SpA interface have been characterized as intolerant to substitutions (residues Gly15, Arg19, Tyr59, Gly65, and Arg66), tolerant to specific substitutions (residues Thr / Lys / Arg57, Thr68, Gln81, Asn82a, and Ser82b), or generally tolerant to multiple substitutions (residues Ser17, Lys64, and Ser70) (all residue positions refer to Kabat numbering) (Henry et al., Plos One, 2016). Therefore, the SpAc binding motif in single-domain antibodies (e.g., VHH) included in this disclosure may comprise one or more of the thirteen residues or all of the thirteen residues.

[0398] In some embodiments, the single-domain antibody of this disclosure (e.g., VHH) includes one or more modifications at the N-terminus to prevent the formation of pyroglutamic acid (salt / ester) and product heterogeneity. In one embodiment, the amino acid residue Glu at the first position of the single-domain antibody (e.g., VHH) is replaced by Asp (E1D).

[0399] In some embodiments, the single-domain antibody of this disclosure (e.g., VHH) comprises a subset selected from SEQ ID NO: The amino acid sequence of any one of the following: 4, 5, 9, 10, 14, 15, 17, 18, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 45, 46, 50, 51, 55, 56, 65, 66, 70, 71, 75, 76, 79, 80, 84, 85, 89, 90, 93, 94, 98, 99, 146 to 761, 2192, 2194, 2198, 2200, 2204, 2206 and 2214 to 2251, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity with the same sequence, wherein the amino acid residue Glu at the first position of the single-domain antibody (e.g., VHH) is replaced by Asp substitution (E1D).

[0400] In some embodiments, the single-domain antibody of this disclosure (e.g., VHH) comprises an amino acid sequence selected from any one of SEQ ID NO: 4, 9, 14, 17, 21, 26, 31, 36, 41, 45, 50, 55, 65, 70, 75, 79, 84, 89, 93, 98, 2192, 2198, and 2204, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with such sequence, wherein the amino acid residue Glu at the first position of the single-domain antibody (e.g., VHH) is replaced by Asp (E1D).

[0401] In some embodiments, the single-domain antibody of this disclosure (e.g., VHH) comprises an amino acid sequence selected from any one of SEQ ID NO: 31, 55, 2192, 2198, and 2204 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with it, wherein the amino acid residue Glu at the first position of the single-domain antibody (e.g., VHH) is replaced by Asp (E1D).

[0402] In some embodiments, the single-domain antibody of this disclosure (e.g., VHH) comprises an amino acid sequence selected from any of SEQ ID NO: 5, 10, 15, 18, 22, 27, 32, 37, 42, 46, 51, 56, 66, 71, 76, 80, 85, 90, 94, 99, 2194, 2200, 2206 and 2354 to 2404 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity with the same sequence, wherein the amino acid residue Glu at the first position of the single-domain antibody (e.g., VHH) is replaced with Asp.

[0403] In some embodiments, the single-domain antibody of this disclosure (e.g., VHH) comprises an amino acid sequence selected from any one of SEQ ID NO: 32, 56, 2200, 2206 and 2194 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity with it, wherein the amino acid residue Glu at the first position of the single-domain antibody (e.g. VHH) is replaced by Asp.

[0404] In some embodiments, the single-domain antibody (e.g., VHH) of this disclosure is modified by replacing one or more of any non-Ala(A) amino acid residues at any position with Ala(A). In some embodiments, the single-domain antibody (e.g., VHH) of this disclosure is modified by replacing one or more of any Ala(A) residues at any position with Gly(G). In some embodiments, the single-domain antibody (e.g., VHH) described herein is modified by replacing one or more of any non-Ala(A) amino acid residues at any position with Ala(A) residues and / or by replacing one or more of any Ala(A) residues with Gly(G).

[0405] In some embodiments, the single-domain antibody of this disclosure (e.g., VHH) comprises a single amino acid residue substituted with Ala (A), or, in the case where the original (i.e. wild-type) residue is Ala (A), a single amino acid residue substituted with Gly (G). Non-limiting examples of single-domain antibodies (e.g., VHH) modified with amino acid substitutions according to the above description include SEQ ID NO: 2354 to 2404.

[0406] In some embodiments, the complementarity-determining region 3 (CDR3) of the single-domain antibody (e.g., VHH) described herein contains a single amino acid residue that has been replaced with Ala (A), or a single amino acid residue that has been replaced with Gly (G) in the case where the original (i.e. wild-type) residue is Ala (A). As an example, CDR3 containing the amino acid sequence AAASSWSRGGVPYGMDY (SEQ ID NO: 30) can be modified according to the above-mentioned amino acid substitutions for, for example, the following amino acid sequences: GAASSWSRGGVPYGMDY (SEQ ID NO: 2298); AGASSWSRGGVPYGMDY (SEQ ID NO: 2299); AAGSSWSRGGVPYGMDY (SEQ ID NO: 2300); AAAASWSRGGVPYGMDY (SEQ ID NO: 2301); AAASAWSRGGVPYGMDY (SEQ ID NO: 2302); AAASSASRGGVPYGMDY (SEQ ID NO: 2303); AAASSWARGGVPYGMDY (SEQ ID NO: 2304); AAASSWSAGGVPYGMDY (SEQ ID NO: 2305); AAASSWSRAGVPYGMDY (SEQ ID NO: 2306). 2306); AAASSWSRGAVPYGMDY (SEQ ID NO: 2307); AAASSWSRGGAPYGMDY (SEQ ID NO: 2308); AAASSWSRGVAYGMDY (SEQ ID NO: 2309); AAASSWSRGVPAGMDY (SEQ ID NO: 2310); AAASSWSRGGAVPYAMDY (SEQ ID NO: 2311); AAASSWSRGGVPYGADY (SEQ ID NO: 2312); AAASSWSRGGVPYGMAY (SEQ ID NO: 2313); or AAASSWSRGGVPYGMDA (SEQ ID NO: 2314).As another example, CDR3 containing the amino acid sequence TAGPTMSYSRGGEF (SEQ ID NO: 54) can be modified according to the above-described amino acid substitutions for, for example, the following amino acid sequences: AAGPTMSYSRGGEF (SEQ ID NO: 2315); TGGPTMSYSRGGEF (SEQ ID NO: 2316); TAAPTMSYSRGGEF (SEQ ID NO: 2317); TAGATMSYSRGGEF (SEQ ID NO: 2318); TAGPAMSYSRGGEF (SEQ ID NO: 2319); TAGPTASYSRGGEF (SEQ ID NO: 2320); TAGPTMAYSRGGEF (SEQ ID NO: 2321); TAGPTMSASRGGEF (SEQ ID NO: 2322); TAGPTMSYARGGEF (SEQ ID NO: 2323); TAGPTMSYSAGGEF (SEQ ID NO: 2324); TAGPTMSYSRAGEF (SEQ ID NO: 2325); TAGPTMSYSRAGEF (SEQ ID NO: 2326); TAGPTMSYSRAGEF (SEQ ID NO: 2327); TAGPTMSYSRGGEF (SEQ ID NO: 2328); TAGPTMSYSRAGEF (SEQ ID NO: 2329); TAGPTMSYSRGGEF (SEQ ID NO: 2320); TAGPTMAYSRGGEF (SEQ ID NO: 2321); TAGPTMSASRGGEF (SEQ ID NO: 2322); TAGPTMSYARGGEF (SEQ ID NO: 2323); TAGPTMSYSAGGEF (SEQ ID NO: 2324); TAGPTMSYSRAGEF (SEQ ID NO: 2325); TAGPTMSYSRAGEF (SEQ ID NO: 2326); TAGPTMSYSRAGEF (SEQ ID NO: 2327); TAGPTMSYSRAGEF (SEQ ID NO: 232 2325); TAGPTMSYSRGAEF (SEQ ID NO: 2326); TAGPTMSYSRGGAF (SEQ ID NO: 2327); or TAGPTMSYSRGGEA (SEQ ID NO: 2328). As another example, CDR3 containing the amino acid sequence RAYNDGGEY (SEQ ID NO: 2191) can be modified according to the above-mentioned amino acid substitutions for, for example, the following amino acid sequences: AAYNDGGEY (SEQ ID NO: 2329); RGYNDGGEY (SEQ ID NO: 2330); RAANDGGEY (SEQ ID NO: 2331); RAYADGGEY (SEQ ID NO: 2332); RAYNAGGEY (SEQ ID NO: 2333); RAYNDAGEY (SEQ ID NO: 2334); RAYNDGAEY (SEQ ID NO: 2335); RAYNDGGAY (SEQ ID NO: 2336) or RAYNDGGEA (SEQ ID NO: 2337).As another example, CDR3 containing the amino acid sequence HSRWYNL (SEQ ID NO: 2197) can be modified according to the above-described amino acid substitutions for, for example, the following amino acid sequences: ASRWYNL (SEQ ID NO: 2338); HARWYNL (SEQ ID NO: 2339); HSAWYNL (SEQ ID NO: 2340); HSRAYNL (SEQ ID NO: 2341); HSRWANL (SEQ ID NO: 2342); HSRWYAL (SEQ ID NO: 2343); or HSRWYNA (SEQ ID NO: 2344). As another example, CDR3 containing the amino acid sequence FTADY (SEQ ID NO: 2203) can be modified according to the above-mentioned amino acid substitutions for, for example, the following amino acid sequences: ATADY (SEQ ID NO: 2345); FAADY (SEQ ID NO: 2346); FTGDY (SEQ ID NO: 2347); or FTAAY (SEQ ID NO: 2348).

[0407] Alternative protein backbone

[0408] In some embodiments, the anti-TRAILR2 antigen-binding protein of this disclosure may employ an alternative protein scaffold. Such an alternative protein scaffold may be a single-chain polypeptide framework optionally having a reduced size (e.g., less than about 200 amino acids) containing a highly structured core associated with variable domains that allow for high conformational tolerance to insertion, deletion, or other substitution. Such an antigen-binding protein can be generated by grafting the CDR or variable region described herein onto a suitable protein scaffold. The structure of the alternative scaffold may vary, but for scaffolds developed as therapeutic agents, human-derived scaffolds are preferred.

[0409] The alternative protein backbones disclosed herein can be based on conventional immunoglobulin (Ig) backbones or derived from completely unrelated proteins. These variable domains can be modified to generate novel binding interfaces against any target antigen. In some embodiments, the alternative protein backbone of this disclosure may be derived from protein A, such as its Z domain (affinity), ImmE7 (immunoprotein), BPTI / APPI (Kunitz domain), Ras-binding protein AF-6 (PDZ domain), charybdotoxin (scorpion venom), CTLA-4, Min-23 (knotting peptide), lipid carrier protein (anticarrier protein), neokarzinostatin, fibronectin domain (used in "adnectin"), ankyrin repeat (AR) domain (used in "DARPin"), affinity multimer (also known as "avimer"), or thioredoxin (Skerra, A., Curr. Opin. Biotechnol. 18:295-304 (2005); Hosse et al., Protein Sci. 15:14-27 (2006); Nicaise et al., Protein Sci. 13:1882-1891 (2004); Nygren and Uhlen, Curr. Opin. Struc. Biol. 7:463-469 (1997), all of which are hereby incorporated in full by reference.

[0410] Anticarrier proteins are suitable types of non-Ig-based alternative backbones for the antigen-binding molecules of this disclosure. Anticarrier proteins are a class of engineered ligand-binding proteins based on a lipid carrier backbone. Lipid carrier proteins are a family of proteins that transport small hydrophobic molecules such as steroids, choline, retinoids, and lipids. Lipid carrier proteins have limited sequence homology but share a common tertiary architecture based on eight antiparallel β-barrel structures. Lipid carrier proteins contain four exposed loops constructed on rigid β-barrel structures. Commonly used exemplary anticarrier proteins have a size of approximately 180 amino acids and a mass of approximately 20 kDa.

[0411] DARPin is another suitable non-Ig-based alternative backbone that can be used in the antigen-binding molecules disclosed herein. DARPin is a genetically engineered antibody mimic protein that typically exhibits highly specific and high-affinity binding to target proteins. They are derived from natural ankyrin repeat (AR) proteins, which typically contain a 33-amino acid protein motif consisting of two loop-separated α-helices, the repeat mediating protein-protein interactions. DARPin can be generated using a combined AR library constructed based on a 33-amino acid AR motif with seven random positions. DARPin libraries can be screened using ribosome display, and library members are generally well generated in *E. coli*, do not aggregate, and exhibit high thermodynamic stability. Preferably, DARPin contains two to four such motifs with N-terminal and C-terminal covering motifs to shield hydrophobic regions and allow for increased solubility.

[0412] Avimer structures can also be used as protein backbones to generate suitable non-Ig-based alternative backbones. Avimers typically consist of two or more peptide sequences, each 30 to 35 amino acids, linked by peptide linkers. Individual sequences are derived from the A domains of various membrane receptors and have a rigid structure, stabilized by disulfide bridges and calcium. Each A domain can bind to a specific epitope of the target protein. The combination of domains binding to different epitopes of the same protein increases the affinity for that protein; this effect is called avidity.

[0413] Proteins derived from the fibronectin III (FN3) domain can also be used to generate suitable non-Ig-based alternative backbones (also known as “monofunctional antibodies”). For example, the tenth fibronectin type III domain (FN10) of human fibronectin corresponds to a β-disassembly with seven β chains and three linker loops, showing structural homology with Ig domains that do not contain disulfide bridges. In some cases, the linker loops of FN10 (each approximately 15 to 21 amino acids in length) can be randomized, and the domains are displayed in bacteriophages and yeast to select a backbone with desired properties. Adnectins TM This is an exemplary skeleton generated using the 10th FN3 domain, which is randomized and displayed in this manner. Another exemplary skeleton containing an FN3 domain is Centyrin. TM Centryrins TM A shared sequence containing the FN3 domain of human tendinogenic protein C (TNC), which is present in the extracellular matrix of various tissues. Centyrin TMThe backbone has loops structurally homologous to the antibody variable domains (i.e., CDR1, CDR2, and CDR3) and is a small (approximately 10 kDa), simple, and highly stable single-domain protein free of cysteine, disulfide bonds, or glycosylated residues. Centyrin TM It possesses excellent biophysical properties, such as stability to heat, pH, denaturants, and organic solvents, reversible unfolding, and monodispersity. Another recent exemplary FN3-based backbone that can be used in this disclosure is the wave-regulated affinity protein (FLAP), as described in See et al., 2020. Biotechnology Journal 15(12):e2000078, which is incorporated herein by reference in its entirety.

[0414] Fusion proteins and conjugates

[0415] In one aspect, this document provides fusion proteins and conjugates comprising at least one anti-TRAILR2 antigen-binding protein (e.g., an antibody, such as a single-domain antibody) directly or indirectly linked to one or more additional domains or portions. At least one anti-TRAILR2 antigen-binding protein specifically binds to TRAILR2. In some embodiments, the fusion proteins or conjugates of this disclosure comprise a single polypeptide. In other embodiments, the fusion proteins or conjugates of this disclosure comprise more than one polypeptide. In some embodiments, the fusion proteins or conjugates of this disclosure comprise two polypeptides.

[0416] In some embodiments, the fusion protein or conjugate of this disclosure comprises at least one anti-TRAILR2 antigen-binding protein (e.g., an antibody, such as a single-domain antibody) described herein. In some embodiments, the fusion protein or conjugate is multivalent. For example, the fusion protein or conjugate of this disclosure may be at least bivalent, but may also be, for example, trivalent, tetravalent, pentavalent, hexavalent, etc. The terms “bivalent,” “trivalent,” “tetravalent,” “pentavalent,” or “hexavalent” are all part of the term “multivalent” and respectively indicate the presence of two, three, four, five, or six binding units (e.g., VHH).

[0417] In some embodiments, the fusion protein or conjugate is multispecific. For example, in some cases, one or more additional domains or portions may be one or more additional binding domains that bind one or more other antigens or proteins. The fusion protein or conjugate of this disclosure may be, for example, bispecific, trispecific, tetraspecific, pentaspecific, etc. The terms “bispecific,” “trispecific,” “tetraspecific,” “pentaspecific,” etc., all fall under the term “multispecific,” and respectively refer to binding to two, three, four, five, etc., different target molecules.

[0418] In some embodiments, the fusion protein or conjugate of this disclosure comprises one or more of the anti-TRAILR2 antigen-binding proteins described herein. In some embodiments, the fusion protein or conjugate of this disclosure comprises two or more of the anti-TRAILR2 antigen-binding proteins described herein. In some embodiments, the fusion protein or conjugate of this disclosure comprises two or more of the anti-TRAILR2 antigen-binding proteins described herein. In some embodiments, the fusion protein or conjugate of this disclosure comprises four or more of the anti-TRAILR2 antigen-binding proteins described herein. In some embodiments, the fusion protein or conjugate of this disclosure comprises one, two, three, four, five, six, seven, eight, nine, or ten or more of the anti-TRAILR2 antigen-binding proteins described herein.

[0419] In some embodiments, one or more antigen-binding proteins may bind to the same epitope on TRAILR2. In some embodiments, one or more antigen-binding proteins may bind to different epitopes on TRAILR2.

[0420] In various embodiments, one or more antigen-binding proteins may be one or more single-domain antibodies disclosed herein, such as one or more VHHs disclosed herein.

[0421] When two or more anti-TRAILR2 antigen-binding proteins are included in a fusion protein or conjugate, the two or more anti-TRAILR2 antigen-binding proteins may contain the same sequence or different sequences. In such embodiments, the two or more anti-TRAILR2 antigen-binding proteins may bind to the same epitope on TRAILR2 or different epitopes on TRAILR2. For example, the fusion protein or conjugate of this disclosure may be biparatopic, such as in the case where two VHHs bind to two different epitopes on TRAILR2.

[0422] An exemplary design of a multivalent anti-TRAILR2 fusion construct containing two or more anti-TRAILR2 binding units (e.g., VHH) is shown in Figure 13 middle.

[0423] Fusion or coupling with Fc region

[0424] In some embodiments, the fusion protein or conjugate of this disclosure comprises at least one anti-TRAILR2 antigen-binding protein (e.g., an antibody, such as a single-domain antibody) operatively linked to a dimerizing domain, such as an immunoglobulin Fc region. The immunoglobulin Fc region may be indirectly or directly linked to at least one anti-TRAILR2 antigen-binding protein (e.g., an antibody, such as a single-domain antibody). In some embodiments, the fusion protein or conjugate of this disclosure comprises one, two, three, four, five, six, or more anti-TRAILR2 antigen-binding proteins operatively linked to an Fc region as provided herein.

[0425] As used herein, "Fc region" refers to the portion of the heavy chain constant region containing CH2 and CH3. In some embodiments, the Fc region includes a hinge, CH2, and CH3. In various embodiments, when the Fc region includes a hinge, the hinge can mediate dimerization between two Fc-containing polypeptides. The Fc region included in the fusion protein or conjugate can be an Fc region from any species, or derived from any species, including (but not limited to) humans, mice, rats, monkeys (e.g., cynomolgus monkeys), camels, llamas, sharks, goats, rabbits, and / or cattle. In various embodiments, the Fc region included in the fusion protein or conjugate included in this disclosure is a human immunoglobulin Fc region or is derived from a human immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region is an IgG, IgE, IgM, IgD, IgA, or IgY isotype. In some embodiments, the immunoglobulin Fc region is an IgG isotype, such as IgG1, IgG2, IgG3, or IgG4 subclasses. The Fc region of immunoglobulins may contain variants or fragments of the native IgG Fc region.

[0426] In some embodiments, the Fc region included in the fusion protein or conjugate described herein may be a murine (e.g., mouse or rat) immunoglobulin Fc region, or a murine immunoglobulin Fc region derived therefrom. In some embodiments, the Fc region included in the fusion protein or conjugate described herein may be a cynomolgus monkey immunoglobulin Fc region, or a cynomolgus monkey immunoglobulin Fc region derived therefrom.

[0427] Natural Fc regions typically possess effector functions, including but not limited to: Fc receptor binding; Clq binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phage activity; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. Such effector functions generally require the Fc region to be combined with a binding domain (e.g., antibody variable domain) and can be assessed using various assays.

[0428] In some embodiments, the fusion protein or conjugate of this disclosure may comprise an Fc region dimer. In some embodiments, the Fc region mediates the dimerization of TRAILR2 binding units under physiological conditions (such as when expressed by an autologous cell) to form a dimer that doubles the number of TRAILR2 binding units. For example, a fusion polypeptide comprising a TRAILR2-binding VHH domain and an Fc region is monovalent as a monomer, but the Fc region can mediate dimerization; therefore, the fusion protein is divalent (i.e., each molecule has two anti-TRAILR2 VHH domains). Similarly, in some embodiments, two anti-TRAILR2 VHH domains (2×) are fused to the IgG Fc region, and due to dimerization, the fusion protein is tetravalent (i.e., each molecule has four anti-TRAILR2 VHH domains). In some implementations, three anti-TRAILR2 VHH domains (3×) are fused to the IgG Fc region, and due to dimerization, the fusion protein is hexavalent (i.e., each molecule has six anti-TRAILR2 VHH domains).

[0429] In some embodiments, the fusion protein or conjugate of this disclosure may comprise two polypeptide chains, each polypeptide chain having the following structure: (anti-TRAILR2 VHH)n-linker-Fc, where n can be any integer (e.g., 1, 2, 3, 4, 5, etc.). When n ≥ 2, each anti-TRAILR2 VHH may optionally be operatively linked to another anti-TRAILR2 VHH via a linker.

[0430] In some embodiments, the fusion protein or conjugate of this disclosure may comprise two polypeptide chains, each having the following structure: (anti-TRAILR2 VHH)n-linker-Fc-(anti-TRAILR2 VHH)m, where n and m may be independently any integer (e.g., 1, 2, 3, 4, 5, etc.). When n ≥ 2 or m ≥ 2, each anti-TRAILR2 VHH may optionally be operatively linked to another anti-TRAILR2 VHH via a linker.

[0431] In some embodiments, the fusion protein or conjugate of this disclosure is bivalent. In some embodiments, the bivalent fusion protein or conjugate of this disclosure comprises two polypeptide chains, each polypeptide chain having the following structure: (anti-TRAILR2VHH)-linker-Fc.

[0432] In some embodiments, the fusion protein or conjugate of this disclosure is tetravalent. In some embodiments, the tetravalent fusion protein or conjugate of this disclosure comprises two polypeptide chains, each having the following structure: (anti-TRAILR2VHH)-linker-(anti-TRAILR2 VHH)-linker-Fc. In some embodiments, the tetravalent fusion protein or conjugate of this disclosure comprises two polypeptide chains, each having the following structure: (anti-TRAILR2 VHH)-linker-Fc-linker-(anti-TRAILR2 VHH). The multiple linkers used in the fusion protein may not be identical.

[0433] In some embodiments, the fusion protein or conjugate of this disclosure is hexavalent. In some embodiments, the hexavalent fusion protein or conjugate of this disclosure comprises two polypeptide chains, each having the following structure: (anti-TRAILR2VHH)-linker-(anti-TRAILR2 VHH)-linker-(anti-TRAILR2 VHH)-linker-Fc. In some embodiments, the hexavalent fusion protein or conjugate of this disclosure comprises two polypeptide chains, each having the following structure: (anti-TRAILR2VHH)-linker-(anti-TRAILR2 VHH)-linker-Fc-linker-(anti-TRAILR2 VHH). In some embodiments, the hexavalent fusion protein or conjugate of this disclosure comprises two polypeptide chains, each having the following structure: (anti-TRAILR2VHH)-linker-Fc-linker-(anti-TRAILR2 VHH)-linker-(anti-TRAILR2 VHH). The multiple linkers used in the fusion protein may not be the same.

[0434] In some implementations, the CH3 domain of the Fc region can be used as a homodimerization domain so that the resulting fusion protein is formed from two identical polypeptides. In other cases, the CH3 dimer interface region of the Fc region can be mutated to achieve heterodimerization. For example, the heterodimerization domain can be incorporated into the fusion protein so that the construct is a heterodimeric fusion protein.

[0435] When the Fc region dimer is used in the fusion protein or conjugate of this disclosure, the first Fc region and the second Fc region can be the same IgG isotype, such as IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4. Alternatively, the first Fc region and the second Fc region can be different IgG isotypes, such as IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0436] In some embodiments, the Fc region in the fusion protein or conjugate included in this disclosure may be mutated or modified. In some embodiments, the mutation comprises one or more amino acid substitutions to reduce the effector function of the Fc region. Various examples of mutations into the Fc region to alter (such as reduce) effector function are known, including any of the examples described below. Generally, residues in the immunoglobulin heavy chain or portions thereof (such as the Fc region) are numbered according to the EU index of Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0437] In some implementations, the human IgG Fc region is modified to alter antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). Non-limiting examples of amino acid modifications that can alter ADCC and / or CDC are described in Alegre et al., 1992 J Immunol, 148: 3461-3468; Idusogie et al., 2001 J Immunol, 166(4): 2571-5; Shields et al., 2001 JBC, 276(9): 6591-6604; Lazar et al., 2006 PNAS, 103(11): 4005-4010; Stavenhagen et al., 2007 Cancer Res, 67(18): 8882-8890; Natsume et al., 2008 Cancer Res, 68(10): 3863-72; Stavenhagen et al., 2008 Advan. EnzymeRegul., 48: 152-164; Moore et al., 2010 mAbs, 2(2): 181-189; and Kaneko and Niwa, 2011 Biodrugs, 25(1):1-11, each of which is incorporated herein by reference in its entirety.

[0438] In some embodiments, the Fc region in the fusion protein or conjugate included in this disclosure exhibits reduced effector functions (such as CDC and ADCC). Various in vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the fusion protein construct and / or its cleaved components lack FcγR binding (and therefore may lack ADCC activity), but retain FcRn binding capacity. Primary cells used to modulate ADCC are NK cells expressing only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Non-limiting examples of in vitro assays for assessing ADCC activity of molecules of interest are described, for example, in US 5,500,362; US 5,821,337; Hellstrom et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986); and Hellstrom et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); Bruggemann et al., J. Exp. Med. 166:1351-1361 (1987). Alternatively, non-radioactive assays may be used, such as the ACTITM non-radioactive cytotoxicity assay or the CytoTox96TM non-radioactive cytotoxicity assay for flow cytometry. Effector cells suitable for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo (e.g., in animal models disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998)). C1q binding assays can also be performed to confirm that the fusion protein construct or its cleavage fractions do not bind C1q and therefore lack CDC activity (see, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402). To assess complement activation, CDC assays can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)).FcRn binding and in vivo clearance / half-life determination can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769(2006)).

[0439] Examples of mutations that enhance ADCC include modifications at Ser239 and Ile332, such as Ser239Asp and Ile332Glu (S239D, I332E). Examples of mutations that enhance CDC include modifications at Lys326 and Glu333. In some implementations, the Fc region is modified at one or both of these locations, for example, using the Kabat numbering system, Lys326Ala and / or Glu333Ala (K326A and E333A).

[0440] In some implementations, the Fc region of the fusion protein is altered at one or more of the following locations to reduce Fc receptor binding: Leu 234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Ser298 (S298), Asn297 (N297), Asn325 (N325), Ala327 (A327), or Pro329 (P329). Examples include Leu234Ala (L234A), Leu235Ala (L235A), Leu235Glu (L235E), Asp265Asn (D265N), Asp265Ala (D265A), Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala (N297A), Pro329Ala (P329A) or Pro239Gly (P329G), Asn325Glu (N325E) or Ala327Ser (A327S). In some embodiments, modifications within the Fc region reduce binding to the Fc receptor-γ receptor (FcγR) while having minimal impact on binding to the neonatal Fc receptor (FcRn).

[0441] In some embodiments, the human IgG1 Fc region is modified at amino acid Asn297 (Kabat number) to prevent fusion protein glycosylation, for example, Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu235 (Kabat number) to alter Fc receptor interaction, for example, Leu235Glu (L235E) or Leu235Ala (L235A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu234 (Kabat number) to alter Fc receptor interaction, for example, Leu234Ala (L234A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu234 (Kabat number) to alter Fc receptor interaction, for example, Leu235Glu (L235E). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234 and 235, for example, Leu234Ala and Leu235Ala (L234A / L235A) or Leu234Val and Leu235Ala (L234V / L235A). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 297, for example, Leu234Ala, Leu235Ala, and Asn297Ala (L234A / L235A / N297A). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 329, for example, Leu234Ala, Leu235Ala, and Pro239Ala (L234A / L235A / P329A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Asp265 (Kabat number) to alter Fc receptor interaction, for example, Asp265Ala (D265A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Pro329 (Kabat number) to alter Fc receptor interaction, for example, Pro329Ala (P329A) or Pro329Gly (P329G). In some embodiments, the Fc region of the fusion protein is altered at amino acids 265 and 329, for example, Asp265Ala and Pro329Ala (D265A / P329A) or Asp265Ala and Pro329Gly (D265A / P329G). In some implementations, the Fc region of the fusion protein is altered at amino acid 234, 235, and 265, for example, Leu234Ala, Leu235Ala, Asp265Ala (L234A / L235A / D265A).In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 329, for example, Leu234Ala, Leu235Ala, Pro329Gly (L234A / L235A / P329G). In other embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, 265, and 329, for example, Leu234Ala, Leu235Ala, Asp265Ala, Pro329Gly (L234A / L235A / D265A / P329G).

[0442] In some embodiments, the Fc region of the fusion protein is altered at Gly235 to reduce Fc receptor binding. For example, the Gly235 self-fusion protein is absent. In some embodiments, the human IgG1 Fc region is modified at amino acid Gly236 to enhance interaction with CD32A, for example, Gly236Ala (G236A). In some embodiments, the human IgG1 Fc region lacks Lys447 (EU index of Sequences of Proteins of Immunological Interest, Kabat et al., 1991).

[0443] In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 236, for example, Leu234Gly, Leu235Ser, Gly236Arg (L234G / L235S / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 236, for example, Leu234Ser, Leu235Thr, Gly236Arg (L234S / L235T / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 236, for example, Leu234Ser, Leu235Val, Gly236Arg (L234S / L235V / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 236, for example, Leu234Thr, Leu235Gln, Gly236Arg (L234T / L235Q / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 236, for example, Leu234Thr, Leu235Thr, Gly236Arg (L234T / L235T / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 329, for example, Leu234Thr, Leu235Thr, Pro329Gly (L234A / L235A / P329G). In some implementations, the Fc region of the fusion protein is altered at amino acids 252, 254, and 256, for example, Met252Tyr, Ser254Thr, Thr256Glu (M252Y / S254T / T256E).

[0444] In some embodiments, the Fc region of the fusion protein lacks one or more amino acids at one of the following positions to reduce Fc receptor binding: Glu233 (E233), Leu234 (L234), or Leu235 (L235). In other embodiments, the Fc region of the fusion protein lacks one or more amino acids at one of the following positions: Glu233 (E233), Leu234 (L234), or Leu235 (L235), and is modified at one or more of Asp265 (D265), Asn297 (N297), or Pro329 (P329) to reduce Fc receptor binding. For example, the Fc region included in the TRAILR2 binding peptide is derived from the human Fc domain and contains a three-amino acid deletion of E233, L234, and L235 in the downstream hinge corresponding to IgG1. In some embodiments, such Fc peptides do not bind to FcγR and are therefore referred to as "effector silent" or "effector null". For example, the deletion of these three amino acids in Fc will reduce the binding of complement protein C1q. In some embodiments, peptides in the Fc region with these three amino acids deleted retain binding to FcRn, thus exhibiting prolonged half-life and endocytic transport associated with FcRn-mediated recycling.

[0445] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG1 Fc region, having the following amino acid sequence:

[0446] IgG1 L234A, L235A (also known as the "LALA" variant) (Mutations in the following sequences are shown in bold)

[0447]

[0448] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG1 Fc region, having the following amino acid sequence:

[0449] IgG1 L234A, L235A, and P329A (also known as the "LALAPA" variant) (Mutations in the following sequences are shown in bold)

[0450]

[0451] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG1 Fc region, having the following amino acid sequence:

[0452] IgG1 D265A, N297A, and P329A (also known as the "DANAPA" variant) (Mutations in the following sequences are shown in bold)

[0453]

[0454] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG1 Fc region, having the following amino acid sequence:

[0455] IgG1 L234A, L235A, and G237A (also known as the "LALAGA" variant) (Mutations in the following sequences are shown in bold)

[0456]

[0457] In one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of the human IgG1 Fc region, having the following amino acid sequence:

[0458] IgG1 L234G / L235S / G236R (Mutations in the following sequences are shown in bold)

[0459]

[0460] In one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of the human IgG1 Fc region, having the following amino acid sequence:

[0461] IgG1 L234S / L235T / G236R (Mutations in the following sequences are shown in bold)

[0462]

[0463] In one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of the human IgG1 Fc region, having the following amino acid sequence:

[0464] IgG1 L234S / L235V / G236R (Mutations in the following sequences are shown in bold)

[0465]

[0466] In one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of the human IgG1 Fc region, having the following amino acid sequence:

[0467] IgG1 L234T / L235Q / G236R (Mutations in the following sequences are shown in bold)

[0468]

[0469] In one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of the human IgG1 Fc region, having the following amino acid sequence:

[0470] IgG1 L234T / L235T / G236R (Mutations in the following sequences are shown in bold)

[0471]

[0472] In one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of the human IgG1 Fc region, having the following amino acid sequence:

[0473] IgG1 L234A / L235A / P329G (Mutations in the following sequences are shown in bold)

[0474]

[0475] In one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of the human IgG1 Fc region, having the following amino acid sequence:

[0476] IgG1 M252Y / S254T / T256E (Mutations in the following sequences are shown in bold)

[0477]

[0478] In some implementations, the human IgG Fc region is modified to enhance FcRn binding. Examples of Fc mutations that enhance binding to FcRn include Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S254T, T256E, respectively) (Kabat ID, Dall'Acqua et al. 2006, J. Biol Chem Vol. 281(33) 23514-23524), Met428Leu and Asn434Ser (M428L, N434S) (Zalevsky et al. 2010 Nature Biotech, Vol. 28(2) 157-159), Met252Ile, Thr256Asp, Met428Leu (M252I, T256D, M428L, respectively) (Kabat et al. 1991 Sequences of Proteins of Immunological Interest EU index), and Asn434Ala. (N434A), Asn434Trp (N434W), Thr256Asp, Thr307Gln (T256D / T307Q), Thr256Asp, Thr307Trp (T256D / T307W), Met252Tyr, Thr256Asp (M252Y / T256D), Thr307Gln, Gln311Val, Ala378Val (T307Q / Q311V / A378V), Thr256Asp, His286Asp, Thr307Arg, Gln311Val, Ala378Val (T256D / H286D / T307R / Q311V / A378V), or Leu309Asp, Gln311His, Asn434Ser (L309D / Q311H / N434S) (see Ko et al., BioDrugs (2021) 35:147-157).

[0479] In some implementations, the Fc region lacks or reduces fucose attached to the N-linked glycan chain at N297. Numerous methods exist to prevent fucosylation, including, but not limited to, generation in FUT8-deficient cell lines; addition of inhibitors to mammalian cell culture media, such as chestnut extract; and metabolic engineering of production cell lines.

[0480] In some embodiments, the Fc domain included in the fusion protein or conjugate of this disclosure is derived from the human Fc domain and includes the mutants M252Y and M428V. In some embodiments, the mutated or modified Fc peptide includes the mutants M252Y and M428L using the Kabat numbering system. In some embodiments, such mutations enhance binding to FcRn at an acidic pH (close to 6.5) in the cytosome and result in loss of detectable binding at a neutral pH (about 7.2), thereby enhancing FcRn-mediated recycling and prolonging the half-life.

[0481] In some embodiments, the Fc domain included in the fusion protein or conjugate is derived from the human Fc domain and contains mutations that induce heterodimerization. In some embodiments, such mutations include those referred to as "knob" and "hole" mutations. For example, an amino acid modification within the CH3 domain at Thr366 allows it to preferentially pair with a second CH3 domain having amino acid modifications at Thr366, Leu368, and Tyr407 (e.g., Ser, Ala, and Val (T366S / L368A / Y407V), respectively), when replaced by a larger amino acid (e.g., Try (T366W)). In some embodiments, the "knob" Fc domain contains the mutated T366W. In some embodiments, the "hole" Fc domain contains the mutated T366S, L368A, and Y407V. Heterodimerization via CH3 modification can be further stabilized by introducing disulfide bonds (e.g., by changing Ser354 to Cys (S354C) and Y349 to Cys (Y349C) on the relative CH3 domains) (reviewed in Carter, 2001 Journal of Immunological Methods, 248: 7-15). In some embodiments, the Fc domain used for heterodimerization contains additional mutations, such as the mutation S354C on the first member of the heterodimeric Fc pair forming the asymmetric disulfide bond and the corresponding mutation Y349C on the second member of the heterodimeric Fc pair. In some embodiments, one member of the heterodimeric Fc pair contains a modification of H435R or H435K to prevent protein A binding while maintaining FcRn binding. In some embodiments, one member of the heterodimeric Fc pair contains a modification of H435R or H435K, while the second member of the heterodimeric Fc pair is unmodified at H435. In various embodiments, the acetyl Fc domain contains a modification of H435R or H435K (in some cases referred to as "acetyl-R" when modified to H435R), while the mortar Fc domain does not contain said modification. In some cases, the acetyl-R mutation improves the purification of the heterodimer relative to the possible homodimeric acetyl Fc domain.

[0482] In some embodiments, the human IgG Fc region is modified to prevent dimerization. In such embodiments, the fusion protein of this disclosure is a monomer. For example, the Thr366 residue is modified with charged residues, such as Thr366Lys, Thr366Arg, Thr366Asp, or Thr366Glu (T366K, T366R, T366D, or T366E, respectively), to prevent CH3-CH3 dimerization.

[0483] In some embodiments, the immunoglobulin Fc region of the fusion protein is a human IgG3 isotype or a variant thereof. In one embodiment, the IgG3 Fc region is modified at amino acid Asn297 (Kabat number) to prevent antibody glycosylation, for example, Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the human IgG3 Fc region is modified at amino acid 435 to prolong its half-life, for example, Arg435His (R435H). In some embodiments, the human IgG3 Fc region lacks Lys447 (EU index of Kabat et al., 1991).

[0484] In some embodiments, the immunoglobulin Fc region of the fusion protein is a human IgG4 isotype or a variant thereof. As a non-limiting example, the immunoglobulin Fc region of the human IgG4 isotype may have the following amino acid sequence:

[0485] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 2091)

[0486] In some embodiments, the Fc region of the human IgG4 isotype immunoglobulin may contain the amino acid sequence shown in SEQ ID NO: 2091 or a similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it.

[0487] In some implementations, the human IgG4 Fc region contains one or more mutations selected from the following: Ser228Pro (S228P), Leu235Glu (L235E), Leu235Ala (L235A), Phe234Ala (F234A) and / or Pro329Gly (P329G) according to EU numbers.

[0488] In some embodiments, the IgG4 Fc region of the fusion protein is altered at amino acids 228 and 235, for example, Ser228Pro, Leu235Glu, or Leu235Ala (S228P / L235E or S228P / L235A). In some embodiments, the IgG4 Fc region of the fusion protein is altered at amino acids 228, 234, and 235, for example, Ser228Pro, Phe234Ala, Leu235Glu, or Leu235Ala (S228P / F234A / L235E or S228P / F234A / L235A). In some embodiments, the IgG4 Fc region of the fusion protein is altered at amino acids 228, 235, and 329, for example, Ser228Pro, Leu235Glu, and P329G (S228P / L235E / P329G).

[0489] In one embodiment, the human IgG4 Fc region is modified at amino acid 235 to alter Fc receptor interaction, for example, Leu235Glu (L235E). In some embodiments, the human IgG4 Fc region is modified at amino acid Asn297 (Kabat number) to prevent antibody glycosylation, for example, Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the human IgG4 Fc region lacks Lys447 (EU index of Kabat et al. 1991).

[0490] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG4 Fc region, having the following amino acid sequence:

[0491] IgG4 S228P, L235E (Mutations in the following sequences are shown in bold)

[0492]

[0493] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG4 Fc region, having the following amino acid sequence:

[0494] IgG4 S228P, L235A (Mutations in the following sequences are shown in bold)

[0495]

[0496] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG4 Fc region, having the following amino acid sequence:

[0497] IgG4 S228P, F234A, L235E (Mutations in the following sequences are shown in bold)

[0498]

[0499] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG4 Fc region, having the following amino acid sequence:

[0500] IgG4 S228P, F234A, L235A (Mutations in the following sequences are shown in bold)

[0501]

[0502] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG4 Fc region, having the following amino acid sequence:

[0503] IgG4 P329G, S228P, L235E (Mutations in the following sequences are shown in bold)

[0504]

[0505] Additional IgG4 heavy chain modifications suitable for use in the fusion proteins or conjugates of this disclosure include those described in Tables 1 and 2 of Dumet et al., mAbs, 11:8, 1341-1350, which are incorporated herein by reference in their entirety.

[0506] In some embodiments, the fusion protein or conjugate contains an immunoglobulin hinge region. In some embodiments, the hinge region acts as a connector to link one or more TRAILR2 binding units (e.g., VHH) to the Fc region. In other embodiments, the fusion protein may contain a connector other than the hinge region to link one or more TRAILR2 binding units (e.g., VHH) to the Fc region. The hinge region may be selected from any human IgG subclass. For example, the fusion protein may contain a modified IgG1 hinge having the sequence EPKSSDKTHTCPPC (SEQ ID NO: 2161), wherein Cys220, which normally forms a disulfide bond with the C-terminal cysteine ​​residue of the light chain, is mutated to a serine residue, such as Cys220Ser (C220S). In other embodiments, the fusion protein contains a truncated hinge having the sequence DKTHTCPPC (SEQ ID NO: 2162).

[0507] In some embodiments, the fusion protein or conjugate has a modified hinge from IgG4 that is modified to prevent or reduce chain exchange, such as Ser228Pro (S228P) having the sequence ESKYGPPCPPC (SEQ ID NO: 2163).

[0508] In alternative embodiments, the fusion protein or conjugate of this disclosure may include sequences other than the Fc region to achieve polymerization (e.g., dimerization). For example, an amino acid sequence containing at least one cysteine ​​residue may be included to promote dimerization of the two polypeptides by forming a disulfide bond between them. In some embodiments, such a polymerization domain may contain one or more cysteine ​​residues or a short cysteine-containing peptide. Other polymerization domains include peptides or polypeptides that contain or are composed of leucine zippers, helical loop motifs, or coiled helical motifs.

[0509] Suitable Fc mutations for the fusion proteins disclosed herein are also discussed in, for example, Wilkinson et al., Fc-engineered antibodies with immune effector functions completely abolished. PLoS One. 2021, WO2021234402A2, US 8,969,526, EP3692065B1, and US 7,083,784, each of which is incorporated herein by reference.

[0510] Fusion or coupling with the extended half-life portion

[0511] In some embodiments, the fusion protein or conjugate of this disclosure may comprise one or more other portions that provide an increased (in vivo) half-life. An increased in vivo half-life refers to an increase in the half-life of the fusion protein or conjugate in mammals (such as human subjects) after administration.

[0512] Non-limiting examples suitable for use in the half-life extension portion of this disclosure include polyethylene glycol (PEG) molecules, serum proteins or fragments thereof, binding units that can bind to serum proteins, Fc moieties, and small proteins or peptides that can bind to serum proteins.

[0513] In some embodiments, the fusion protein or conjugate of this disclosure may include a binding moiety capable of binding to serum albumin (such as human serum albumin) or serum immunoglobulin (such as IgG). In one embodiment, the fusion protein or conjugate of this disclosure may include a binding moiety capable of binding to human serum albumin. In one embodiment, the binding moiety is a single-domain antibody (e.g., VHH).

[0514] For example, but not limited to, albumin conjugates described in, for example, WO 04 / 041865, WO 06 / 122787, WO2012 / 175400, WO 2012 / 175741, WO2015 / 173325, WO2017 / 080850, WO2017 / 085172, WO2018 / 104444, WO2018 / 134235, WO2018 / 134234, each of which is incorporated herein by reference in its entirety, may be used for fusion proteins or conjugates disclosed herein.

[0515] Integration or coupling with other parts

[0516] The anti-TRAILR2 antigen-binding protein (e.g., antibody, such as a single-domain antibody) provided herein may be directly or indirectly operably linked to a second part, such as, but not limited to, detectable markers, drugs, toxins, radionuclides, enzymes, immunomodulators, cytokines, cytotoxic agents, small molecule drugs, chemotherapeutic agents, therapeutic agents, diagnostic agents, or combinations thereof.

[0517] In some embodiments, the conjugates of this disclosure comprise a label that generates a detectable signal. Such conjugates can be used for research or diagnostic purposes, such as for in vivo cancer detection. Preferably, the label is capable of generating a detectable signal directly or indirectly. For example, the label may be a radiopaque or radioactive isotope (such as 3H, 14C, 32P, 35S, 123I, 125I, 131I); a fluorescent (fluorophore) or chemiluminescent (chromophore) compound (such as fluorescein isothiocyanate, rhodamine, or fluorescein); an enzyme (such as β-galactosidase, alkaline phosphatase, or horseradish peroxidase); a contrast agent; or a metal ion. In some implementations, radioactive atoms, such as 99Tc or 123I, are labeled for scintigraphic studies, or spin labels, such as zirconium-89, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron, are used for nuclear magnetic resonance (NMR) imaging. Zirconium-89 can also be conjugated with various metal chelating agents and antibodies, for example, for PET imaging (WO 2011 / 056983).

[0518] The anti-TRAILR2 antigen-binding proteins (e.g., antibodies, such as single-domain antibodies) of this disclosure can bind to another component (such as an epitope tag) for purposes such as purification or detection. Examples of such molecules useful for protein purification include those that present structural epitopes that can be recognized by a second molecule. This is commonly used for protein purification via affinity chromatography, where molecules are immobilized on a solid support and exposed to a heterogeneous mixture containing a target protein that can bind to a molecule capable of binding the immobilized compound. Non-limiting examples of epitope tag molecules that can be coupled to the anti-TRAILR2 antigen-binding proteins (e.g., antibodies, such as single-domain antibodies) of this disclosure for purposes such as molecular recognition include polyhistidine tags (His-tags), myc-tags, human influenza hemagglutinin (HA) tags, FLAG-tags, maltose-binding proteins, glutathione S-transferases, biotin, and streptavidin. Conjugates containing epitopes presented by these molecules can be recognized by complementary molecules, such as maltose, glutathione, nickel-containing complexes, anti-FLAG antibodies, anti-myc antibodies, anti-HA antibodies, streptavidin, or biotin. For example, the anti-TRAILR2 antigen-binding protein of this disclosure, conjugated to epitope tags from complexes of other proteins and biomolecules (e.g., DNA, RNA, carbohydrates, phospholipids, etc.), can be purified by treating a mixture with a solid-phase resin containing complementary molecules that selectively recognize and bind to epitope tags of TRAILR2 antibodies or fragments thereof. Examples of solid-phase resins include agarose beads that are compatible with purification in aqueous solutions.

[0519] In some embodiments, the conjugates disclosed herein may comprise one or more anti-TRAILR2VHH domains described herein conjugated to a therapeutic agent that may be cytotoxic, have cell growth inhibitory properties, or otherwise provide some therapeutic benefit. In some embodiments, the cytotoxic agent is a drug, a chemotherapeutic agent, a growth inhibitor, a toxin (e.g., an enzyme-active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope (e.g., a radioconjugate). Such conjugates may be suitable for, for example, treating or preventing cancer. In some embodiments, the antibody-drug conjugates described herein may allow for partial targeted delivery of a drug to a target tissue (e.g., a tumor).

[0520] In some embodiments, the conjugates disclosed herein contain toxins. In some implementations, the toxins include, for example, bacterial toxins such as diphtheria toxin; plant toxins such as ricin; small molecule toxins such as geldanamycin (Mandler et al., J. Nat. Cancer Inst. 92(19):1573-1581 (2000); Mandler et al., Bioorganic & Med. Chem. Letters 10:1025-1028 (2000); Mandler et al., Bioconjugate Chem. 13:786-791 (2002)); maytansinoids (EP 1391213; Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996)); and calicheamycin (Lode et al., Cancer Res. 58:2928). (1998); Hinman et al., Cancer Res. 53:3336-3342 (1993). The toxin exerts its cytotoxic and growth-inhibiting effects through mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Examples of other therapeutic agents that can be conjugated to the anti-TRAILR2 antigen-binding protein of this disclosure are described herein (see the "Therapeutic Uses and Other Applications" section).

[0521] In some embodiments, the anti-TRAILR2 antigen-binding protein of this disclosure (e.g., an antibody, such as a single-domain antibody) may be fused to or conjugated to one or more portions that facilitate delivery to the central nervous system (CNS) / brain. The portion that facilitates delivery of the anti-TRAILR2 antigen-binding protein to the CNS / brain may be, for example, a peptide, polypeptide, small molecule, lipid, or synthetic polymer. Various methods for delivering single-domain antibodies to the brain are described in Pothin et al., Pharmaceutics 2020, 12(10), 937, which is incorporated herein by reference in its entirety.

[0522] As a non-limiting example, the anti-TRAILR2 antigen-binding protein of this disclosure (e.g., an antibody, such as a single-domain antibody) may be fused to or conjugated to a portion (e.g., an antibody) that binds to the transferrin receptor (TfR) or insulin receptor. The transferrin receptor (TfR) is highly expressed by brain capillary endothelial cells (BCECs) forming the blood-brain barrier (BBB) ​​and has been used as a target for brain drug delivery. Monoclonal antibodies that bind to TfR (such as clone Ri7) have been shown to internalize into BCECs in vivo. As another example, the anti-TRAILR2 antigen-binding protein of this disclosure (e.g., an antibody, such as a single-domain antibody) may be coupled to: a hydrophobic fatty acid moiety, such as a C18 fatty acid (stearic acid), a C16 fatty acid (palmitic acid), or a C8 fatty acid (caprylic acid) moiety; or an amphiphilic block copolymer moiety, such as poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (pluronics or poloxamers) or poly(2-oxazoline). Various fatty acid moieties and block copolymer moieties that can be used for transbrain delivery of proteins are described, for example, in Yi and Kabanov, J Drug Target. 2013; 21(10): 940-955, which are incorporated herein by reference in their entirety.

[0523] Exemplary methods for linking a portion (such as a label) to a binding protein include those described in: Hunter et al., Nature 144:945 (1962); David et al., Biochemistry 13:1014 (1974); Pain et al., J. Immunol. Meth. 40:219 (1981); Nygren, J. Histochem. and Cytochem. 30:407 (1982); Wensel and Meares, Elsevier, NY (1983); and Colcher et al., Meth. Enzymol., 121:802-16 (1986). Other suitable methods for preparing conjugates of this disclosure include, for example, those described in WO 2009 / 067800, WO 2011 / 133886, and US2014322129, which are incorporated herein by reference in their entirety.

[0524] In some embodiments, the link between the anti-TRAILR2 antigen-binding protein and the second part can be covalent or non-covalent, for example via a biotin-streptavidin non-covalent interaction. In some embodiments, the second part can be linked to the anti-TRAILR2 antigen-binding protein using any of the various molecular biological or chemical couplings and linkages known in the art and described below. In some embodiments, a linker (such as a peptide linker, a cleavable linker, a cleavable linker, or a linker that facilitates the coupling reaction) can be used to link or couple the second part to the anti-TRAILR2 antigen-binding protein described herein.

[0525] In some embodiments, an anti-TRAILR2 antigen-binding protein (e.g., an antibody, such as a single-domain antibody) is optionally coupled to one or more second parts (e.g., about 1 to about 20 parts per molecule) via a linker. In some embodiments, the one or more second parts may be the same or different. The linker may consist of one or more linker components. For the covalent linking of an antibody to a second part, the linker typically has two reactive functional groups, i.e., it is divalent in a reactive sense. Divalent linker reagents suitable for linking two or more functional or biologically active parts (such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups) have been described, for example, in Hermanson, GT (1996) Bioconjugate Techniques; Academic Press: New York, pp. 234-242.

[0526] In some embodiments, the linker used in the couplings of this disclosure may include 6-maleimide hexanoyl (“MC”), maleimide propionyl (“MP”), valine-citrulline (“val-cit”), alanine-phenylalanine (“ala-phe”), p-aminobenzoxycarbonyl (“PAB”), 4-(2-pyridylthio)valerate N-succinimide ester (“SPP”), 4-(N-maleimidemethyl)cyclohexane-I-carboxylic acid N-succinimide ester (“SMCC”), or (4-iodo-acetyl)aminobenzoate N-succinimide ester (“STAB”), or combinations thereof.

[0527] In some embodiments, the linker used in the conjugates of this disclosure may comprise amino acid residues. Exemplary amino acid linker components include dipeptides, tripeptides, tetrapeptides, or pentapeptides. Exemplary dipeptides include valine-citrulline (VC or val-cit) and alanine-phenylalanine (AF or ala-phe). Exemplary tripeptides include glycine-valine-citrulline (Gly-val-cit) and glycine-glycine (Gly-gly-gly). The amino acid residues used in the amino acid linker components may include naturally occurring amino acids, as well as minute amino acids and non-naturally occurring amino acid analogs, such as citrulline. The amino acid linker components may be designed and optimized for their selectivity against enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C, and D, or fibrinolytic enzymes.

[0528] Conjugates of anti-TRAILR2 antigen-binding proteins (e.g., antibodies, such as single-domain antibodies) with a second part (e.g., cytotoxic agents) can be prepared using a variety of bifunctional protein conjugates, such as N-succinimide-3-(2-pyridyldithiool)propionate (SPDP), iminothiones (IT), bifunctional derivatives of imine esters (such as dimethyl diimide adipate HCl), active esters (such as bissuccinimide matrix), aldehydes (such as glutaraldehyde), diazid compounds (such as bis(p-azidobenzoyl)hexamethylenediamine), diazide derivatives (such as bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bifunctional fluorinated compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

[0529] The conjugates disclosed herein can be prepared by a variety of methods. For example, the conjugation method may include: (1) reacting the nucleophilic group of the VHH domain with a divalent linker reagent to form a VHH-linker via a covalent bond, and then reacting it with the drug moiety; or (2) reacting the nucleophilic group of the drug moiety with a divalent linker reagent to form a drug-linker via a covalent bond, and then reacting it with the nucleophilic group of the VHH domain.

[0530] Nucleophilic groups on proteins including antibodies (e.g., VHH domains) include, but are not limited to: (i) N-terminal amino groups, (ii) side-chain amino groups (e.g., lysine), (iii) side-chain thiols (e.g., cysteine), and (iv) glycosyl hydroxyl or amino groups, wherein the antibody is glycosylated. Amines, thiols, and hydroxyl groups are nucleophilic and capable of reacting with electrophilic groups on linker moieties and linker reagents to form covalent bonds, said electrophilic groups including: (i) active esters, such as NHS esters, HOBt esters, halocarbamates, and acid halides; (ii) alkyl and benzyl halides, such as haloacetamides; and (iii) aldehydes, ketones, carboxyl groups, and maleimide groups. Additional nucleophilic groups can be introduced into proteins (e.g., antibodies, such as VHH domains) via the reaction of lysine with 2-iminothione (Traut's reagent), causing the amine to convert to a thiol. Reactive thiol groups can be introduced into proteins (e.g., antibodies, such as VHH domains) by introducing one, two, three, four or more cysteine ​​residues.

[0531] Conjugates (such as antibody-drug conjugates) can also be generated by modifying antibodies (such as VHH domains) to introduce electrophilic moieties, which can react with nucleophilic substituents on the linker reagent or drug. The sugar in a glycosylated antibody can be oxidized, for example, with a periodate oxidizing agent to form an aldehyde or ketone group, which can react with the amino group of the linker reagent or drug moiety. The resulting iminoschiff base group can form a stable bond, or can be reduced, for example, with a borohydride reagent to form a stable amine bond. In one embodiment, the carbohydrate moiety of a glycosylated antibody reacts with galactose oxidase or sodium periodate to produce a carbonyl group (aldehyde and ketone) in the protein, which can react with a suitable group on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, a protein containing an N-terminal serine or threonine residue can react with sodium periodate to produce an aldehyde replacing the first amino acid. Such an aldehyde can react with the drug moiety or a linker nucleophilic reagent.

[0532] Similarly, nucleophilic groups on the pharmaceutical moiety include, but are not limited to, amine, thiol, hydroxyl, acylhydrazine, oxime, hydrazine, thiourea, hydrazine carboxylate, and aryl acylhydrazine groups capable of reacting with electrophilic groups on the linker moiety and linker reagent to form covalent bonds, wherein the electrophilic groups include: (i) active esters, such as NHS esters, HOBi esters, haloformates, and acid halides; (ii) alkyl and benzyl halides, such as haloacetamides; and (iii) aldehydes, ketones, carboxyl groups, and maleimide groups.

[0533] Alternatively, the fusion protein containing the VHH domain and the cytotoxic agent can be prepared, for example, by recombinant DNA technology or peptide synthesis. The DNA sequence can be engineered to include respective regions encoding two parts of the fusion protein, said two parts being adjacent to each other or separated by regions encoding a linker peptide that does not impair the desired properties of the fusion protein. The DNA sequence can then be transfected into host cells expressing the fusion protein. The fusion protein can be recovered from cell culture and purified using techniques known in the art.

[0534] connector

[0535] In some embodiments, one or more polypeptides of the fusion protein of this disclosure are operably linked via peptide linkers. The length of the peptide linker can range from 2 amino acids to 60 or more amino acids, and in some aspects, the length of the peptide linker is in the range of 3 to 50 amino acids, 4 to 30 amino acids, 5 to 25 amino acids, 10 to 25 amino acids, 10 to 60 amino acids, 12 to 20 amino acids, 20 to 50 amino acids, or 25 to 35 amino acids.

[0536] In some embodiments, the peptide linker (e.g., a peptide linker separating two VHH domains or a VHH domain from a heavy chain constant region) is at least 5 amino acids, at least 6 amino acids, or at least 7 amino acids in length, and optionally at most 30 amino acids, at most 40 amino acids, at most 50 amino acids, or at most 60 amino acids in length.

[0537] In some embodiments, the length of the linker ranges from 5 to 50 amino acids, for example, from 5 to 50, 5 to 45, 5 to 40, 5 to 35, 5 to 30, 5 to 25, or 5 to 20 amino acids. In other embodiments described above, the length of the linker ranges from 6 to 50 amino acids, for example, from 6 to 50, 6 to 45, 6 to 40, 6 to 35, 6 to 30, 6 to 25, or 6 to 20 amino acids. In other embodiments described above, the length of the linker ranges from 7 to 50 amino acids, for example, from 7 to 50, 7 to 45, 7 to 40, 7 to 35, 7 to 30, 7 to 25, or 7 to 20 amino acids.

[0538] In some embodiments, charged (e.g., charged hydrophilic linkers) and / or flexible linkers are used. Examples of flexible linkers that can be used in the fusion proteins of this disclosure include those disclosed by Chen et al., 2013, Adv Drug Deliv Rev. 65(10): 1357-1369 and Klein et al., 2014, Protein Engineering, Design & Selection 27(10): 325-330. Particularly suitable flexible linkers are or contain repeating sequences of glycine and serine (referred to herein as “GS linkers”), such as G... n S (SEQ ID NO: 2136) or SG n A monomer or polymer of (SEQ ID NO: 2137), where n is an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6 or 7, 8, 9 or 10. In one embodiment, the connector is or contains a repeating sequence of G4S (SEQ ID NO: 2093) (e.g., (GGGGS)). n (SEQ ID NO: 2138)) monomers or polymers.

[0539] Polyglycine linkers are suitable for use in the fusion proteins of this disclosure. In some embodiments, the peptide linkers used herein comprise two consecutive glycines (2 Gly), three consecutive glycines (3 Gly), four consecutive glycines (4 Gly) (SEQ ID NO: 2090), five consecutive glycines (5 Gly) (SEQ ID NO: 2139), six consecutive glycines (6 Gly) (SEQ ID NO: 2140), seven consecutive glycines (7 Gly) (SEQ ID NO: 2141), eight consecutive glycines (8 Gly) (SEQ ID NO: 2142), or nine consecutive glycines (9 Gly) (SEQ ID NO: 2143).

[0540] In some embodiments, the GS linker used herein comprises an amino acid sequence selected from the following: GGSGGS, i.e., (GGS)2 (SEQ ID NO: 2092); GGSGGSGGS, i.e., (GGS)3 (SEQ ID NO: 2144); GGSGGSGGSGGS, i.e., (GGS)4 (SEQ ID NO: 2145); and GGSGGSGGSGGSGGS, i.e., (GGS)5 (SEQ ID NO: 2146). In some embodiments, the fusion protein may comprise a combination of the GS linker and the glycine linker.

[0541] In one embodiment, two or more VHHs are connected via a GGGGSGGGGSGGGGS (SEQ ID NO: 2094) connector. In one embodiment, two or more VHHs are connected via a GGGGSGGGGS (SEQ ID NO: 2147) connector. In one embodiment, the VHH and Fc region are connected via a GGGGSESKYGPPCPSCP (SEQ ID NO: 2131) connector. In one embodiment, the VHH and Fc region are connected via a GGGGS (SEQ ID NO: 2093) connector.

[0542] In some embodiments, one or more polypeptides of the fusion protein of this disclosure are operatively linked via a “rigid” peptide linker. Such peptide linkers may comprise proline-rich peptides. In one embodiment, the rigid peptide linker comprises PAPAPAPAPAPAPAPAP (SEQ ID NO: 2132). In one embodiment, the rigid peptide linker comprises GGGGSPAPAPAPAPAPAPAPAPAP (SEQ ID NO: 2133). In one embodiment, the rigid peptide linker comprises PAPAPAPAPAPAPAPAPGGGGS (SEQ ID NO: 2134). In one embodiment, the rigid peptide linker comprises GGGGSPAPAPAPAPAPAPAPAPGGGGS (SEQ ID NO: 2135). In one embodiment, the rigid peptide linker comprises GGGGSPAPAPAPAPAPAPAPAPAPGGGS (SEQ ID NO: 2088).

[0543] In one embodiment, the rigid peptide linker comprises A(EAAAK)nA (SEQ ID NO: 2148), where n is any integer, such as 1, 2, 3, 4, 5, 6 or 7, 8, 9 or 10.

[0544] Other exemplary peptide linkers that can be used in the fusion proteins described herein are shown in Table 2.

[0545] Table 2. Exemplary peptide linker sequences

[0546]

[0547] signal sequence

[0548] In some embodiments, the fusion protein described herein may further include a signal sequence at its N-terminus. The signal sequence may be present in a precursor molecule of the fusion protein and may be removed during production after the protein is secreted by the host cell. In some embodiments, the signal sequence is MAVMAPRTLVLLLSGALALTQTWA (SEQ ID NO: 2164) or a fragment or variant thereof. In some embodiments, the signal sequence is MYRMQLLSCIALSLALVTNS (SEQ ID NO: 2165) or a fragment or variant thereof.

[0549] Exemplary fusion proteins of this disclosure

[0550] Non-limiting examples of fusion proteins (e.g., bivalent, trivalent, or tetravalent constructs with / without an Fc region) are disclosed in the “Examples” and “Sequence Listing” below.

[0551] Non-limiting examples of amino acid sequences of fusion proteins are described in Table 3. VHH amino acid sequences are shown in bold; linker sequences are underlined with a single line; and Fc regions are shown in italics.

[0552] Table 3. Non-limiting examples of amino acid sequences of fusion proteins

[0553]

[0554]

[0555]

[0556]

[0557] In various embodiments, the fusion protein of this disclosure comprises any one of SEQ ID NO: 2054 to 2070 or a similar sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it.

[0558] In various embodiments, the fusion protein of this disclosure comprises any one of SEQ ID NO: 2054 to 2070 or a sequence having at least 75% identity with it.

[0559] It should be understood that although the exemplary fusion proteins described herein contain non-humanized VHH amino acid sequences, these non-humanized VHH amino acid sequences may be replaced by any of the humanized VHH amino acid sequences described herein (e.g., in Tables 1-1 and 1-2).

[0560] Any of the various fusion proteins described in this article can have an agonistic effect when bound to TRAILR2.

[0561] Polynucleotide molecules

[0562] On the other hand, this document provides polynucleotide molecules encoding anti-TRAILR2 antigen-binding proteins (e.g., antibodies, including single-domain antibodies) or fusion proteins as described herein. Polynucleotide molecules encoding one or more polypeptide portions of conjugates of this disclosure are also covered within this disclosure.

[0563] In some embodiments, the polynucleotide molecules of this disclosure encode anti-TRAILR2 VHH amino acid sequences selected from SEQ ID NO: 4, 9, 14, 17, 21, 26, 31, 36, 41, 45, 50, 55, 65, 70, 75, 79, 84, 89, 93, 98, 146 to 468, 2192, 2198, 2204 and 2214 to 2233, or similar sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with them.

[0564] In some embodiments, the polynucleotide molecule of this disclosure encoding anti-TRAILR2 VHH comprises a nucleotide sequence of any one of SEQ ID NO: 57 to 61, 131 to 145, 1731 to 2053, 2193, 2199, 2205 and 2278 to 2297 or a similar sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with it.

[0565] In some embodiments, the polynucleotide molecule of this disclosure encoding anti-TRAILR2 VHH comprises a nucleotide sequence of any one of SEQ ID NO: 57 to 61, 131 to 145, 2071 to 2087, 2193, 2199 and 2205 or a similar sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with it.

[0566] In some embodiments, the polynucleotide molecule of this disclosure encoding anti-TRAILR2 VHH comprises a nucleotide sequence of any one of SEQ ID NO: 132, 137, 2193, 2199 and 2205 or a similar sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with it.

[0567] In the embodiments provided herein, the polynucleotide molecules of this disclosure are encoded by sequences selected from the humanized VHH amino acid sequences of SEQ ID NO: 5, 10, 15, 18, 22, 27, 32, 37, 42, 46, 51, 56, 66, 71, 76, 80, 85, 90, 94, 99, 469 to 761, 2194, 2200, 2206, 2234 to 2251 and 2354 to 2404 or similar sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with them.

[0568] In the embodiments provided herein, the polynucleotide molecules of this disclosure encode fusion proteins comprising an amino acid sequence selected from SEQ ID NO:2054 to 2070 or similar sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with them.

[0569] In the embodiments provided herein, the polynucleotide molecule of this disclosure encoding the fusion protein comprises a nucleotide sequence of any one of SEQ ID NO: 2071 to 2087 or a similar sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it.

[0570] Polynucleotide molecules can be used to transform / transfect host cells or host organisms, for example, for the expression and / or production of peptides. Suitable hosts or host cells for producing the anti-TRAILR2 peptides described herein include any suitable fungus, prokaryotic or eukaryotic cell or cell line, or any suitable fungus, prokaryotic or eukaryotic organism. This disclosure also covers host cells or host organisms containing polynucleotide molecules encoding the anti-TRAILR2 antigen-binding protein, peptide, or fusion protein described herein.

[0571] Polynucleotide molecules can be, for example, DNA, RNA, or hybrids thereof, and may also contain (e.g., chemically modified) nucleotides, such as locked nucleic acids (LNAs) or peptide nucleic acids (PNAs). In some embodiments, the polynucleotide is single-stranded. In some embodiments, the polynucleotide is double-stranded. In one embodiment, the polynucleotide is in the form of double-stranded DNA (e.g., a plasmid). In some embodiments, the polynucleotide is in the form of single-stranded RNA (e.g., mRNA).

[0572] Techniques for generating polynucleotides may include, for example (but not limited to), automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and / or synthetic sequences (or two or more portions thereof) to introduce mutations that result in the expression of a truncated expression product; introducing one or more restriction sites (e.g., to generate cassettes and / or regions that can be readily digested and / or linked using suitable restriction enzymes) and / or introducing mutations using a PCR reaction with one or more “mismatched” primers. Alternatively, the polynucleotides of this disclosure may be isolated from suitable natural sources. Polynucleotide sequences encoding naturally occurring (poly)peptides may, for example, be site-directed mutagenesis to produce polynucleotide molecules encoding polypeptides with sequence changes.

[0573] carrier

[0574] This document also provides vectors comprising polynucleotide molecules encoding anti-TRAILR2 antigen-binding proteins (e.g., antibodies, including single-domain antibodies), fusion proteins, or other related polypeptides disclosed herein. As used herein, “vector” refers to a medium suitable for carrying genetic material into a host cell. Vectors may include nucleic acid vectors, such as plasmids or mRNA, or nucleic acids embedded in larger structures, such as liposomes or viral vectors.

[0575] Vectors may include one or more of the following components: an origin of replication, one or more regulatory sequences (e.g., promoters, enhancers, terminators) that regulate the expression of the polypeptide of interest, and / or one or more optional marker genes (such as antibiotic resistance genes and genes that can be used in colorimetric assays, such as β-galactosidase). For DNA-based vectors, this typically includes the presence of transcriptional components (e.g., promoters and polyA signals) and translational components (e.g., Kozak sequences). In some embodiments, the vector is an expression vector, i.e., a vector suitable for expressing an encoded polypeptide or construct in a host cell under suitable conditions.

[0576] To express the anti-TRAILR2 antigen-binding protein or fusion protein (or fragments thereof) of this disclosure, polynucleotides encoding partial or full-length polypeptide chains (e.g., VHH, VHH-Fc) as described above can be inserted into an expression vector, such that the gene is operatively linked to one or more transcriptional and translational control sequences. An expression vector and expression control sequences compatible with the host cell used for expression are selected. Polynucleotides encoding two or more polypeptide chains (if present and distinct from each other) of the anti-TRAILR2 antigen-binding protein or fusion protein of this disclosure can be inserted into different vectors or optionally incorporated into the same expression vector.

[0577] In addition to polynucleotides encoding polypeptide chains that encode anti-TRAILR2 antigen-binding proteins or fusion proteins, the recombinant expression vectors of the present invention may include regulatory sequences that control the expression of genes encoding polypeptide chains in host cells. The design of the expression vector (including the selection of regulatory sequences) may depend on the selection of host cells to be transformed and / or the desired protein expression level. For example, regulatory sequences suitable for expression in mammalian host cells include viral components that guide high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenoviruses (e.g., the adenovirus major late promoter (AdMLP)), and polyomaviruses. Other examples of viral regulatory components and their sequences include those described, for example, in U.S. Patent Nos. 5,168,062, 4,510,245, and 4,968,615; the disclosure of each of these is incorporated herein by reference.

[0578] The recombinant expression vectors disclosed herein may carry additional sequences, such as sequences regulating vector replication in host cells (e.g., origin of replication) and optional marker genes. Optional marker genes facilitate the selection of host cells into which the vector has been introduced (see, for example, US 4,399,216, US 4,634,665, and US 5,179,017; the disclosures of each of these are incorporated herein by reference in their entirety). For example, optional marker genes typically confer resistance in host cells into which the vector has been introduced to antibiotics (such as ampicillin, chloramphenicol, kanamycin, or nourseothricin) or cytotoxic agents (such as G418, puromycin, blasticidin, hygromycin, or methotrexate). Suitable alternative marker genes may include the dihydrofolate reductase (DHFR) gene (for DHFR-deficient host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0579] The vectors disclosed herein may further include sequence components that enhance the translation rate of these genes or improve the stability of mRNA induced by gene transcription or nuclear export. These sequence components include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signaling sites to guide efficient transcription of the genes carried on the expression vector.

[0580] Viral vectors can be used to efficiently deliver exogenous genes into the genome of cells (e.g., eukaryotic or prokaryotic cells). Viral vectors are particularly suitable for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the genome of the target cell via general or specific transduction. This process occurs as part of the natural viral replication cycle and does not require the addition of proteins or reagents to induce gene integration. Examples of suitable viral vectors include retroviruses; adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48); parvoviruses (e.g., adeno-associated viruses (AAVs), such as AAV2, AAV8, and AAV9); negative-strand RNA viruses, such as orthomyxoviruses (e.g., influenza virus); rhabdoviruses (e.g., rabies virus and herpestostomatitis virus); paramyxoviruses (e.g., measles virus and Sendai virus); positive-strand RNA viruses (e.g., picoviruses and alphaviruses); and double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), baculoviruses, coronaviruses, and poxviruses (e.g., vaccinia virus, modified Ankara vaccinia virus). vaccinia Ankara (MVA), fowlpox virus, and canarypox virus. Other viruses suitable for delivering polynucleotides encoding the polypeptides disclosed herein include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include, but are not limited to, avian leukosis sarcoma virus, mammalian type C virus, mammalian type B virus, mammalian type D virus, HTLV-BLV group, lentivirus, foamy virus (Coffin, JM 1996. Fundamental Virology, DMKDN Fields, PM Howley ed. (Philadelphia, Lippincott-Raven Publishers: 763-843., the disclosure of which is incorporated herein by reference).Other examples of viral genomes applicable to the compositions and methods of this disclosure include murine leukemia virus, murine sarcoma virus, mouse mammary cancer virus, bovine leukemia virus, feline sarcoma virus, feline leukemia virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentiviruses.

[0581] host cells

[0582] In one aspect, this disclosure also provides a host cell or host organism comprising a polynucleotide or vector encoding an anti-TRAILR2 antigen-binding protein (e.g., an antibody, including a single-domain antibody), a fusion protein, or other related polypeptide as described herein. Suitable host cells or host organisms may be any suitable fungus, prokaryotic or eukaryotic cell or cell line, or any suitable fungus, prokaryotic or eukaryotic organism. Host cells include progeny of a single host cell, and said progeny may be attributable to natural, accidental, or intentional mutations and are not necessarily completely identical to the original parent cell (in terms of morphology or genomic DNA complement). Host cells may also include cells transfected in vivo via the polynucleotide or vector provided herein.

[0583] Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast (e.g., Saccharomyces cerevisiae or Pichia pastoris); plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6 cells, etc. ® Crucells, COS cells, SP2 / 0 cells, and 293 and CHO cells, and their derivatives, such as 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells. Exemplary prokaryotic cells include bacterial cells, such as Escherichia coli.

[0584] Preparation method

[0585] This disclosure also provides methods for generating anti-TRAILR2 antigen-binding proteins (e.g., antibodies, including single-domain antibodies), fusion proteins, or conjugates as described herein.

[0586] In some embodiments, the method may include transforming / transfecting a host cell or host organism with a polynucleotide encoding an anti-TRAILR2 antigen-binding protein (e.g., an antibody, such as a single-domain antibody), a fusion protein, or other related peptide as described herein; expressing the anti-TRAILR2 antigen-binding protein (e.g., an antibody, such as a single-domain antibody), the fusion protein, or other related peptide in the host; optionally followed by one or more isolation and / or purification steps.

[0587] When a recombinant expression vector encoding one or more polypeptides of the disclosed anti-TRAILR2 antigen-binding protein (e.g., an antibody, such as a single-domain antibody), fusion protein, or conjugate is introduced into a mammalian host cell, the host cell is cultured for a period sufficient to allow expression of one or more proteins or polypeptides in the host cell, or to secrete one or more proteins or polypeptides into the host cell into the culture medium in which they are grown. One or more proteins or polypeptides can be recovered from the culture medium using standard protein purification methods. The host cell can also be used to produce portions of intact antibodies, such as the VHH domain.

[0588] After the proteins or peptides of this disclosure are generated through recombinant expression, they can be purified by any method known in the art for purifying proteins or peptides, such as chromatography (e.g., ion exchange, affinity, particularly affinity for TRAILR2 after selection of protein A or protein G, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins. Furthermore, the proteins or peptides of this disclosure can be fused to heterologous peptide sequences (e.g., His tags) described herein or otherwise known in the art to facilitate purification or to generate the therapeutic conjugates described below. After separation, if necessary, the proteins or peptides of this disclosure can be purified, for example, by high-performance liquid chromatography or by gel filtration chromatography (such as in Superdex). TM Further purification was carried out on a column.

[0589] Pharmaceutical compositions and formulations

[0590] This disclosure also provides a composition comprising an anti-TRAILR2 antigen-binding protein of the present technology (e.g., an antibody, such as a single-domain antibody), a fusion protein or conjugate, at least one polynucleotide molecule encoding thereon, at least one carrier comprising such a polynucleotide molecule, or at least one host cell comprising said polynucleotide molecule or carrier. The composition may be a pharmaceutical composition. The composition may further comprise at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally comprise one or more other pharmaceutically active peptides and / or compounds.

[0591] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotropic and absorption-delaying agents, and analogues compatible with drug administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, which is incorporated herein by reference. Suitable examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextran solutions, and 5% human serum albumin. Liposomes and non-aqueous media, such as fixed oils, may also be used. Complementary active compounds may also be incorporated into the composition.

[0592] Examples of suitable formulations include, but are not limited to, solutions, suspensions, powders, pastes, ointments, gels, waxes, oils, lipids, and lipid-containing vesicles (such as LIPOFECTIN). TM Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., “Compendium of excipients for parenteral formulations”, PDA (1998), J PhdomainSci Technol 52:238-311.

[0593] The pharmaceutical compositions disclosed herein can be formulated according to their intended route of administration. Examples of suitable routes of administration include, for example, intravenous, subcutaneous, intratumoral, oral (e.g., buccal, sublingual), intranasal, inhalation, intraocular, intradermal, percutaneous (i.e., local), intraperitoneal, mucosal, vaginal, and rectal administration, or injection into the CNS / brain (e.g., intraspinal, intracerebral, or intrathecal administration). Solutions or suspensions intended for parenteral, intradermal, or subcutaneous administration may include the following components: sterile diluents, such as water for injection, saline solution, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; fixing oils; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as phosphates, acetates, or citrates; and tonic modifiers, such as sodium chloride or dextran. pH may be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be packaged in ampoules, disposable syringes, or multi-dose vials made of plastic or glass.

[0594] Suitable pharmaceutical compositions for injectable use include sterile aqueous solutions (in the water-soluble case) or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include, for example, physiological saline, antibacterial water, Cremophor EL®, or phosphate-buffered saline (PBS). The composition is preferably sterile and has suitable flowability. In most embodiments, the composition is stable under manufacturing and storage conditions and is protected against contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Suitable flowability can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. Prevention of microbial contamination can be achieved by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, the composition preferably includes an isotonic agent, such as sugar, polyol (e.g., mannitol, sorbitol), or sodium chloride. Extended absorption in injectable compositions can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0595] Sterile injectable solutions can be prepared by combining the desired amount of the active compound, as needed, with one or more of the ingredients or combinations thereof described above in a suitable solvent, followed by filtration and sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile medium containing a base dispersion medium and other desired ingredients from the ingredients listed above. In the case of sterile powders used to prepare sterile injectable solutions, preparation methods include vacuum drying and / or freeze-drying, which produce a powder of the active ingredient plus any additional desired ingredients from its previously sterile filtered solution.

[0596] Oral compositions may include inert diluents or edible carriers. They may be encapsulated in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound may contain excipients and be used in tablet, lozenge, capsule, or liquid form. Formulations in tablet and liquid forms may be used for protease-insensitive VHH. Oral compositions may also be prepared using fluid carriers used as mouthwashes, wherein the compound in the fluid carrier is applied orally and rinsed and spat out or swallowed. Pharmaceutically compatible binders and / or adjuvants may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following ingredients or compounds with similar properties: conjugates, such as microcrystalline cellulose, astragalus gum, or gelatin; excipients, such as starch or lactose; disintegrants, such as alginic acid, Primogel, or corn starch; lubricants, such as magnesium stearate or sterotes; gliding agents, such as colloidal silica; sweeteners, such as sucrose or saccharin; or flavoring agents, such as peppermint, methyl salicylate, or orange flavoring.

[0597] For administration by inhalation, the compound may be delivered in the form of an aerosol spray from a pressurized container or dispenser or sprayer containing a suitable propellant (e.g., a gas such as carbon dioxide).

[0598] Systemic administration is also possible via mucosal or transdermal routes. For mucosal or transdermal administration, a penetrant suitable for penetrating barriers is used in the formulation. Such penetrants are generally known in the art, and for mucosal administration, they include, for example, detergents, bile salts, and clostridial acid derivatives. Mucosal administration can be achieved via nasal sprays or suppositories. For transdermal administration, the active compound is formulated as an ointment, cream, gel, or lotion, as is generally known in the art.

[0599] The compound can also be used in the preparation of suppositories (e.g., having a conventional suppository base, such as cocoa soybean oil and other glycerides) or retention enemas.

[0600] For brain delivery, the compounds of this disclosure can be formulated to facilitate crossing the blood-brain barrier. For example, the anti-TRAILR2 antigen-binding proteins (e.g., antibodies, such as single-domain antibodies), fusion proteins, or conjugates of this disclosure can be encapsulated into brain-targeting liposomes, lipid nanoparticles, lipid microparticles, or lipid microcapsules for brain delivery. Exemplary liposome delivery systems are described in Pothin et al., Pharmaceutics 2020, 12(10), 937, which is incorporated herein by reference in its entirety.

[0601] In some embodiments, the active compound is prepared using a delivery vehicle that prevents rapid elimination of the compound from the body, such as a controlled-release formulation, including implants and microencapsulation delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, and polyanhydride can be used. Liposome suspensions can also be used as pharmaceutically acceptable delivery vehicles. These can be prepared according to methods known to those skilled in the art, for example, as described in US 4,522,811, which is incorporated herein by reference in its entirety.

[0602] For ease of administration and dosage uniformity, it is particularly advantageous to formulate oral or parenteral compositions in unit dosage forms. As used herein, a unit dosage form refers to a physically discrete unit suitable as a unit dose for a subject to be treated; each unit contains a predetermined amount of the active compound, which is calculated to bind with the desired drug delivery vehicle to produce the desired therapeutic effect. The specifications of the unit dosage forms disclosed herein depend on: the unique characteristics of the active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations in the techniques for mixing such active compounds for treating an individual.

[0603] Pharmaceutical compositions (or components thereof) may be included in kits, containers, packages, or dispensers along with instructions for use. These pharmaceutical compositions may also be included in diagnostic kits along with instructions for use.

[0604] The pharmaceutical composition is administered in an amount effective for the treatment or prevention of a specific indication. The effective therapeutic amount typically depends on the weight of the subject being treated, the subject's physical or health condition, the extent of the condition to be treated, or the age of the subject being treated. In some embodiments, the pharmaceutical composition may be administered in amounts ranging from about 50 μg / kg body weight to about 50 mg / kg body weight per dose. In some embodiments, the pharmaceutical composition may be administered in amounts ranging from about 100 μg / kg body weight to about 50 mg / kg body weight per dose. In some embodiments, the pharmaceutical composition may be administered in amounts ranging from about 100 μg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the pharmaceutical composition may be administered in amounts ranging from about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose. The frequency and duration of treatment may be adjusted depending on the severity of the condition. The effective dose and schedule for administering the pharmaceutical compositions of this disclosure may be determined empirically; for example, patient progress may be monitored through periodic assessments, and the dose adjusted accordingly. In addition, interspecies ratio adjustments for dosage can be made using methods well known in the art (e.g., Mordenti et al., 1991, Phdomainaceut. Res. 8:1351).

[0605] In some embodiments, the pharmaceutical composition may be administered in amounts ranging from about 10 mg to about 1,000 mg per dose. In some embodiments, the pharmaceutical composition may be administered in amounts ranging from about 20 mg to about 500 mg per dose. In some embodiments, the pharmaceutical composition may be administered in amounts ranging from about 20 mg to about 300 mg per dose. In some embodiments, the pharmaceutical composition may be administered in amounts ranging from about 20 mg to about 200 mg per dose.

[0606] In some embodiments of the antigen-binding protein disclosed herein administered in the form of a viral vector (e.g., AAV), the dose range and frequency of administration of the viral vector described herein may vary depending on the nature of the viral vector and the medical condition, as well as the parameters of the specific patient and the route of administration used. In some embodiments, the viral vector composition may be administered at a dose of approximately 1 × 10⁻⁶. 5 Plaque-forming units (pfu) to approximately 1 × 10 15 The dosage within the PFU range is administered to the subject, depending on the mode of administration, route of administration, and the nature of the subject's disease and condition. In some cases, the viral vector composition may be administered at a dose of approximately 1 × 10⁻⁶. 8 pfu to approximately 1×10 15 pfu, or about 1×10 10 pfu to approximately 1×10 15 pfu, or about 1×10 8 pfu to approximately 1×10 12 Dosage administration within the pfu range. More precise dosages may also depend on the subject to which they are administered. For example, a lower dose may be required if the subject is an adolescent, while a higher dose may be required if the subject is an adult. In some embodiments, a more precise dosage may be determined based on the subject's weight. In some embodiments, for example, an adolescent human subject may receive approximately 1 × 10⁻⁶ pfu. 8 pfu to approximately 1×10 10 PFU, while adult human subjects can tolerate approximately 1×10 10 pfu to approximately 1×10 12 The dosage of PFU.

[0607] Various delivery systems are known and can be used to administer the pharmaceutical compositions disclosed herein, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, intraocular, epidural, intraspinal, intracerebral, intrathecal, and oral routes. The compositions can be administered via any convenient route, such as by infusion or bolus injection, by absorption through the epithelial or mucosal lining of the skin (e.g., oral mucosa, rectal mucosa, and intestinal mucosa), and can be administered together with other biologically active agents. Administration can be systemic or local.

[0608] The disclosed pharmaceutical compositions can be delivered subcutaneously or intravenously using standard needles and syringes. Furthermore, regarding subcutaneous delivery, pen-type delivery devices are readily applicable when delivering the disclosed pharmaceutical compositions. Such pen-type delivery devices can be reusable or disposable. Reusable pen-type delivery devices typically utilize a replaceable sleeve containing the pharmaceutical composition. Once all the pharmaceutical composition in the sleeve has been administered and the sleeve is empty, the empty sleeve can be easily discarded and replaced with a new sleeve containing the pharmaceutical composition. The pen-type delivery device can then be reused. In disposable pen-type delivery devices, there is no replaceable sleeve. Instead, the disposable pen-type delivery device is pre-filled with the pharmaceutical composition contained in a reservoir within the device. Once the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0609] In some cases, the pharmaceutical composition may be delivered in a controlled-release system. In one embodiment, a pump may be used (see Langer, above; Sefton 1987 CRC Crit. Ref. Biomed. Eng. 14: 201). In another embodiment, a polymeric material may be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, the controlled-release system may be placed close to the target of the composition, thus requiring only a portion of the systemic dose (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, above, Vol. 2, pp. 115–138). Other controlled-release systems are discussed in the review in Langer, 1990, Science 249:1527–1533.

[0610] Injectable formulations may include dosage forms for intravenous, subcutaneous, intradermal, intramuscular, intratumoral, intraperitoneal, intraspinal, intracerebral, and intrathecal injections, intravenous infusions, etc. In one embodiment, the injectable formulation may be prepared, for example, by dissolving, suspending, or emulsifying the aforementioned antibody or its salt in a conventional sterile aqueous or oily medium for injection. As an aqueous medium for injection, there are, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, which may be used in combination with suitable solubilizers, such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (a polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Oily media, such as sesame oil or soybean oil, may be used in combination with solubilizers (e.g., benzyl benzoate, benzyl alcohol, etc.). Preferably, the injection thus prepared is filled into a suitable ampoule.

[0611] Advantageously, the above-described pharmaceutical compositions for oral or parenteral use are prepared in unit dose dosage forms suitable for combining with the active ingred...

Claims

1. An antigen-binding protein that specifically binds to TNF-associated apoptosis-inducing ligand receptor 2 (TRAILR2), comprising a complementarity-determining region 3 (CDR3), said CDR3 comprising an amino acid sequence selected from: a). (A / V)ASRL(P / V)FNSRSA(I / V)YTDRIYDS (SEQ ID NO: 100); b). AVRRSAWY(S / T)DSIYTVSQYDY (SEQ ID NO: 101); c).NAARSYSR(D / G / N)(G / Y)(E / R)PL(E / K)P(A / D)Y (SEQ ID NO: 102); d).AAASSWSRGG(A / G / I / V)PYGMDY (SEQ ID NO: 103); e).AADS(H / R)FRR(P / Y)(A / T / V)PG(I / Q)QYEY (SEQ ID NO: 104); f).NAA(K / R)SYHRDY(K / S)PL(K / S)(G / P)DY (SEQ ID NO: 105); g).AAAPSFGM(M / R / T)(I / N)PESYVHS (SEQ ID NO: 106); h). AANRGIMSMRLSRYDD (SEQ ID NO: 49); i). T(A / V)GP(A / T)MSYSRGGEF (SEQ ID NO: 107); j). (A / V)ADRGAISRSGAGM(D / N)Y (SEQ ID NO: 108); k).TAGP(A / S)IS(L / Y)SRGGEY (SEQ ID NO: 109); l). A(A / T)NGWGLDP(S / T)TYH(Y / D) (SEQ ID NO: 110); m). SAGWTRRIFQY (SEQ ID NO: 88); n).TAGQSISLSQGGE(H / Y) (SEQ ID NO: 111); o).KAGIRGE(T / V)Y (SEQ ID NO: 112); p). RAYNDGGEY (SEQ ID NO: 2191); q). HS(N / R)WYNL (SEQ ID NO: 2208); and r). (F / M / N)T(A / S)DY, One or more non-alanine residues in the CDR3 sequence are optionally replaced with alanine, and / or one or more alanine residues in the CDR3 sequence are optionally replaced with glycine.

2. The antigen-binding protein of claim 1, wherein the CDR3 comprises an amino acid sequence selected from the following: s). (A / G)(A / G)(A / G)(A / S)(A / S)(A / W)(A / S)(A / R)(A / G)(A / G)(A / G / I / V)(A / P)(A / Y)(A / G)(A / M)(A / D)(A / Y); t). (A / T)(A / G / V)(A / G)(A / P)(A / T)(A / M)(A / S)(A / Y)(A / S)(A / R)(A / G)(A / G)(A / E)(A / F); u). (A / R)(A / G)(A / Y)(A / N)(A / D)(A / G)(A / G)(A / E)(A / Y); v). (A / H)(A / S)(A / N / R)(A / W)(A / Y)(A / N)(A / L); and w). (A / F / M / N)(A / T)(A / G / S)(A / D)Y.

3. The antigen-binding protein according to claim 1, wherein the CDR3 comprises an amino acid sequence selected from the following: VASRPFNSRSAIYTDRIYDS (SEQ ID NO: 3); AVRRSAWYSDSIYTVSQYDY (SEQ ID NO: 8); NAARSYSRDYEPLKPDY (SEQ ID NO: 13); NAARSYSRNYEPLKPDY (SEQ ID NO: 16); NAARSYSRGGEPLKPDY (SEQ ID NO: 20); NAARSYSRGGRPLEPAY (SEQ ID NO: 25); AAASSWSRGGVPYGMDY (SEQ ID NO: 30); AADSHFRRYTPGQQYEY (SEQ ID NO: 35); NAAKSYHRDYSPLSPDY (SEQ ID NO: 40); AAAPSFGMRNPESYVHS (SEQ ID NO: 44); AANRGIMSMRLSRYDD (SEQ ID NO: 30); NAARSYSRDYEPLKPDY (SEQ ID NO: 45); NAARSYSRDYEPLKPDY (SEQ ID NO: 46 ... 49); TAGPTMSYSRGGEF (SEQ ID NO: 54); VADRGAISRSGAGMDY (SEQ ID NO: 64); AADRGAISRSGAGMDY (SEQ ID NO: 69); TAGPAISLSRGGEY (SEQ ID NO: 74); TAGPSISYSRGGEY (SEQ ID NO: 78); AANGWGLDPTTYHY (SEQ ID NO: 83); SAGWTRRIFQY (SEQ ID NO: 88); TAGQSISLSQGGEY (SEQ ID NO: 92); KAGIRGEVY (SEQ ID NO: 97); RAYNDGGEY (SEQ ID NO: 2191); HSRWYNL (SEQ ID NO: 2197); FTADY (SEQ ID NO: 2203); GAASSWSRGGVPYGMDY (SEQ ID NO: 2298); AGASSWSRGGVPYGMDY (SEQ ID NO: 2299); AAGSSWSRGGVPYGMDY (SEQ ID NO: 2300); AAAASWSRGGVPYGMDY (SEQ ID NO: 2301); AAASAWSRGGVPYGMDY (SEQ ID NO: 2302); AAASSASRGGVPYGMDY (SEQ ID NO: 2303);AAASSWARGGVPYGMDY (SEQ ID NO: 2304);AAASSWSAGGVPYGMDY (SEQ ID NO: 2305);AAASSWSRAGVPYGMDY (SEQ ID NO: 2306);AAASSWSRGAVPYGMDY (SEQ ID NO: 2307) (SEQ ID NO: 2308);AAASSWSRGGVAYGMDY (SEQ ID NO: 2309);AAASSWSRGGVPAGMDY (SEQ ID NO: 2310);AAASSWSRGGVPYAMDY (SEQ ID NO: 2311); 2312);AAASSWSRGGVPYGMAY (SEQ ID NO: 2313);AAASSWSRGGVPYGMDA (SEQ ID NO: 2314);AAGPTMSYSRGGEF (SEQ ID NO: 2315);TGGPTMSYSRGGEF (SEQ ID NO: 2315); 2316);TAAPTMSYSRGGEF (SEQ ID NO: 2317);TAGATMSYSRGGEF (SEQ ID NO: 2318);TAGPAMSYSRGGEF (SEQ ID NO: 2319);TAGPTASYSRGGEF (SEQ ID NO: 2320); NO: 2321);TAGPTMSASRGGEF (SEQ ID NO: 2322);TAGPTMSYARGGEF (SEQ ID NO: 2323);TAGPTMSYSAGGEF (SEQ ID NO: 2324);TAGPTMSYSAGGEF (SEQ ID NO: 2325); NO: 2326);TAGPTMSYSRGGAF (SEQ ID NO: 2327);TAGPTMSYSRGGEA (SEQ ID NO: 2328);AAYNDGGEY(SEQ ID NO: 2329);RGYNDGGEY (SEQ ID NO: 2330);SEQ ID NO: SEQ ID NO: 2331);RAYADGGEY (SEQ ID NO: 2332);RAYNAGGEY (SEQ ID NO: 2333);RAYNDAGEY (SEQ ID NO:2334); RAYNDGAEY (SEQ ID NO: 2335); RAYNDGGAY (SEQ ID NO: 2336); RAYNDGGEA (SEQID NO: 2337); ASRWYNL (SEQ ID NO: 2338); HARWYNL (SEQ ID NO: 2339); HSAWYNL (SEQID NO: 2340); HSRAYNL (SEQ ID NO: 2341); HSRWANL (SEQ ID NO: 2342); HSRWYAL (SEQID NO: 2343); HSRWYNA (SEQ ID NO: 2344); ATADY (SEQ ID NO: 2345); FAADY (SEQ IDNO: 2346); FTGDY (SEQ ID NO: 2347); and FTAAY (SEQ ID NO: 2348).; 4. The antigen-binding protein according to any one of claims 1 to 3, which further comprises CDR1, and the CDR1 comprises an amino acid sequence selected from the following: a). GRTFSSNL (SEQ ID NO: 1); b). GGT(F / L)(A / S)N(D / N)G (SEQ ID NO: 113); c). GRTL(D / N / S)(A / D / E)Y(A / G) (SEQ ID NO: 114); d). GRTFS(N / S)YA (SEQ ID NO: 115); ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 5. The antigen-binding protein according to claim 4, wherein the CDR1 comprises an amino acid sequence selected from the following: GRTFSSNL (SEQ ID NO: 1); GGTLANNG (SEQ ID NO: 6); GRTLDAYG (SEQ ID NO: 11); GRTLSDYA (SEQ ID NO: 23); GRTFSSYA (SEQ ID NO: 28); GRDFSNYV (SEQ ID NO: 33); GRSFSSYA (SEQ ID NO: 38); GRTFSTLA (SEQ ID NO: 43); GRTFSSDI (SEQ ID NO: 47); GRSFGDFA (SEQ ID NO: 52); GGTLSNYA (SEQ ID NO: 62); GRTLSNYA (SEQ ID NO: 67); GRSFGAQGMEG (SEQ ID NO: 72); GFTLDLGAYA (SEQ ID NO: 81); GFTFGALA (SEQ ID NO: 86); GFTLSGYA (SEQ ID NO: 95); GSIFGGYN (SEQ ID NO: 2189); GSNFRILS (SEQ ID NO: 2195); and GFTFSRYA (SEQ ID NO: 2201).

6. The antigen-binding protein according to any one of claims 1 to 5, further comprising CDR2, said CDR2 comprising an amino acid sequence selected from the following: a).VSWNGAST (SEQ ID NO: 2); b). DHR(S / T)GT (SEQ ID NO: 121); c). I(N / S)W(N / S / T)G(T / V)(D / G)T (SEQ ID NO: 122); d). LNW(N / S)G(D / E)ST (SEQ ID NO: 123); e). INWAD(E / T)T (SEQ ID NO: 124); f). INWSGG(S / T)T (SEQ ID NO: 125); g). ISWSDMSA (SEQ ID NO: 48); h). I(N / R)W(A / D / T)G(D / N)T (SEQ ID NO: 126); i). ISQ(S / T)S(D / S)T (SEQ ID NO: 127); j). (I / M)KWTGNT (SEQ ID NO: 128); k). ISN(S / T)GTTT (SEQ ID NO: 129); l). ISNDGEHI (SEQ ID NO: 87); m).ISWNGDIT (SEQ ID NO: 91); n).IT(G / S)(A / S)G(G / S)(N / S)T (SEQ ID NO: 130); o). IFISGN(D / N) (SEQ ID NO: 2207); p). (I / L)T(K / M / S)D(D / G)TT (SEQ ID NO: 2210); and q).ISS(A / G / S)(G / S)G(I / Y)(I / T / V) (SEQ ID NO: 2213).

7. The antigen-binding protein according to claim 6, wherein the CDR2 comprises an amino acid sequence selected from the following: VSWNGAST (SEQ ID NO: 2); DHRSGT (SEQ ID NO: 7); ISWTGVDT (SEQ ID NO: 12); ISWTGTDT (SEQ ID NO: 19); IWSGVDT (SEQ ID NO: 24); LNWSGEST (SEQ ID NO: 29); INWADET (SEQ ID NO: 34); INWSGGST (SEQ ID NO: 39); ISWSDMSA (SEQ ID NO: 48); IRWTGDT (SEQ ID NO: 53); ISQTSST (SEQ ID NO: 63); ISQSSDT (SEQ ID NO: 68); MKWTGNT (SEQ ID NO: 73); IKWTGNT (SEQ ID NO: 77); ISNTGTTT (SEQ ID NO: 82); ISNDGEHI (SEQ ID NO: 83); MKWTGNT (SEQ ID NO: 73); IKWTGNT (SEQ ID NO: 77); ISNTGTTT (SEQ ID NO: 82); ISNDGEHI (SEQ ID NO: 83); MKWTGNT (SEQ ID NO: 84); MKWTGNT (SEQ ID NO: 83); MKWTGNT (SEQ ID NO: 84); MKWTGNT (SEQ ID NO: 85); MKWTGNT (SEQ ID NO: 86); MKWTGNT (SEQ ID NO: 87); MKWTGNT (SEQ ID NO: 8 ... 87); ISWNGDIT (SEQ ID NO: 91); ITSAGGST (SEQ ID NO: 96); IFISGNN (SEQ ID NO: 2190); ITSDDTT (SEQ ID NO: 2196); and ISSAGGYI (SEQ ID NO: 2202).

8. An antigen-binding protein that specifically binds to TNF-associated apoptosis-inducing ligand receptor 2 (TRAILR2), comprising CDR1, which comprises an amino acid sequence selected from the following: SEQ ID NO: 1, 6, 11, 23, 28, 33, 38, 43, 47, 52, 62, 67, 72, 81, 86, 95, 762 to 1084, 2189, 2195, 2201 and 2252 to 2261; and CDR2, which comprises an amino acid sequence selected from the following: SEQ ID NO: 2, 7, 12, 19, 24, 29, 34, 39, 48, 53, 63, 68, 73, 77, 82, 87, 91, 96, 1085 to 1407, 2190, 2196, 2202 and 2262 to 2271; and / or CDR3, which comprises an amino acid sequence selected from the following: SEQ ID NO: 3, 8, 13, 16, 20, 25, 30, 35, 40, 44, 49, 54, 64, 69, 74, 78, 83, 88, 92, 97, 1408 to 1730, 2191, 2197, 2203, 2272 to 2277 and 2298 to 2348.

9. The antigen-binding protein according to any one of claims 1, 3 to 8, wherein the antigen-binding protein comprises: i) CDR1 containing the amino acid sequence GRTFSSNL (SEQ ID NO: 1), CDR2 containing the amino acid sequence VSWNGAST (SEQ ID NO: 2), and CDR3 containing the amino acid sequence VASRPPFNSRSAIYTDRIYDS (SEQ ID NO: 3); ii) CDR1 containing the amino acid sequence GGTLANNG (SEQ ID NO: 6), CDR2 containing the amino acid sequence DHRSGT (SEQ ID NO: 7), and CDR3 containing the amino acid sequence AVRRSAWYSDSIYTVSQYDY (SEQ ID NO: 8); iii) CDR1 containing the amino acid sequence GRTLDAYG (SEQ ID NO: 11), CDR2 containing the amino acid sequence ISWTGVDT (SEQ ID NO: 12), and CDR3 containing the amino acid sequence NAARSYSRDYEPLKPDY (SEQ ID NO: 13); iv). CDR1 containing the amino acid sequence GRTLDAYG (SEQ ID NO: 11), CDR2 containing the amino acid sequence ISWTGVDT (SEQ ID NO: 12), and CDR3 containing the amino acid sequence NAARSYSRNYEPLKPDY (SEQ ID NO: 16); v). CDR1 containing the amino acid sequence GRTLDAYG (SEQ ID NO: 11), CDR2 containing the amino acid sequence ISWTGTDT (SEQ ID NO: 19), and CDR3 containing the amino acid sequence NAARSYSRGGEPLKPDY (SEQ ID NO: 20); vi). CDR1 containing the amino acid sequence GRTLSDYA (SEQ ID NO: 23), CDR2 containing the amino acid sequence IWSSGVDT (SEQ ID NO: 24), and CDR3 containing the amino acid sequence NAARSYSRGGRPLEPAY (SEQ ID NO: 25); vii). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSRGGVPYGMDY (SEQ ID NO: 30); viii). CDR1 containing the amino acid sequence GRDFSNYV (SEQ ID NO: 33), CDR2 containing the amino acid sequence INWADET (SEQ ID NO: 34), and CDR3 containing the amino acid sequence AADSHFRRYTPGQQYEY (SEQ ID NO: 35); ix). CDR1 containing the amino acid sequence GRSFSSYA (SEQ ID NO: 38), CDR2 containing the amino acid sequence INNWSGGST (SEQ ID NO: 39), and CDR3 containing the amino acid sequence NAAKSYHRDYSPLSPDY (SEQ ID NO: 40); x). CDR1 containing the amino acid sequence GRTFSTLA (SEQ ID NO: 43), CDR2 containing the amino acid sequence INNWSGGST (SEQ ID NO: 39), and CDR3 containing the amino acid sequence AAAPSFGMRNPESYVHS (SEQ ID NO: 44); xi). CDR1 containing the amino acid sequence GRTFSSDI (SEQ ID NO: 47), CDR2 containing the amino acid sequence ISWSDMSA (SEQ ID NO: 48), and CDR3 containing the amino acid sequence AANRGIMSMRLSRYDD (SEQ ID NO: 49); xii). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPTMSYSRGGEF (SEQ ID NO: 54); xiii). CDR1 containing the amino acid sequence GGTLSNYA (SEQ ID NO: 62), CDR2 containing the amino acid sequence ISQTSST (SEQ ID NO: 63), and CDR3 containing the amino acid sequence VADRGAISRSGAGMDY (SEQ ID NO: 64); xiv). CDR1 containing the amino acid sequence GRTLSNYA (SEQ ID NO: 67), CDR2 containing the amino acid sequence ISQSSDT (SEQ ID NO: 68), and CDR3 containing the amino acid sequence AADRGAISRSGAGMDY (SEQ ID NO: 69); xv). CDR1 containing the amino acid sequence GRSFGAQGMEG (SEQ ID NO: 72), CDR2 containing the amino acid sequence MKWTGNT (SEQ ID NO: 73), and CDR3 containing the amino acid sequence TAGPAISLSRGGEY (SEQ ID NO: 74); xvi). CDR1 containing the amino acid sequence GRSFGAQGMEG (SEQ ID NO: 72), CDR2 containing the amino acid sequence IKWTGNT (SEQ ID NO: 77), and CDR3 containing the amino acid sequence TAGPSISYSRGGEY (SEQ ID NO: 78); xvii). CDR1 containing the amino acid sequence GFTLDLGAYA (SEQ ID NO: 81), CDR2 containing the amino acid sequence ISNTGTTT (SEQ ID NO: 82), and CDR3 containing the amino acid sequence AANGWGLDPTTYHY (SEQ ID NO: 83); xviii). CDR1 containing the amino acid sequence GFTFGALA (SEQ ID NO: 86), CDR2 containing the amino acid sequence ISNDGEHI (SEQ ID NO: 87), and CDR3 containing the amino acid sequence SAGWTRRIFQY (SEQ ID NO: 88); xix). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence ISWNGDIT (SEQ ID NO: 91), and CDR3 containing the amino acid sequence TAGQSISLSQGGEY (SEQ ID NO: 92); xx). CDR1 containing the amino acid sequence GFTLSGYA (SEQ ID NO: 95), CDR2 containing the amino acid sequence ITSAGGST (SEQ ID NO: 96), and CDR3 containing the amino acid sequence KAGIRGEVY (SEQ ID NO: 97); xxi). CDR1 containing the amino acid sequence GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence RAYNDGGEY (SEQ ID NO: 2191); xxii). CDR1 containing the amino acid sequence GSNFRILS (SEQ ID NO: 2195), CDR2 containing the amino acid sequence ITSDDTT (SEQ ID NO: 2196), and CDR3 containing the amino acid sequence HSRWYNL (SEQ ID NO: 2197); xxiii). CDR1 containing the amino acid sequence GFTFSRYA (SEQ ID NO: 2201), CDR2 containing the amino acid sequence ISSAGGYI (SEQ ID NO: 2202), and CDR3 containing the amino acid sequence FTADY (SEQ ID NO: 2203); xxiv). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence GAASSWSRGGVPYGMDY (SEQ ID NO: 2298); xxv). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AGASSWSRGGVPYGMDY (SEQ ID NO: 2299); xxvi). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAGSSWSRGGVPYGMDY (SEQ ID NO: 2300); xxvii). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAAASWSRGGVPYGMDY (SEQ ID NO: 2301); xxviii). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASAWSRGGVPYGMDY (SEQ ID NO: 2302); xxix). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSASRGGVPYGMDY (SEQ ID NO: 2303); xxx). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWARGGVPYGMDY (SEQ ID NO: 2304); xxxi). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSAGGVPYGMDY (SEQ ID NO: 2305); xxxii). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSRAGVPYGMDY (SEQ ID NO: 2306); xxxiii). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSRGAVPYGMDY (SEQ ID NO: 2307); xxxiv). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSRGGAPYGMDY (SEQ ID NO: 2308); xxxv). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSRGGVAYGMDY (SEQ ID NO: 2309); xxxvi). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSRGGVPAGMDY (SEQ ID NO: 2310); xxxvii). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSRGGVPYAMDY (SEQ ID NO: 2311); xxxviii). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSRGGVPYGADY (SEQ ID NO: 2312); xxxix). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSRGGVPYGMAY (SEQ ID NO: 2313); xl). CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSRGGVPYGMDA (SEQ ID NO: 2314); xli). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence AAGPTMSYSRGGEF (SEQ ID NO: 2315); xlii). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TGGPTMSYSRGGEF (SEQ ID NO: 2316); xliii). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAAPTMSYSRGGEF (SEQ ID NO: 2317); xliv). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGATMSYSRGGEF (SEQ ID NO: 2318); xlv). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPAMSYSRGGEF (SEQ ID NO: 2319); xlvi). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPTASYSRGGEF (SEQ ID NO: 2320); xlvii). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPTMAYSRGGEF (SEQ ID NO: 2321); xlviii). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPTMSASRGGEF (SEQ ID NO: 2322); xlix). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPTMSYARGGEF (SEQ ID NO: 2323); l) CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPTMSYSAGGEF (SEQ ID NO: 2324); li). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPTMSYSRAGEF (SEQ ID NO: 2325); lii). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPTMSYSRGAEF (SEQ ID NO: 2326); liii). CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPTMSYSRGGAF (SEQ ID NO: 2327); CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPTMSYSRGGEA (SEQ ID NO: 2328); lv). CDR1 containing the amino acid sequence GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence AAYNDGGEY (SEQ ID NO: 2329); lvi). CDR1 containing the amino acid sequence GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence RGYNDGGEY (SEQ ID NO: 2330); lvii). CDR1 containing the amino acid sequence GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence RAANDGGEY (SEQ ID NO: 2331); lviii). CDR1 containing the amino acid sequence GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence RAYADGGEY (SEQ ID NO: 2332); CDR1 containing the amino acid sequence GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence RAYNAGGEY (SEQ ID NO: 2333); lx). CDR1 containing the amino acid sequence GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence RAYNDAGEY (SEQ ID NO: 2334); lxi). CDR1 containing the amino acid sequence GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence RAYNDGAEY (SEQ ID NO: 2335); lxii). CDR1 containing the amino acid sequence GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence RAYNDGGAY (SEQ ID NO: 2336); lxiii). CDR1 containing the amino acid sequence GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence RAYNDGGEA (SEQ ID NO: 2337); (lxiv). CDR1 containing the amino acid sequence GSNFRILS (SEQ ID NO: 2195), CDR2 containing the amino acid sequence ITSDDTT (SEQ ID NO: 2196), and CDR3 containing the amino acid sequence ASRWYNL (SEQ ID NO: 2338). lxv). CDR1 containing the amino acid sequence GSNFRILS (SEQ ID NO: 2195), CDR2 containing the amino acid sequence ITSDDTT (SEQ ID NO: 2196), and CDR3 containing the amino acid sequence HARWYNL (SEQ ID NO: 2339); lxvi). CDR1 containing the amino acid sequence GSNFRILS (SEQ ID NO: 2195), CDR2 containing the amino acid sequence ITSDDTT (SEQ ID NO: 2196), and CDR3 containing the amino acid sequence HSAWYNL (SEQ ID NO: 2340); lxvii). CDR1 containing the amino acid sequence GSNFRILS (SEQ ID NO: 2195), CDR2 containing the amino acid sequence ITSDDTT (SEQ ID NO: 2196), and CDR3 containing the amino acid sequence HSRAYNL (SEQ ID NO: 2341); lxviii). CDR1 containing the amino acid sequence GSNFRILS (SEQ ID NO: 2195), CDR2 containing the amino acid sequence ITSDDTT (SEQ ID NO: 2196), and CDR3 containing the amino acid sequence HSRWANL (SEQ ID NO: 2342); lxix). CDR1 containing the amino acid sequence GSNFRILS (SEQ ID NO: 2195), CDR2 containing the amino acid sequence ITSDDTT (SEQ ID NO: 2196), and CDR3 containing the amino acid sequence HSRWYAL (SEQ ID NO: 2343). lxx). CDR1 containing the amino acid sequence GSNFRILS (SEQ ID NO: 2195), CDR2 containing the amino acid sequence ITSDDTT (SEQ ID NO: 2196), and CDR3 containing the amino acid sequence HSRWYNA (SEQ ID NO: 2344); lxxi). CDR1 containing the amino acid sequence GFTFSRYA (SEQ ID NO: 2201), CDR2 containing the amino acid sequence ISSAGGYI (SEQ ID NO: 2202), and CDR3 containing the amino acid sequence ATADY (SEQ ID NO: 2345); lxxii). CDR1 containing the amino acid sequence GFTFSRYA (SEQ ID NO: 2201), CDR2 containing the amino acid sequence ISSAGGYI (SEQ ID NO: 2202), and CDR3 containing the amino acid sequence FAADY (SEQ ID NO: 2346); lxxiii). CDR1 containing the amino acid sequence GFTFSRYA (SEQ ID NO: 2201), CDR2 containing the amino acid sequence ISSAGGYI (SEQ ID NO: 2202), and CDR3 containing the amino acid sequence FTGDY (SEQ ID NO: 2347); or lxxiv). CDR1 containing the amino acid sequence GFTFSRYA (SEQ ID NO: 2201), CDR2 containing the amino acid sequence ISSAGGYI (SEQ ID NO: 2202), and CDR3 containing the amino acid sequence FTAAY (SEQ ID NO: 2348).

10. The antigen-binding protein according to any one of claims 1 to 9, wherein the antigen-binding protein comprises: i) CDR1 containing the amino acid sequence GRTFSSYA (SEQ ID NO: 28), CDR2 containing the amino acid sequence LNWSGEST (SEQ ID NO: 29), and CDR3 containing the amino acid sequence AAASSWSRGGVPYGMDY (SEQ ID NO: 30); ii) CDR1 containing the amino acid sequence GRSFGDFA (SEQ ID NO: 52), CDR2 containing the amino acid sequence IRWTGDT (SEQ ID NO: 53), and CDR3 containing the amino acid sequence TAGPTMSYSRGGEF (SEQ ID NO: 54); iii) CDR1 containing the amino acid sequence GSIFGGYN (SEQ ID NO: 2189), CDR2 containing the amino acid sequence IFISGNN (SEQ ID NO: 2190), and CDR3 containing the amino acid sequence RAYNDGGEY (SEQ ID NO: 2191); iv) CDR1 containing the amino acid sequence GSNFRILS (SEQ ID NO: 2195), CDR2 containing the amino acid sequence ITSDDTT (SEQ ID NO: 2196), and CDR3 containing the amino acid sequence HSRWYNL (SEQ ID NO: 2197); or v). CDR1 containing the amino acid sequence GFTFSRYA (SEQ ID NO: 2201), CDR2 containing the amino acid sequence ISSAGGYI (SEQ ID NO: 2202), and CDR3 containing the amino acid sequence FTADY (SEQ ID NO: 2203).

11. The antigen-binding protein according to any one of claims 1 to 10, wherein the antigen-binding protein is a single-domain antibody.

12. The antigen-binding protein according to claim 11, wherein the single-domain antibody is a VHH, VNAR, or VH domain.

13. The antigen-binding protein according to claim 12, wherein the VHH is a camel VHH.

14. The antigen-binding protein of claim 13, wherein the VHH comprises an amino acid sequence selected from any one of SEQ ID NO: 4, 9, 14, 17, 21, 26, 31, 36, 41, 45, 50, 55, 65, 70, 75, 79, 84, 89, 93, 98, 146 to 468, 2192, 2198, 2204 and 2214 to 2233, or an amino acid sequence having at least 75% identity with such sequences.

15. The antigen-binding protein according to claim 13 or 14, wherein the VHH comprises an amino acid sequence selected from any one of SEQ ID NO: 4, 9, 14, 17, 21, 26, 31, 36, 41, 45, 50, 55, 65, 70, 75, 79, 84, 89, 93, 98, 2192, 2198, and 2204, or an amino acid sequence having at least 75% identity with such sequence.

16. The antigen-binding protein according to any one of claims 13 to 15, wherein the VHH comprises an amino acid sequence selected from any one of SEQ ID NO: 31, 55, 2192, 2198 and 2204 or an amino acid sequence having at least 75% identity with it.

17. The antigen-binding protein of claim 12, wherein the VHH is a humanized VHH.

18. The antigen-binding protein of claim 17, wherein the humanized VHH comprises an amino acid sequence selected from any one of SEQ ID NO: 5, 10, 15, 18, 22, 27, 32, 37, 42, 46, 51, 56, 66, 71, 76, 80, 85, 90, 94, 99, 469 to 761, 2194, 2200, 2206, 2234 to 2251, and 2354 to 2404, or an amino acid sequence having at least 75% identity with such sequences.

19. The antigen-binding protein according to claim 17 or 18, wherein the humanized VHH comprises an amino acid sequence selected from any one of SEQ ID NO: 5, 10, 15, 18, 22, 27, 32, 37, 42, 46, 51, 56, 61, 66, 71, 76, 80, 85, 90, 94, 99, 2194, 2200, 2206 and 2354 to 2404 or an amino acid sequence having at least 75% identity with such sequences.

20. The antigen-binding protein according to any one of claims 17 to 19, wherein the humanized VHH comprises an amino acid sequence selected from any one of SEQ ID NO: 32, 56, 2200, 2206, 2194 and 2354 to 2404 or an amino acid sequence having at least 75% identity with it.

21. The antigen-binding protein according to any one of claims 1 to 20, wherein the antigen-binding protein binds to human TRAILR2.

22. The antigen-binding protein of claim 21, wherein the antigen-binding protein has a concentration of less than about 3 × 10⁻⁶. -7 M of K D It combines with human TRAILR2.

23. The antigen-binding protein of claim 22, wherein the antigen-binding protein is expressed in terms of about 1 × 10⁻⁶. -10 Approximately 5×10 -8 M of K D It combines with human TRAILR2.

24. The antigen-binding protein according to any one of claims 1 to 23, wherein the antigen-binding protein binds to TRAILR2 in cynomolgus monkeys.

25. The antigen-binding protein of claim 24, wherein the antigen-binding protein has a concentration of less than about 3 × 10⁻⁶. -7 M of K D It binds to the cynomolgus monkey TRAILR2.

26. The antigen-binding protein of claim 25, wherein the antigen-binding protein is expressed in terms of about 1 × 10⁻⁶. -9 Up to 1×10 -7 M of K D It binds to the cynomolgus monkey TRAILR2.

27. The antigen-binding protein according to any one of claims 1 to 26, wherein the antigen-binding protein does not block the binding of TNF-associated apoptosis-inducing ligand (TRAIL) to TRAILR2.

28. The antigen-binding protein according to any one of claims 1 to 26, wherein the antigen-binding protein blocks the binding of TRAIL to TRAILR2.

29. The antigen-binding protein according to any one of claims 1 to 28, wherein the antigen-binding protein does not specifically bind to TRAILR1, TRAILR3 and / or TRAILR4.

30. The antigen-binding protein according to any one of claims 1 to 28, wherein the antigen-binding protein binds to the cysteine-rich domain (CRD) 1 (CRD1) and / or CRD2 of TRAILR2.

31. The antigen-binding protein of claim 30, wherein the antigen-binding protein binds to CRD1 of TRAILR2.

32. The antigen-binding protein of claim 30, wherein the antigen-binding protein binds to the CRD2 of TRAILR2.

33. The antigen-binding protein of claim 30, wherein the antigen-binding protein binds to CRD1 and CRD2 of TRAILR2.

34. The antigen-binding protein according to any one of claims 1 to 28, wherein the antigen-binding protein binds to CRD2 and CRD3 of TRAILR2.

35. The antigen-binding protein according to any one of claims 1 to 34, wherein the antigen-binding protein comprises one or more modifications that reduce the binding of the antigen-binding protein to pre-existing antibodies present in human blood or serum.

36. A fusion protein that specifically binds to TNF-associated apoptosis-inducing ligand receptor 2 (TRAILR2), comprising one or more antigen-binding proteins according to any one of claims 1 to 35.

37. The fusion protein of claim 36, comprising three or more of the antigen-binding proteins.

38. The fusion protein of claim 37, comprising the four antigen-binding proteins.

39. The fusion protein according to any one of claims 36 to 38, wherein one or more of the antigen-binding proteins bind to the same epitope on TRAILR2.

40. The fusion protein according to any one of claims 36 to 38, wherein one or more of the antigen-binding proteins bind to different epitopes on TRAILR2.

41. The fusion protein according to any one of claims 36 to 40, wherein one or more of the antigen-binding proteins are one or more single-domain antibodies.

42. The fusion protein according to claim 41, wherein the one or more single-domain antibodies are one or more VHHs.

43. The fusion protein according to any one of claims 36 to 42, further comprising an immunoglobulin Fc region.

44. The fusion protein according to claim 43, wherein the immunoglobulin Fc region is the Fc region of human immunoglobulin.

45. The fusion protein of claim 44, wherein the immunoglobulin Fc region is the Fc region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof.

46. ​​The fusion protein of claim 45, wherein the immunoglobulin Fc region is the Fc region of human IgG1 or a variant thereof.

47. The fusion protein of claim 46, wherein the Fc region of said human IgG1 comprises one or more mutations selected from the group consisting of: Leu234Ala (L234A), Leu234Gly (L234G), Leu234Ser (L234S), Leu234Thr (L234T), Leu234Ala (L234A), Leu235Ala (L235A), Leu235Glu (L235E), Leu235Ser (L235S), Leu235Thr (L235T), Leu235Val (L235V), Leu235Gln (L235Q), Gly236Arg (G236R), Met252Tyr (M252Y), Ser254Thr (S254T), Thr256Glu (T256E), Asp265Asn (D265N), Asp265Ala (D265A), Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala (N297A), Pro329Ala (P329A), Pro239Gly (P329G), Asn325Glu (N325E) and / or Ala327Ser (A327S).

48. The fusion protein of claim 47, wherein the Fc region of said human IgG1 comprises a mutation selected from the group consisting of: 1) L234A and L235A; 2) L234A, L235A, and P329A; 3) D265A, N297A, and P329A; 4) L234A, L235A, and G237A; 5) L234G, L235S, and G236R; 6) L234S, L235T, and G236R; 7). L234S, L235V, and G236R; 8). L234T, L235Q, and G236R; 9). L234T, L235T, and G236R; 10) L234A, L235A, and P329G; and 11). M252Y, S254T and T256E.

49. The fusion protein of claim 45, wherein the immunoglobulin Fc region is the Fc region of human IgG4 or a variant thereof.

50. The fusion protein of claim 49, wherein the Fc region of said human IgG4 comprises one or more mutations selected from the group consisting of Ser228Pro (S228P), Leu235Glu (L235E), Leu235Ala (L235A), Phe234Ala (F234A) and / or Pro329Gly (P329G) according to EU designations.

51. The fusion protein of claim 50, wherein the Fc region of said human IgG4 comprises a mutation selected from the group consisting of: 1) S228P and L235E; 2). S228P and L235A; 3) S228P, F234A, and L235E; 4) S228P, F234A, and L235A; and 5). P329G, S228P and L235E.

52. The fusion protein according to any one of claims 36 to 51, further comprising a portion that binds to serum albumin.

53. The fusion protein of claim 36, comprising the amino acid sequence of any one of SEQ ID NO: 2054 to 2070 or a sequence having at least 75% identity with it.

54. The fusion protein according to any one of claims 36 to 53, wherein the fusion protein has an agonist effect when bound to TRAILR2.

55. A conjugate comprising an antigen-binding protein according to any one of claims 1 to 35 or a fusion protein according to any one of claims 36 to 54, wherein the antigen-binding protein or fusion protein is conjugated to a second portion.

56. The conjugate of claim 55, wherein the second portion is selected from detectable markers, drugs, toxins, radionuclides, enzymes, immunomodulators, cytokines, cytotoxic agents, chemotherapeutic agents, diagnostic agents, or combinations thereof.

57. A polynucleotide molecule encoding an antigen-binding protein according to any one of claims 1 to 35 or a fusion protein according to any one of claims 36 to 54.

58. The polynucleotide molecule of claim 57, comprising a nucleotide sequence of any one of SEQ ID NO: 57 to 61, 131 to 145, 1731-2053, 2071 to 2087, 2193, 2199, 2205 and 2278 to 2297 or a nucleotide sequence having at least 70% identity with such sequences.

59. The polynucleotide molecule according to claim 57 or 58, comprising a nucleotide sequence of any one of SEQ ID NO: 57 to 61, 131 to 145, 2071 to 2087, 2193, 2199 and 2205 or a nucleotide sequence having at least 70% identity with it.

60. The polynucleotide molecule according to any one of claims 57 to 59, comprising a nucleotide sequence of any one of SEQ ID NO: 132, 137, 2193, 2199 and 2205 or a nucleotide sequence having at least 70% identity with it.

61. A recombinant vector comprising a polynucleotide molecule according to any one of claims 57 to 60.

62. A host cell comprising a polynucleotide molecule according to any one of claims 57 to 60 or a recombinant vector according to claim 61.

63. A kit comprising an antigen-binding protein according to any one of claims 1 to 35, a fusion protein according to any one of claims 36 to 54, or a conjugate according to any one of claims 55 to 56, a polynucleotide molecule according to any one of claims 57 to 60, or a recombinant vector according to claim 61, and optionally its instructions and / or packaging.

64. A pharmaceutical composition comprising an antigen-binding protein according to any one of claims 1 to 35, a fusion protein according to any one of claims 36 to 54, or a conjugate according to any one of claims 55 to 56, a polynucleotide molecule according to any one of claims 57 to 60, or a recombinant carrier according to claim 61, and a pharmaceutically acceptable carrier and / or excipient.

65. A method for preparing an antigen-binding protein or fusion protein that specifically binds to TNF-associated apoptosis-inducing ligand receptor 2 (TRAILR2), the method comprising the following steps: (a) In a culture medium, under conditions suitable for expressing the antigen-binding protein or fusion protein, the host cells according to claim 62 are cultured, and (b) Isolate the antigen-binding protein or fusion protein from the host cell and / or the culture medium.

66. A method for inducing cell death in cells expressing TNF-associated apoptosis-inducing ligand receptor 2 (TRAILR2), the method comprising contacting the cells with an antigen-binding protein according to claims 1 to 35, a fusion protein according to any one of claims 36 to 54, or a conjugate according to any one of claims 55 to 56.

67. The method of claim 66, wherein the contact is performed in vitro.

68. The method of claim 66, wherein the contact is performed in vivo.

69. The method of claim 68, wherein the method further comprises administering the antigen-binding protein, the fusion protein, or the conjugate to a subject in need.

70. The method according to any one of claims 66 to 69, wherein the cells expressing TRAILR2 are cancer cells.

71. The method of claim 70, wherein the cancer is a solid tumor.

72. The method of claim 70, wherein the cancer is selected from adenoid cystic carcinoma, adrenal tumor, amyloidosis, anal cancer, appendiceal cancer, astrocytoma, ataxia-telangiectasia, Beckwell syndrome, cholangiocarcinoma, Birt-Hogg-Dubé syndrome, bladder cancer, bone cancer (osteosarcoma), brainstem glioma, brain tumor, breast cancer, inflammatory breast cancer, metastatic breast cancer, male breast cancer, Carney complex, central nervous system tumors (brain tumors and spinal cord tumors), cervical cancer, childhood cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, desmoidoma, infantile connective tissue proliferative ganglioglioma, childhood tumors, ependymoma, esophageal cancer, Ewing sarcoma, eye cancer, eyelid cancer, familial polycystic adenoma. Cancer, familial GIST, familial malignant melanoma, familial pancreatic cancer, gallbladder cancer, gastrointestinal stromal tumor (GIST), germ cell tumors, gestational trophoblastic disease, head and neck cancer, hereditary breast and ovarian cancer, hereditary diffuse gastric cancer, hereditary leiomyomatosis and renal cell carcinoma, hereditary mixed polyposis syndrome, hereditary pancreatitis, hereditary papillary renal cell carcinoma, HIV / AIDS-related cancers, juvenile polyposis syndrome, kidney cancer, lacrimal gland tumor, laryngeal and hypopharyngeal cancer, Rife syndrome, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, Lynch syndrome, mastocytosis, myeloma, melanoma, meningioma, mesothelioma, type 1 multiple endocrine tumor, type 2 multiple endocrine tumor, multiple myeloma, MUTYH (or MYH) related polyposis, myelodysplastic syndrome (MDS), nasal cavity and sinus carcinoma, nasopharyngeal carcinoma, neuroblastoma, gastrointestinal neuroendocrine tumor, pulmonary neuroendocrine tumor, pancreatic neuroendocrine tumor, neuroendocrine tumor, neurofibroma type 1, neurofibroma type 2, nevus-like basal cell tumor syndrome, oral and oropharyngeal carcinoma, osteosarcoma, ovarian, fallopian tube and peritoneal carcinoma, pancreatic cancer, parathyroid carcinoma, penile cancer, Boytz-Yage syndrome, pheochromocytoma and paraganglioma, pituitary adenoma, pleural pulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, Kaposi's sarcoma, soft tissue sarcoma, skin cancer (non-melanoma), small intestine cancer, gastric cancer, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis, uterine cancer, vaginal cancer, Shih-Lin syndrome, vulvar cancer, Werner syndrome, nephroblastoma or xeroderma pigmentosum.

73. The method of claim 70, wherein the cancer is gastrointestinal cancer, breast cancer, or lung cancer.

74. The method according to claim 73, wherein the gastrointestinal cancer is colorectal cancer, gastric cancer, esophageal cancer, pancreatic cancer, or cholangiocarcinoma.

75. The method according to any one of claims 66 to 74, wherein the method further comprises contacting the cells with one or more other therapeutic agents.

76. A method of treating or preventing cancer in a subject in need, the method comprising administering to the subject an antigen-binding protein according to claims 1 to 35, a fusion protein according to any one of claims 36 to 54, or a conjugate according to any one of claims 55 to 56.

77. The method of claim 76, wherein the cancer is a solid tumor.

78. The method of claim 76, wherein the cancer is selected from adenoid cystic carcinoma, adrenal tumor, amyloidosis, anal cancer, appendiceal cancer, astrocytoma, ataxia-telangiectasia, Beckwell syndrome, cholangiocarcinoma, Birt-Hogg-Dubé syndrome, bladder cancer, bone cancer (osteosarcoma), brainstem glioma, brain tumor, breast cancer, inflammatory breast cancer, metastatic breast cancer, male breast cancer, Carney complex, central nervous system tumors (brain tumors and spinal cord tumors), cervical cancer, childhood cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, desmoidoma, infantile connective tissue proliferative ganglioglioma, childhood tumors, ependymoma, esophageal cancer, Ewing sarcoma, eye cancer, eyelid cancer, familial polycystic adenoma. Cancer, familial GIST, familial malignant melanoma, familial pancreatic cancer, gallbladder cancer, gastrointestinal stromal tumor (GIST), germ cell tumors, gestational trophoblastic disease, head and neck cancer, hereditary breast and ovarian cancer, hereditary diffuse gastric cancer, hereditary leiomyomatosis and renal cell carcinoma, hereditary mixed polyposis syndrome, hereditary pancreatitis, hereditary papillary renal cell carcinoma, HIV / AIDS-related cancers, juvenile polyposis syndrome, kidney cancer, lacrimal gland tumor, laryngeal and hypopharyngeal cancer, Rife syndrome, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, Lynch syndrome, mastocytosis, myeloma, melanoma, meningioma, mesothelioma, type 1 multiple endocrine tumor, type 2 multiple endocrine tumor, multiple myeloma, MUTYH (or MYH) related polyposis, myelodysplastic syndrome (MDS), nasal cavity and sinus carcinoma, nasopharyngeal carcinoma, neuroblastoma, gastrointestinal neuroendocrine tumor, pulmonary neuroendocrine tumor, pancreatic neuroendocrine tumor, neuroendocrine tumor, neurofibroma type 1, neurofibroma type 2, nevus-like basal cell tumor syndrome, oral and oropharyngeal carcinoma, osteosarcoma, ovarian, fallopian tube and peritoneal carcinoma, pancreatic cancer, parathyroid carcinoma, penile cancer, Boytz-Yage syndrome, pheochromocytoma and paraganglioma, pituitary adenoma, pleural pulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, Kaposi's sarcoma, soft tissue sarcoma, skin cancer (non-melanoma), small intestine cancer, gastric cancer, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis, uterine cancer, vaginal cancer, Shih-Lin syndrome, vulvar cancer, Werner syndrome, nephroblastoma or xeroderma pigmentosum.

79. The method of claim 76, wherein the cancer is gastrointestinal cancer, breast cancer, or lung cancer.

80. The method of claim 79, wherein the gastrointestinal cancer is colorectal cancer, gastric cancer, esophageal cancer, pancreatic cancer, or cholangiocarcinoma.

81. The method according to any one of claims 76 to 80, wherein the method further comprises administering one or more other therapeutic agents.

82. The method according to claim 75 or 81, wherein the one or more other therapeutic agents are selected from chemotherapeutic agents, vascular endothelial growth factor (VEGF) inhibitors, epidermal growth factor receptor (EGFR) inhibitors or apoptosis inducers, immunotherapeutic agents, or combinations thereof.

83. The method according to claim 82, wherein the chemotherapeutic agent is selected from bleomycin, carboplatin, chlorambucil, cisplatin, colchicine, cyclophosphamide, daunorubicin, doxorubicin or doxorubicin liposomes, mitomycin C, actinomycin, diethylstilbestrol, etoposide, 5-fluorouracil, fluorouridine, melphalan, methotrexate, mitomycin, 6-mercaptopurine, teniposide, 6-thioguanine, vincristine and vinblastine, leflunomide, tamoxifen, interferon α-2b, glutamate, purcamycin, Mercaptopurine, 6-thioguanine, carmustine, BCNU, limousine, CCNU, cytosine arabinose, estradiol, hydroxyurea, procarbazine, busulfan, medroxyprogesterone, estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorestradiol, testosterone, melphalan, chlorambucil, nitrogen mustard, thiourea, betamethasone sodium phosphate, dacarbazine, asparagine, mitotane, vincristine sulfate, vinblastine sulfate, FOLFOX (leucovorin, 5-fluorouracil, and oxaliplatin) or FOLFIRI (leucovorin, 5-fluorouracil, and irinotecan) and combinations thereof.

84. The method according to claim 82, wherein the VEGF inhibitor is bevacizumab, ramucirumab, regorafenib, or aflibercept.

85. The method of claim 82, wherein the EGFR inhibitor is selected from cetuximab and / or panitumumab.

86. The method of claim 82, wherein the apoptosis inducer is selected from B-cell lymphoma 2 (BCL2) inhibitors, BCL super-large (BCL-XL) inhibitors, or inhibitors of apoptosis proteins (IAP) inhibitors, or combinations thereof.

87. The method of claim 82, wherein the immunotherapeutic agent is an anti-CTLA4 agent, an anti-PD1 agent, an anti-PD-L1 agent, an anti-LAG3 agent, and an anti-TIM3 agent.

88. The method according to any one of claims 69 to 87, wherein the subject is a mammal.

89. The method of claim 88, wherein the mammal is a human.

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