Anti-trailr2 antigen-binding proteins and uses thereof

Abstract

The present application provides antigen-binding proteins (e.g., antibodies such as single-domain antibodies) that specifically bind TNF-related apoptosis-inducing ligand receptor 2 (TRA5LR2). The application also provides fusion proteins and conjugates comprising the antigen-binding proteins, polynucleotides and recombinant vectors encoding the antigen-binding proteins, as well as host cellsand methods for preparing the antigen-binding proteins. The application further providespharmaceutical compositions comprising the antigen-binding proteins and methods for treating a disease or disorder using the antigen-binding proteins.

Description

Attorney Docket No: 260525.000049 ANTI-TRAILR2 ANTIGEN-BINDING PROTEINS AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.63 / 524,095, filed June 29, 2023, the disclosure of which is herein incorporated by reference in its entirety. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submited electronicaly in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on June 12, 2024, is named 260525_000049_SL.xml and is 2,376,169 bytes in size. FIELD OF THE INVENTION

[0003] The present application relates to antigen-binding proteins (e.g., antibodies such as single- domain antibodies) that specificaly bind TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2), methods for their preparation, and uses thereof. BACKGROUND OF THE INVENTION

[0004] Apoptosis controls celular homeostasis of normal tissue compartments and is tightly regulated on multiple levels. Cancer cels often evade apoptotic cel death through downregulation of pro- apoptotic proteins and / or overexpression of anti-apoptotic proteins which leads to intrinsic resistance to anticancer therapies. Apoptosis can be induced with TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2, also known as death receptor 5 or DR5) agonists or recombinant TNF-related apoptosis- inducing ligand (TRAIL). High TRAILR2 expression is observed across multiple indications. Preclinical data have consistently demonstrated efective activation of the apoptotic pathway in vitro and significant anti-tumor eficacy by TRAILR2 agonists in vivo using xenograft models. However, IgG based TRAILR2 agonists disappointed in the clinic with very limited response rate and this poor translation of preclinical to clinical performance was hypothesized to be due to suboptimal clustering of the receptor agonists and subsequent weak activation of the apoptotic pathway. Accordingly, there is a need in the art to develop therapeutic molecules that can efectively mimic TRAIL-induced activation, e.g., through TRAILR2 multimerization for potent apoptotic signaling downstream.Attorney Docket No: 260525.000049 SUMMARY OF THE INVENTION

[0005] As mentioned in the background section above, there is an unmet need in the art to develop therapeutic molecules designed for optimal multimerization of TRAILR2 and subsequent induction of potent apoptotic signaling. This application provides compositions and methods to address this and other related needs.

[0006] In one aspect, provided herein is an antigen-binding protein that specificaly binds TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2), comprising a complementarity determining region 3 (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, wherein one or more non- alanine residues in the CDR3 sequence is optionaly replaced with an alanine, and / or one or more alanine residues in the CDR3 sequence is optionaly replaced with a glycine.

[0007] In some embodiments, the CDR3 comprises an amino acid sequence selected from 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.

[0008] In some embodiments, the CDR3 comprises an amino acid sequence selected from 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: 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 (SEQAttorney Docket No: 260525.000049 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); AAASSWSRGGAPYGMDY (SEQ ID NO: 2308); AAASSWSRGGVAYGMDY (SEQ ID NO: 2309); AAASSWSRGGVPAGMDY (SEQ ID NO: 2310); AAASSWSRGGVPYAMDY (SEQ ID NO: 2311); AAASSWSRGGVPYGADY (SEQ ID NO: 2312); AAASSWSRGGVPYGMAY (SEQ ID NO: 2313); AAASSWSRGGVPYGMDA (SEQ ID NO: 2314); 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); TAGPTMSYSRGAEF (SEQ ID NO: 2326); TAGPTMSYSRGGAF (SEQ ID NO: 2327); TAGPTMSYSRGGEA (SEQ ID NO: 2328); 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); 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).

[0009] In some embodiments, the antigen-binding protein further comprises a CDR1 comprising an amino acid sequence selected from 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). GFTLDLGAYA (SEQ ID NO: 81); m). GFTFGALA (SEQ ID NO: 86); n). GFTLS(G / S)YA (SEQ ID NO: 120); o). GSIFGGYN (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).

[0010] In some embodiments, the CDR1 comprises an amino acid sequence selected from GRTFSSNL (SEQ ID NO: 1); GGTLANNG (SEQ ID NO: 6); GRTLDAYG (SEQ ID NO: 11); GRTLSDYA (SEQ ID NO: 23);Attorney Docket No: 260525.000049 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).

[0011] In some embodiments, the antigen-binding protein further comprises a CDR2 comprising an amino acid sequence selected from 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).

[0012] In some embodiments, the CDR2 comprises an amino acid sequence selected from VSWNGAST (SEQ ID NO: 2); DHRSGT (SEQ ID NO: 7); ISWTGVDT (SEQ ID NO: 12); ISWTGTDT (SEQ ID NO: 19); ISWSGVDT (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: 91); ITSAGGST(SEQ ID NO: 96); IFISGNN (SEQ ID NO: 2190); ITSDDTT (SEQ ID NO: 2196); and ISSAGGYI (SEQ ID NO: 2202).

[0013] In some embodiments, the antigen-binding protein provided herein comprises a CDR1 comprising an amino acid sequence selected from SEQ ID Nos: 1, 6, 11, 23, 28, 33, 38, 43, 47, 52, 62, 67, 72, 81, 86, 95, 762-1084, 2189, 2195, 2201, and 2252-2261; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 2, 7, 12, 19, 24, 29, 34, 39, 48, 53, 63, 68, 73, 77, 82, 87, 91, 96, 1085-1407, 2190, 2196, 2202, and 2262-2271; and / or a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 3, 8, 13, 16, 20, 25, 30, 35, 40, 44, 49, 54, 64, 69, 74, 78, 83, 88, 92, 97, 1408-1730, 2191, 2197, 2203, 2272-2277, and 2298-2348.

[0014] In some embodiments, the antigen-binding protein comprises i). a CDR1 comprising an amino acid sequence of GRTFSSNL (SEQ ID NO: 1), a CDR2 comprising an amino acid sequence of VSWNGAST (SEQ ID NO: 2), and a CDR3 comprising an amino acid sequence of VASRLPFNSRSAIYTDRIYDS (SEQ ID NO: 3); i). a CDR1 comprising an amino acid sequence of GGTLANNG (SEQ ID NO: 6), a CDR2 comprising an amino acid sequence of DHRSGT (SEQ ID NO: 7), and a CDR3 comprising an amino acid sequence ofAttorney Docket No: 260525.000049 AVRRSAWYSDSIYTVSQYDY (SEQ ID NO: 8); ii). a CDR1 comprising an amino acid sequence of GRTLDAYG (SEQ ID NO: 11), a CDR2 comprising an amino acid sequence of ISWTGVDT (SEQ ID NO: 12), a CDR3 comprising an amino acid sequence of NAARSYSRDYEPLKPDY (SEQ ID NO: 13); iv). a CDR1 comprising an amino acid sequence of GRTLDAYG (SEQ ID NO: 11), a CDR2 comprising an amino acid sequence of ISWTGVDT (SEQ ID NO: 12), and a CDR3 comprising an amino acid sequence of NAARSYSRNYEPLKPDY (SEQ ID NO: 16); v). a CDR1 comprising an amino acid sequence of GRTLDAYG (SEQ ID NO: 11), a CDR2 comprising an amino acid sequence of ISWTGTDT (SEQ ID NO: 19), and a CDR3 comprising an amino acid sequence of NAARSYSRGGEPLKPDY (SEQ ID NO: 20); vi). a CDR1 comprising an amino acid sequence of GRTLSDYA (SEQ ID NO: 23) , a CDR2 comprising an amino acid sequence of ISWSGVDT (SEQ ID NO: 24), and a CDR3 comprising an amino acid sequence of NAARSYSRGGRPLEPAY (SEQ ID NO: 25); vi). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRGGVPYGMDY (SEQ ID NO: 30); vii). a CDR1 comprising an amino acid sequence of GRDFSNYV (SEQ ID NO: 33), a CDR2 comprising an amino acid sequence of INWADET (SEQ ID NO: 34), a CDR3 comprising an amino acid sequence of AADSHFRRYTPGQQYEY (SEQ ID NO: 35); ix). a CDR1 comprising an amino acid sequence of GRSFSSYA (SEQ ID NO: 38), a CDR2 comprising an amino acid sequence of INWSGGST (SEQ ID NO: 39), and a CDR3 comprising an amino acid sequence of NAAKSYHRDYSPLSPDY (SEQ ID NO: 40); x). a CDR1 comprising an amino acid sequence of GRTFSTLA (SEQ ID NO: 43), a CDR2 comprising an amino acid sequence of INWSGGST (SEQ ID NO: 39), a CDR3 comprising an amino acid sequence of AAAPSFGMRNPESYVHS (SEQ ID NO: 44); xi). a CDR1 comprising an amino acid sequence of GRTFSSDI (SEQ ID NO: 47), a CDR2 comprising an amino acid sequence of ISWSDMSA (SEQ ID NO: 48), and a CDR3 comprising an amino acid sequence of AANRGIMSMRLSRYDD (SEQ ID NO: 49); xi). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTMSYSRGGEF (SEQ ID NO: 54); xii). a CDR1 comprising an amino acid sequence of GGTLSNYA (SEQ ID NO: 62), a CDR2 comprising an amino acid sequence of ISQTSST (SEQ ID NO: 63), and a CDR3 comprising an amino acid sequence of VADRGAISRSGAGMDY (SEQ ID NO: 64); xiv). a CDR1 comprising an amino acid sequence of GRTLSNYA (SEQ ID NO: 67), a CDR2 comprising an amino acid sequence of ISQSSDT (SEQ ID NO: 68), and a CDR3 comprising an amino acid sequence of AADRGAISRSGAGMDY (SEQ ID NO: 69); xv). a CDR1 comprising an amino acid sequence of GRSFGAQGMEG (SEQ ID NO: 72), a CDR2 comprising an amino acid sequence of MKWTGNT (SEQ ID NO: 73), and a CDR3 comprising an amino acid sequence of TAGPAISLSRGGEY (SEQ ID NO: 74); xvi). a CDR1 comprising an amino acid sequence ofAttorney Docket No: 260525.000049 GRSFGAQGMEG (SEQ ID NO: 72), a CDR2 comprising an amino acid sequence of IKWTGNT (SEQ ID NO: 77), and a CDR3 comprising an amino acid sequence of TAGPSISYSRGGEY (SEQ ID NO: 78); xvi). a CDR1 comprising an amino acid sequence of GFTLDLGAYA (SEQ ID NO: 81), a CDR2 comprising an amino acid sequence of ISNTGTTT (SEQ ID NO: 82), and a CDR3 comprising an amino acid sequence of AANGWGLDPTTYHY (SEQ ID NO: 83); xvii). a CDR1 comprising an amino acid sequence of GFTFGALA (SEQ ID NO: 86), a CDR2 comprising an amino acid sequence of ISNDGEHI (SEQ ID NO: 87), and a CDR3 comprising an amino acid sequence of SAGWTRRIFQY (SEQ ID NO: 88); xix). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of ISWNGDIT (SEQ ID NO: 91), and a CDR3 comprising an amino acid sequence of TAGQSISLSQGGEY (SEQ ID NO: 92); xx). a CDR1 comprising an amino acid sequence of GFTLSGYA (SEQ ID NO: 95), a CDR2 comprising an amino acid sequence of ITSAGGST (SEQ ID NO: 96), and a CDR3 comprising an amino acid sequence of KAGIRGEVY (SEQ ID NO: 97); xxi). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of RAYNDGGEY (SEQ ID NO: 2191); xxi). a CDR1 comprising an amino acid sequence of GSNFRILS (SEQ ID NO: 2195), a CDR2 comprising an amino acid sequence of ITSDDTT (SEQ ID NO: 2196), a CDR3 comprising an amino acid sequence of HSRWYNL (SEQ ID NO: 2197); or xxii). a CDR1 comprising an amino acid sequence of GFTFSRYA (SEQ ID NO: 2201), a CDR2 comprising an amino acid sequence of ISSAGGYI (SEQ ID NO: 2202), and a CDR3 comprising an amino acid sequence of FTADY (SEQ ID NO: 2203); xxiv). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of GAASSWSRGGVPYGMDY (SEQ ID NO: 2298); xxv). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AGASSWSRGGVPYGMDY (SEQ ID NO: 2299); xxvi). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAGSSWSRGGVPYGMDY (SEQ ID NO: 2300); xxvi). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAAASWSRGGVPYGMDY (SEQ ID NO: 2301); xxvii). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASAWSRGGVPYGMDY (SEQ ID NO: 2302); xxix). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQAttorney Docket No: 260525.000049 ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSASRGGVPYGMDY (SEQ ID NO: 2303); xxx). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWARGGVPYGMDY (SEQ ID NO: 2304); xxxi). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSAGGVPYGMDY (SEQ ID NO: 2305); xxxi). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRAGVPYGMDY (SEQ ID NO: 2306); xxxii). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRGAVPYGMDY (SEQ ID NO: 2307); xxxiv). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRGGAPYGMDY (SEQ ID NO: 2308); xxxv). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRGGVAYGMDY (SEQ ID NO: 2309); xxxvi). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRGGVPAGMDY (SEQ ID NO: 2310); xxxvi). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRGGVPYAMDY (SEQ ID NO: 2311); xxxvii). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRGGVPYGADY (SEQ ID NO: 2312); xxxix). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRGGVPYGMAY (SEQ ID NO: 2313); xl). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRGGVPYGMDA (SEQ ID NO: 2314); xli). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of AAGPTMSYSRGGEF (SEQ ID NO: 2315); xli). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acidAttorney Docket No: 260525.000049 sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TGGPTMSYSRGGEF (SEQ ID NO: 2316); xlii). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAAPTMSYSRGGEF (SEQ ID NO: 2317); xliv). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGATMSYSRGGEF (SEQ ID NO: 2318); xlv). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPAMSYSRGGEF (SEQ ID NO: 2319); xlvi). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTASYSRGGEF (SEQ ID NO: 2320); xlvi). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTMAYSRGGEF (SEQ ID NO: 2321); xlvii). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTMSASRGGEF (SEQ ID NO: 2322); xlix). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTMSYARGGEF (SEQ ID NO: 2323); l). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTMSYSAGGEF (SEQ ID NO: 2324); li). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTMSYSRAGEF (SEQ ID NO: 2325); li). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTMSYSRGAEF (SEQ ID NO: 2326); lii). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTMSYSRGGAF (SEQ ID NO: 2327); liv). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTMSYSRGGEA (SEQ ID NO: 2328); lv). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acidAttorney Docket No: 260525.000049 sequence of AAYNDGGEY (SEQ ID NO: 2329); lvi). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of RGYNDGGEY (SEQ ID NO: 2330); lvi). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of RAANDGGEY (SEQ ID NO: 2331); lvii). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of RAYADGGEY (SEQ ID NO: 2332); lix). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of RAYNAGGEY (SEQ ID NO: 2333); lx). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of RAYNDAGEY (SEQ ID NO: 2334); lxi). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of RAYNDGAEY (SEQ ID NO: 2335); lxi). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of RAYNDGGAY (SEQ ID NO: 2336); lxii). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of RAYNDGGEA (SEQ ID NO: 2337); lxiv). a CDR1 comprising an amino acid sequence of GSNFRILS (SEQ ID NO: 2195), a CDR2 comprising an amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and a CDR3 comprising an amino acid sequence of ASRWYNL (SEQ ID NO: 2338); lxv). a CDR1 comprising an amino acid sequence of GSNFRILS (SEQ ID NO: 2195), a CDR2 comprising an amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and a CDR3 comprising an amino acid sequence of HARWYNL (SEQ ID NO: 2339); lxvi). a CDR1 comprising an amino acid sequence of GSNFRILS (SEQ ID NO: 2195), a CDR2 comprising an amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and a CDR3 comprising an amino acid sequence of HSAWYNL (SEQ ID NO: 2340); lxvi). a CDR1 comprising an amino acid sequence of GSNFRILS (SEQ ID NO: 2195), a CDR2 comprising an amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and a CDR3 comprising an amino acid sequence of HSRAYNL (SEQ ID NO: 2341); lxvii). a CDR1 comprising an amino acid sequence of GSNFRILS (SEQ ID NO: 2195), a CDR2 comprising an amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and a CDR3 comprising an amino acid sequence of HSRWANL (SEQ ID NO: 2342); lxix). a CDR1 comprising an amino acid sequence of GSNFRILS (SEQ ID NO: 2195), a CDR2 comprising an amino acidAttorney Docket No: 260525.000049 sequence of ITSDDTT (SEQ ID NO: 2196), and a CDR3 comprising an amino acid sequence of HSRWYAL (SEQ ID NO: 2343); lxx). a CDR1 comprising an amino acid sequence of GSNFRILS (SEQ ID NO: 2195), a CDR2 comprising an amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and a CDR3 comprising an amino acid sequence of HSRWYNA (SEQ ID NO: 2344); lxxi). a CDR1 comprising an amino acid sequence of GFTFSRYA (SEQ ID NO: 2201), a CDR2 comprising an amino acid sequence of ISSAGGYI (SEQ ID NO: 2202), and a CDR3 comprising an amino acid sequence of ATADY (SEQ ID NO: 2345); lxxi). a CDR1 comprising an amino acid sequence of GFTFSRYA (SEQ ID NO: 2201), a CDR2 comprising an amino acid sequence of ISSAGGYI (SEQ ID NO: 2202), and a CDR3 comprising an amino acid sequence of FAADY (SEQ ID NO: 2346); lxxii). a CDR1 comprising an amino acid sequence of GFTFSRYA (SEQ ID NO: 2201), a CDR2 comprising an amino acid sequence of ISSAGGYI (SEQ ID NO: 2202), and a CDR3 comprising an amino acid sequence of FTGDY (SEQ ID NO: 2347); orlxxiv). a CDR1 comprising an amino acid sequence of GFTFSRYA (SEQ ID NO: 2201), a CDR2 comprising an amino acid sequence of ISSAGGYI (SEQ ID NO: 2202), and a CDR3 comprising an amino acid sequence of FTAAY (SEQ ID NO: 2348).

[0015] In some embodiments, the antigen-binding protein comprises i). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRGGVPYGMDY (SEQ ID NO: 30) ; i). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTMSYSRGGEF (SEQ ID NO: 54) ; ii). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of RAYNDGGEY (SEQ ID NO: 2191); iv). a CDR1 comprising an amino acid sequence of GSNFRILS (SEQ ID NO: 2195), a CDR2 comprising an amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and a CDR3 comprising an amino acid sequence of HSRWYNL (SEQ ID NO: 2197) ; or v). a CDR1 comprising an amino acid sequence of GFTFSRYA (SEQ ID NO: 2201), a CDR2 comprising an amino acid sequence of ISSAGGYI (SEQ ID NO: 2202), and a CDR3 comprising an amino acid sequence of FTADY (SEQ ID NO: 2203).

[0016] In some embodiments, the antigen-binding protein is a single-domain antibody.

[0017] In some embodiments, the single-domain antibody is a VHH, a VNAR, or a VH domain.

[0018] In some embodiments, the VHH is a camelid VHH.

[0019] In some embodiments, the VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 4, 9, 14, 17, 21, 26, 31, 36, 41, 45, 50, 55, 65, 70, 75, 79, 84, 89, 93, 98, 146-468, 2192, 2198, 2204, and 2214-2233, or an amino acid sequence having at least 75% identity thereto.Attorney Docket No: 260525.000049

[0020] In some embodiments, the VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 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 thereto.

[0021] In some embodiments, the VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 31, 55, 2192, 2198, and 2204, or an amino acid sequence having at least 75% identity thereto.

[0022] In some embodiments, the VHH is a humanized VHH.

[0023] In some embodiments, the humanized VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 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, 2234-2251, and 2354-2404, or an amino acid sequence having at least 75% identity thereto.

[0024] In some embodiments, the humanized VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 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 2354-2404, or an amino acid sequence having at least 75% identity thereto.

[0025] In some embodiments, the humanized VHH comprises an amino acid sequence selected from any one of 32, 56, 2200, 2206, 2194, and 2354-2404, or an amino acid sequence having at least 75% identity thereto.

[0026] In various embodiments, the antigen-binding protein binds to human TRAILR2. In some embodiments, the antigen-binding protein binds to human TRAILR2 with a K −7 D of less than about 3×10 M, for example, about 1×10−10 to 5×10−8 M.

[0027] In various embodiments, the antigen-binding protein binds to cyno TRAILR2. In some embodiments, the antigen-binding protein binds to cyno TRAILR2 with a K of le −7 D ss than about 3×10 M. In some embodiments, the antigen-binding protein binds to cyno TRAILR2 with a K −9 D of about 1×10 to 1×10−7 M.

[0028] In various embodiments, the antigen-binding protein does not block binding of TNF-related apoptosis-inducing ligand (TRAIL) to TRAILR2.

[0029] In various embodiments, the antigen-binding protein blocks binding of TRAIL to TRAILR2.

[0030] In various embodiments, the antigen-binding protein comprises one or more modifications that reduce binding of said antigen-binding protein by pre-existing antibodies found in human blood or serum.

[0031] In another aspect, provided herein is a fusion protein that specificaly binds TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2), comprising one or more of said antigen-binding proteinsAttorney Docket No: 260525.000049 described herein. In some embodiments, the fusion protein comprises three or more said antigen- binding proteins. In some embodiments, the fusion protein comprises four said antigen-binding proteins. In some embodiments, the one or more antigen-binding proteins bind to the same epitope on TRAILR2. In some embodiments, the one or more antigen-binding proteins bind to diferent epitopes on TRAILR2. In some embodiments, the 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.

[0032] In some embodiments, the fusion protein further comprises an immunoglobulin Fc region. The immunoglobulin Fc region can be an Fc region of a human immunoglobulin. The immunoglobulin Fc region can be an Fc region of human IgG1, IgG2, IgG3 or IgG4, or a variant thereof.

[0033] In some embodiments, the immunoglobulin Fc region is an Fc region of human IgG1, or a variant thereof. In some embodiments, the Fc region of human IgG1 comprises one or more mutations selected from 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) according to EU numbering. In some embodiments, the Fc region of human IgG1 comprises a set of mutations selected from 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.

[0034] In some embodiments, the immunoglobulin Fc region is an Fc region of human IgG4, or a variant thereof. In some embodiments, the Fc region of human IgG4 comprises one or more mutations selected from Ser228Pro (S228P), Leu235Glu (L235E), Leu235Ala (L235A), Phe234Ala (F234A), and / or Pro329Gly (P329G) according to EU numbering. In some embodiments, the Fc region of human IgG4 comprises a set of mutations selected from 1). S228P and L235E; 2). S228P and L235A; 3). S228P, F234A, and L235E; 4). S228P, F234A, and L235A; and 5). P329G, S228P, and L235E.

[0035] In some embodiments, the fusion protein further comprises a moiety that binds to serum albumin.

[0036] In some embodiments, the fusion protein comprises the amino acid sequence of any one of SEQ ID Nos: 2054-2070, or a sequence having at least 75% identity thereto.Attorney Docket No: 260525.000049

[0037] In various embodiments, the fusion protein has an agonist efect upon binding to TRAILR2.

[0038] In another aspect, provided herein is a conjugate comprising the antigen-binding protein described herein or the fusion protein described herein, wherein the antigen-binding protein or fusion protein is conjugated to a second moiety. In some embodiments, the second moiety is selected from a detectable label, a drug, a toxin, a radionuclide, an enzyme, an immunomodulatory agent, a cytokine, a cytotoxic agent, a chemotherapeutic agent, a diagnostic agent, or a combination thereof.

[0039] In another aspect, provided herein is a polynucleotide molecule encoding the antigen-binding protein described herein or the fusion protein described herein. In some embodiments, the polynucleotide molecule comprises the nucleotide sequence of any one of SEQ ID NOs: 57-61, 131-145, 1731-2053, 2071-2087, 2193, 2199, 2205, and 2278-2297, or a nucleotide sequence having at least 70% identity thereto. In some embodiments, the polynucleotide molecule comprises the nucleotide sequence of any one of SEQ ID NOs: 57-61, 131-145, 2071-2087, 2193, 2199, and 2205, or a nucleotide sequence having at least 70% identity thereto. In some embodiments, the polynucleotide molecule comprises the nucleotide sequence of any one of SEQ ID NOs: 132, 137, 2193, 2199, and 2205, or a nucleotide sequence having at least 70% identity thereto.

[0040] In another aspect, provided herein is a recombinant vector comprising the polynucleotide molecule described herein.

[0041] In another aspect, provided herein is a host cel comprising polynucleotide molecule described herein, or the recombinant vector described herein.

[0042] In another aspect, provided herein is a kit comprising the antigen-binding protein, the fusion protein described herein, the conjugate, the polynucleotide molecule, or the recombinant vector described herein, and optionaly, instructions and / or packaging for the same.

[0043] In another aspect, provided herein is a pharmaceutical composition comprising the antigen- binding protein, the fusion protein, the conjugate, the polynucleotide molecule, or the recombinant vector described herein, and a pharmaceuticaly acceptable carrier and / or excipient.

[0044] In another aspect, provided herein is a method for preparing an antigen-binding protein or a fusion protein that specificaly binds TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2), comprising the steps of:(a) culturing a host cel described herein in a culture medium under conditions suitable for expression of the antigen-binding protein or fusion protein, and (b) isolating the antigen- binding protein or fusion protein from the host cel and / or culture medium.

[0045] In another aspect, provided herein is a method for inducing cel death in a cel expressing TNF- related apoptosis-inducing ligand receptor 2 (TRAILR2) comprising contacting the cel with the antigen-Attorney Docket No: 260525.000049 binding protein, the fusion protein, or the conjugate described herein. In some embodiments, the contacting occurs in vitro. In some embodiments, the contacting occurs in vivo. In some embodiments, the method further comprises administering the antigen-binding protein, the fusion protein, or the conjugate into a subject in need thereof.

[0046] In some embodiments, the cel expressing TRAILR2 is a cel of a cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is selected from adenoid cystic carcinoma, adrenal gland tumor, amyloidosis, anal cancer, appendix cancer, astrocytoma, ataxia-telangiectasia, Beckwith-Wiedemann syndrome, bile duct cancer (cholangiocarcinoma), Birt-Hogg-Dubé syndrome, bladder cancer, bone cancer (sarcoma of bone), brain stem glioma, brain tumor, breast cancer, inflammatory breast cancer, metastatic breast cancer, male breast cancer, Carney complex, central nervous system tumors (brain and spinal cord), cervical cancer, childhood cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, desmoid tumor, desmoplastic infantile ganglioglioma, childhood tumor, ependymoma, esophageal cancer, Ewing sarcoma, eye cancer, eyelid cancer, familial adenomatous polyposis, familial GIST, familial malignant melanoma, familial pancreatic cancer, galbladder cancer, gastrointestinal stromal tumor (GIST), germ cel tumor, gestational trophoblastic disease, head and neck cancer, hereditary breast and ovarian cancer, hereditary difuse gastric cancer, hereditary leiomyomatosis and renal cel cancer, hereditary mixed polyposis syndrome, hereditary pancreatitis, hereditary papilary renal carcinoma, HIV / AIDS-related cancer, juvenile polyposis syndrome, kidney cancer, lacrimal gland tumor, laryngeal and hypopharyngeal cancer, Li-Fraumeni syndrome, liver cancer, lung cancer, non-smal cel lung cancer, smal cel lung cancer, lynch syndrome, mastocytosis, meduloblastoma, melanoma, meningioma, mesothelioma, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, MUTYH (or MYH)-associated polyposis, myelodysplastic syndromes (MDS), nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine tumor of the gastrointestinal tract, neuroendocrine tumor of the lung, neuroendocrine tumor of the pancreas, neuroendocrine tumors, neurofibromatosis type 1, neurofibromatosis type 2, nevoid basal cel carcinoma syndrome, oral and oropharyngeal cancer, osteosarcoma, ovarian, falopian tube, and peritoneal cancer, pancreatic cancer, parathyroid cancer, penile cancer, Peutz-Jeghers syndrome, pheochromocytoma and paraganglioma, pituitary gland tumor, pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Kaposi sarcoma, soft tissue sarcomas, skin cancer (non-melanoma), smal bowel cancer, stomach cancer, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis complex, uterine cancer, vaginal cancer, Von Hippel-Lindau syndrome, vulvar cancer, Werner syndrome, Wilms tumor, orAttorney Docket No: 260525.000049 xeroderma pigmentosum. In some embodiments, the cancer is gastrointestinal cancer, breast cancer, or lung cancer. In some embodiments, the gastrointestinal cancer is colorectal cancer, gastric cancer, esophageal cancer, pancreatic cancer, or cholangiocarcinoma cancer.

[0047] In some embodiments, the method further comprises contacting the cel with one or more additional therapeutic agents.

[0048] In another aspect, provided herein is a method of treating or preventing a cancer in a subject in need thereof, said method comprising administering to the subject the antigen-binding protein, the fusion protein, or the conjugate described herein. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is selected from adenoid cystic carcinoma, adrenal gland tumor, amyloidosis, anal cancer, appendix cancer, astrocytoma, ataxia-telangiectasia, Beckwith-Wiedemann syndrome, bile duct cancer (cholangiocarcinoma), Birt-Hogg-Dubé syndrome, bladder cancer, bone cancer (sarcoma of bone), brain stem glioma, brain tumor, breast cancer, inflammatory breast cancer, metastatic breast cancer, male breast cancer, Carney complex, central nervous system tumors (brain and spinal cord), cervical cancer, childhood cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, desmoid tumor, desmoplastic infantile ganglioglioma, childhood tumor, ependymoma, esophageal cancer, Ewing sarcoma, eye cancer, eyelid cancer, familial adenomatous polyposis, familial GIST, familial malignant melanoma, familial pancreatic cancer, galbladder cancer, gastrointestinal stromal tumor (GIST), germ cel tumor, gestational trophoblastic disease, head and neck cancer, hereditary breast and ovarian cancer, hereditary difuse gastric cancer, hereditary leiomyomatosis and renal cel cancer, hereditary mixed polyposis syndrome, hereditary pancreatitis, hereditary papilary renal carcinoma, HIV / AIDS-related cancer, juvenile polyposis syndrome, kidney cancer, lacrimal gland tumor, laryngeal and hypopharyngeal cancer, Li-Fraumeni syndrome, liver cancer, lung cancer, non-smal cel lung cancer, smal cel lung cancer, lynch syndrome, mastocytosis, meduloblastoma, melanoma, meningioma, mesothelioma, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, MUTYH (or MYH)-associated polyposis, myelodysplastic syndromes (MDS), nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine tumor of the gastrointestinal tract, neuroendocrine tumor of the lung, neuroendocrine tumor of the pancreas, neuroendocrine tumors, neurofibromatosis type 1, neurofibromatosis type 2, nevoid basal cel carcinoma syndrome, oral and oropharyngeal cancer, osteosarcoma, ovarian, falopian tube, and peritoneal cancer, pancreatic cancer, parathyroid cancer, penile cancer, Peutz-Jeghers syndrome, pheochromocytoma and paraganglioma, pituitary gland tumor, pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer,Attorney Docket No: 260525.000049 Kaposi sarcoma, soft tissue sarcomas, skin cancer (non-melanoma), smal bowel cancer, stomach cancer, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis complex, uterine cancer, vaginal cancer, Von Hippel-Lindau syndrome, vulvar cancer, Werner syndrome, Wilms tumor, or xeroderma pigmentosum. In some embodiments, the cancer is gastrointestinal cancer, breast cancer, or lung cancer. In some embodiments, the gastrointestinal cancer is colorectal cancer, gastric cancer, esophageal cancer, pancreatic cancer, or cholangiocarcinoma cancer.

[0049] In some embodiments, the method further comprises administering one or more additional therapeutic agents.

[0050] In various embodiments where one or more additional therapeutic agents are used, the one or more additional therapeutic agents can be selected from a chemotherapeutic agent, a vascular endothelial growth factor (VEGF) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, or an apoptosis-inducing agent, an immunotherapeutic agent, or a combination thereof. In some embodiments, the chemotherapeutic agent is selected from bleomycin, carboplatin, chlorambucil, cisplatin, colchicine, cyclophosphamide, daunorubicin, doxorubicin or liposomal doxorubicin, mitomycin C, actinomycin, diethylstilbestrol, etoposide, 5-fluorouracil, floxuridine, melphalan, methotrexate, mitomycin, 6-mercaptopurine, teniposide, 6-thioguanine, vincristine and vinblastine, leflunomide, tamoxifen, interferon α-2b, glutamic acid, plicamycin, mercaptopurine, 6-thioguanine, carmustine, BCNU, limousine, CCNU, cytosine arabinose, estramustine, hydroxyurea, procarbazine, busulfan, medroxyprogesterone, estramustine phosphate sodium, ethenyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, testolactone, melphalan, chlorambucil, mechlorethamine, thiourea, bethamethasone sodium phosphate, dicarbazine, asparagine, mitotane, vincristine sulfate, vinblastine sulfate, FOLFOX (folinic acid, 5-fluorouracil and oxaliplatin) or FOLFIRI (folinic acid, 5-fluorouracil, and irinotecan), and a combination thereof. In some embodiments, the VEGF inhibitor is bevacizumab, ramucirumab, regorafenib, ziv-aflibercept. In some embodiments, the EGFR inhibitor is selected from cetuximab and / or panitumumab. In some embodiments, the apoptosis-inducing agent is selected from a B-cel lymphoma 2 (BCL2) inhibitor, a BCL-extra large (BCL- XL) inhibitor, or an inhibitor of apoptosis proteins (IAP) inhibitor, or a combination thereof. In some embodiments, the immunotherapeutic agent is an anti-CTLA4 agent, anti-PD1 agent, anti-PD-L1 agent, anti-LAG3 agent, and anti-TIM3 agent.

[0051] In various embodiments, the subject is a mammal, e.g., a human.Attorney Docket No: 260525.000049 BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 depicts an exemplary general panning strategy for immune library construction and panning strategy for discovery of TRAILR2 binders.

[0053] Figure 2 shows sample selection for next generation sequencing (NGS) throughout the phage display process. For the three initial libraries, 12 TRAILR2 samples of the first panning round, and 18 TRAILR2 samples of the second panning round were sequenced with 20 milion, 2 milion, and 2 milion reads, respectively. Comparison of V-body enrichment from the initial library to the first and second round of panning enabled the identification of potential V-body candidates.

[0054] Figure 3 shows a schematic diagram of an exemplary NGS workflow. Folowing phage display, the VHH region of the phage elutions was polymerase chain reaction (PCR) amplified, unique and sample-specific barcodes were fused, and NGS was performed with the Ilumina NovaSeq platform from Genewiz. The raw data were demultiplexed, and then processed by the Pipebio NGS analysis pipeline. Forward and reverse sequence pairs were merged via overlapping regions and the VHHs, including CDRs, were annotated. Based on CDR3 identity, V-body sequences were clustered, alowing for a detailed analysis of V-body enrichment during phage display, sequence diversity, CDR3 length distribution and cluster abundance. Based on these analyses, up to 500 candidates were selected for DNA synthesis by Twist and further characterization.

[0055] Figures 4A-4B ilustrate on-cel binding of anti-TRAILR2 V-body monomers to an engineered TRAILR2 overexpressing cel line.

[0056] Figures 5A-5B ilustrate on-cel binding of anti-TRAILR2 V-body monomers to an endogenous TRAILR2 expressing cel line.

[0057] Figure 6 shows a schematic diagram of an exemplary experimental setup for determination of binding afinities of the V-bodies for their respective target via surface plasmon resonance (SPR). Figure discloses “HHHHHH” as SEQ ID NO: 2407.

[0058] Figures 7A-7G depict surface plasmon resonance (SPR) sensorgrams of VHH binding to human, cynomolgus (cyno), and mouse TNFR2. Fited binding curves and calculated dissociation constants (KD) are included. The first panel shows binding afinities of single V-bodies to human TRAILR2. Each tile represents the global analysis of human TRAILR2 (extracelular domain) binding to a single V-body captured onto a discrete spot, with KDvalues reported. The lines representing ascending TRAILR2 concentration are fitted globaly indicating that the binding kinetics are wel-described by a simple Langmuir mode. Spots with non-ideal behaviors are boxed (e.g., insuficient on-rate / of-rate information, inactive or barely binding). Binding afinities were measured under physiological conditionsAttorney Docket No: 260525.000049 (50 mM HEPES pH 7.4, 150 mM NaCl, 0.1 % (w / v) BSA, 0.05% (v / v) Tween20, 25°C) using 11 diferent antigen concentrations (2‑fold serial dilution, starting at 1 µM). The second panel shows binding afinities of single V-bodies to cyno TRAILR2 extracelular domain. The third panel shows binding afinities of single V-bodies to mouse TRAILR2 extracelular domain. Two competitor molecules were used as controls in Figure 7G. Figure discloses “HHHHHH” as SEQ ID NO: 2407.

[0059] Figure 8 shows a schematic diagram of an exemplary experimental setup for determination of ligand competition with TRAIL.

[0060] Figures 9A-9I depict surface plasmon resonance (SPR) sensorgrams for determination of TRAIL blocking. Each tile represents the sensorgram overlay plot for a single V-body captured onto a discrete spot. In Figures 9A-9G, the solid-lined sensorgram displays TRAIL competition: association of human TRAILR2 (extracelular domain) folowed either by additional binding by TRAIL, indicating an unoccupied epitope (non-overlapping epitopes), or no TRAIL binding, indicating epitope blocking (overlapping epitopes); while the dotted-lined sensogram displays the bufer control: association and dissociation of human TRAILR2 in the absence of TRAIL). In Figures 9H-9I, the ligand and bufer control sensograms are as indicated. Human TRAILR2 (extracelular domain) was injected (500 nM) under physiological conditions (50 mM HEPES pH 7.4, 150 mM NaCl, 0.1 % (w / v) BSA, 0.05% (v / v) Tween20, 25°C) folowed by human TRAIL (1 µM).

[0061] Figure 10 depicts potency of necrosis induction by TRAILR2 V-body agonists of distinct valency in an endogenous TRAILR2 expressing cel line, compared to a tetravalent competitor molecule (competitor #1 in SPR characterization data).

[0062] Figure 11 depicts the time course of Annexin V induction by TRAILR2 V-body agonists of distinct valency in an endogenous TRAILR2 expressing cel line.

[0063] Figures 12A-12B depict that distinct tetravalent formats display comparable kinetics and potency.

[0064] Figure 13 ilustrates exemplary designs of multivalent anti-TRAILR2 V-body constructs. Diferent V-body formats for testing of TRAILR2 signal induction are depicted. Anti-TRAILR2 V-bodies are shown as oval shapes, linkers are shown in lines with Glycine-Serine (GS) linkers as curved lines, rigid linkers as straight lines and Fc domains as dimeric bars. A first set of constructs comprises anti-TRAILR2 V-bodies connected via flexible GS linkers. A second set of constructs with the same anti-TRAILR2 valencies comprises a mixture of flexible GS linkers and a rigid linker. A rigid proline-rich linker included in some of the constructs can mimic the distance of ~90-100Å that can be spanned by a conventional antibody (e.g., a monoclonal antibody, mAb) and roughly equals the size of the death-inducing signaling complexAttorney Docket No: 260525.000049 (DISC) complex. V-bodies fused to a silenced antibody Fc domain are also included. The rigidity of an Fc domain and the distance spanned indicates its suitability for TRAILR2 signal induction. A set of Fc mounted V-bodies have also been designed, including N- and C-terminal fusions of V-bodies to the Fc domain with diferent valencies. Figure discloses “PAPAP” as SEQ ID NO: 2408.

[0065] Figure 14 depicts high throughput kinetic analysis of ODY-N2280Hu1 and ODY-28B1Hu1. Binding afinities of human TRAILR2 are shown in the top panel. Each tile represents the global analysis of human TRAILR2 (extracelular domain) binding to a single V-body captured onto a discrete spot, with KD values reported. The lines representing ascending TRAILR2 concentration were fited globaly, indicating that the binding kinetics were wel-described by a simple Langmuir mode. Binding afinities were 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 diferent antigen concentrations (2‑fold serial dilution, starting at 200 nM (human TRAILR2), 300 nM (cyno TRAILR2) or 1 µM (mouse TRAILR2). Binding afinities of cyno TRAILR2 are shown in the middle panel. Binding afinities of mouse TRAILR2 are shown in the botom panel. Figure discloses “HHHHHH” as SEQ ID NO: 2407.

[0066] Figure 15 depicts high throughput kinetic analysis of ODY-N1039Hu1 and ODY-N1047Hu1. Binding afinities of human TRAILR2 are shown in the top panel. Each tile represents the global analysis of human TRAILR2 (extracelular domain) binding to a single V-body captured onto a discrete spot, with KDvalues reported. The lines representing ascending TRAILR2 concentration are fitted globaly, indicating that the binding kinetics were wel-described by a simple Langmuir mode. Binding afinities were 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 diferent antigen concentrations (2-fold serial dilution, starting at 62.5 nM (human TRAILR2), 125 nM (cyno TRAILR2) or 1 µM (mouse TRAILR2). Binding afinities of cyno TRAILR2 are shown in the middle panel. Binding afinities of mouse TRAILR2 are shown in the bottom panel. Figure discloses “HHHHHH” as SEQ ID NO: 2407.

[0067] Figure 16 shows of-target binding analysis of ODY-30A9Hu1, ODY-N1047Hu1, ODY-28B1Hu1, ODY-N2280Hu1, and ODY-N1039Hu1. Binding afinities of V-bodies for human TRAILR2 compared to homolog proteins TRAILR1, TRAILR3 and TRAILR4 were assessed. Each tile represents the global analysis of a receptor binding to a single V-body captured onto a discrete spot, with KD values reported. The lines representing ascending receptor concentrations were fitted globaly, indicating that the binding kinetics were wel-described by a simple Langmuir mode. Spots with non-ideal behaviors are displayed in boxes (e.g., insuficient on-rate / of-rate information, inactive or barely binding). Binding afinities were measured under physiological conditions (50 mM HEPES pH 7.4, 150 mM NaCl, 0.1 % (w / v) BSA, 0.05%Attorney Docket No: 260525.000049 (v / v) Tween20, 25°C) using 12 diferent antigen concentrations (2‑fold serial dilution, starting at 500 nM for TRAILR1, TRAILR3 and TRAILR4, and starting at 31.3 nM for TRAILR2).

[0068] Figure 17 shows constructs for V-body domain mapping. Swap constructs were generated to identify the TRAILR2 domain that was targeted by diferent V-bodies. Individual cysteine-rich domains (CRDs) or the stalk region were swapped from the human protein sequence to the camelid protein sequence to generate hybrid TRAILR2 extracelular receptor domains (ECDs).

[0069] Figures 18A-18B ilustrate domain mapping of TRAILR2 binding V-bodies ODY-28B1Hu1, ODY- N829Hu1, ODY-N2280Hu1, ODY-23D12Hu1, ODY-24F10Hu1, and ODY-25C08Hu1 (Figure 18A), and ODY- N1039Hu1 and ODY-N1047Hu1 (Figure 18B). Binding of V-bodies to TRAILR2 human / camelid swap extracelular receptor domain (ECD) proteins was determined. Each tile represents the global analysis of TRAILR2 binding to a single V-body captured onto a discrete spot. The lines represent ascending receptor concentrations injected. Spots where no binding event was observed, when compared to the WT TRAILR2 ECD sensorgram, are displayed in boxes.

[0070] Figures 19A-19B show epitope binning of TRAILR2 binding V-bodies. Simultaneous binding of two V-bodies to TRAILR2 was determined to test if the V-bodies had overlapping epitopes. V-bodies were coupled to the SPR chip, saturated with TRAILR2 and tested for binding of an additional V-body that was injected. An example of an experimental set for competition binding and epitope binning analysis is depicted in Figure 19A. Results of the binning analysis are shown in a matrix representation (Figure 19B). Dotted squares indicate that no simultaneous binding of the 2 V-bodies was possible, showing that their epitope on TRAILR2 is overlapping. Striped squares indicate that a simultaneous binding of the tested V-bodies was possible. Hence, these V-bodies target distinct epitopes on TRAILR2.

[0071] Figures 20A-20G show kinetic analysis of CDR3 alanine (Ala) scan V-body mutants. Binding afinities of CDR3 alanine (Ala) scan mutant V-bodies for human TRAILR2 were determined for Ala scan mutants ODY-25C08Hu1 (Figures 20A-20B), ODY-28B1Hu1 (Figures 20C-20D), ODY-1039Hu1 (Figure 20E), ODY-1047Hu1 (Figure 20F), and ODY-2280Hu1 (Figure 20G). Each tile represents the global analysis of human or cyno TRAILR2 binding to a single V-body captured onto a discrete spot, with KD values reported. The lines representing ascending receptor concentrations were fitted globaly, indicating that the binding kinetics were wel-described by a simple Langmuir mode. Spots with non- ideal behaviors are displayed in boxes (e.g., insuficient on-rate / of-rate information, inactive or barely binding). Binding afinities were 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 7 diferent antigen concentrations (2‑fold serial dilution, starting at 15.6 nM for human and cyno TAILR2).Attorney Docket No: 260525.000049

[0072] Figures 21A-21E display on-cel binding fluorescence-activated cel sorting (FACS) analysis of CDR3 alanine (Ala) scan V-body mutants ODY-25C08Hu1 (Figure 21A), ODY-28B1Hu1 (Figure 21B), ODY- 1039Hu1 (Figure 21C), ODY-1047Hu1 (Figure 21D), and ODY-2280Hu1 (Figure 21E). Al data are represented as percent mean fluorescence intensity (MFI) compared to parental V-body. Figure discloses 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, 2303, 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, respectively, in order of appearance. DETAILED DESCRIPTION OF THE INVENTION Definitions

[0073] Unless defined otherwise, al technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skil in the art to which this disclosure belongs. For purposes of interpreting this specification, the folowing description of terms wil apply and whenever appropriate, terms used in the singular wil also include the plural and vice versa. Al patents, applications, published applications and other publications are incorporated by reference in their entirety. In the event that any description of terms set forth conflicts with any document incorporated herein by reference, the description of term set forth below shal control.

[0074] As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 5%. For example, as used herein, the expression "about 100" includes 95 and 105 and al values in between (e.g., 96, 97, 98, 99, etc.).

[0075] The term “antigen” encompasses any agent (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleotide, portions thereof, or combinations thereof) that may be specificaly bound by the products of specific humoral or celular immunity, such as an antibody molecule or T-cel receptor. In various embodiments of the present disclosure, the antigen described herein is TRAILR2, including human, cynomolgus, and / or mouse TRAILR2.

[0076] The term "epitope" can refer 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. Thus, diferent antibodies may bind to diferent areas on an antigen and may have diferent biological efects (e.g., agnostic or antagonistic efects). Epitopes may be either conformational or linear. A conformationalAttorney Docket No: 260525.000049 epitope is formed by spatialy juxtaposed amino acids from diferent segments of the linear polypeptide chain. A linear epitope is formed by adjacent amino acid residues in a polypeptide chain. In some cases, an epitope may include non-peptidic moieties on the antigen, such as saccharides, phosphoryl groups, or sulfonyl groups.

[0077] The term "antigen-binding protein" refers in its broadest sense to a protein that specificaly binds an antigen (e.g., TRAILR2). In certain embodiments, an antigen-binding protein is an antibody or an antigen-binding fragment of an antibody, such as a human antibody, a humanized antibody; a camelid antibody; a chimeric antibody; a recombinant antibody; a heavy chain antibody; a single-domain antibody (e.g., VHH); a single chain antibody (e.g., single chain fragment variable (scFv); a diabody; a triabody; a tetrabody; a Fab fragment; a F(ab′) 2 fragment; an IgD antibody; an IgE antibody; an IgM antibody; an IgG1 antibody; an IgG2 antibody; an IgG3 antibody; or an IgG4 antibody, and fragments thereof. The term "antigen-binding protein" also encompasses, for example, an alternative protein scafold or artificial scafold with grafted CDRs or CDR derivatives. Such scafolds include, but are not limited to, antibody-derived scafolds comprising mutations introduced to, for example, stabilize the three-dimensional structure of the antigen-binding protein as wel as wholy synthetic scafolds comprising, for example, a biocompatible polymer. In addition, peptide antibody mimetics can be used, as wel as scafolds based on antibody mimetics utilizing fibronectin components (e.g., fibronectin type II domain (FN3) as a scafold.

[0078] The term “TNF-related apoptosis-inducing ligand receptor 2”, or “TRAILR2”, or the like, are used interchangeably herein and can refer to any isoform(s), variant(s), and / or species homolog(s) of TRAILR2 from any source, e.g., mammals including primates (e.g., humans and monkeys) and rodents (e.g., rats and mice). The term encompasses naturaly-occurring variants of TRAILR2 such as but not limited to alelic variants and splice variants. The term also encompasses “ful-length” or unprocessed TRAILR2 in addition to any form of TRAILR2 that can result from processing such as that which may occur within a cel. In some embodiments, TRAILR2 is human TRAILR2. In some embodiments, TRAILR2 is cynomolgus monkey (“cyno”) TRAILR2.

[0079] The term “antibody” and “immunoglobulin” or “Ig” are used interchangeably herein, and is used in the broadest sense and encompasses, for example, individual monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, ful length or intact monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, polyclonal antibodies, monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), single-domain antibodies (e.g., VHH), single chain antibodies, intrabodies, anti-idiotypic (anti-Id) antibodies, andAttorney Docket No: 260525.000049 antigen-binding fragments of antibodies, as described below. An antibody can be human, humanized, camelized, recombinantly produced, chimeric, synthetic, afinity de-matured and / or afinity matured as wel as an antibody from other species, for example mouse, camel, lama, rabbit, etc. In specific embodiments, the specific target antigen that can be bound by an antibody provided herein includes a TRAILR2 polypeptide, TRAILR2 fragment or TRAILR2 epitope. An “antigen-binding fragment” generaly refers a portion of an antibody heavy and / or light chain polypeptide that retains some or al of the binding activity of the antibody from which the fragment was derived. Non-limiting examples of antigen- binding fragments include single-domain antibody (e.g., VHH), single-chain Fvs (scFv), Fab fragments, F(ab′) fragments, F(ab)2 fragments, F(ab′)2 fragments, disulfide-linked Fvs (sdFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody and minibody, or a chemicaly modified derivative thereof. In particular, antibodies provided herein include immunoglobulin molecules and molecules that contain immunologicaly active portion(s) of an immunoglobulin molecule, for example, one or more complementarity determining regions (CDRs) of an antibody that binds to TRAILR2. Such antibody fragments can be found described in, for example, 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., Cel Biophysics, 22:189- 224 (1993); Plückthun and Skerra, Meth. Enzymol., 178:497-515 (1989) and in Day, E.D., Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York, N.Y. (1990). The antibodies provided herein can be of any type (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) of immunoglobulin molecule.

[0080] The term “single-domain antibody” or “sdAb” as used herein, refers to an antibody or antibody fragment containing a single antibody variable domain that is able to bind to a specific antigen alone, without the requirement of another antibody variable domain. The complementary determining regions (CDRs) of a single-domain antibody are part of a single antibody variable domain. Examples of single- domain antibodies include, but are not limited to, heavy chain antibodies, antibodies naturaly devoid of light chains, single domain antibodies derived from conventional four-chain antibodies, engineered antibodies, variable domains derived from the aforementioned antibodies, and single domain scafolds other than those derived from antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, lama, shark, goat, rabbit, and / or bovine. In some embodiments, a single domain antibody as used herein is a naturaly occurring single domain antibody known as heavy chain antibody devoid of light chains. For clarity reasons, the variable domain derived from a heavy chain antibody naturaly devoid of light chain is known herein as a VHH to distinguish itAttorney Docket No: 260525.000049 from the conventional VH of four-chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae species, e.g., camel, lama, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain antibodies naturaly devoid of light chain, which are also within the scope of the invention. For example, cartilaginous fishes such as sharks can produce immunoglobulin-like structures known as VNAR. In some embodiments, a single-domain antibody may be obtained from a Camelidae VH domain. In some embodiments, a single-domain antibody may be obtained from human VH by camelization. See Saerens et al., Current Opinion in Pharmacology, 2008, 8:600-608, the disclosure of which being incorporated by reference, for review of single-domain antibodies.

[0081] The term “specificaly binds” as used herein means that an antigen-binding protein forms a complex with a target antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by a dissociation constant (K -6 -6 D) of about 1x10 M or less (e.g., less than 10 M, less than 5x10-7M, less than 10-7M, less than 5x10-8M, less than 10-8M, less than 5x10-9M, less than 10-9M, or less than 10-10 M). Methods for determining the binding afinity of an antigen-binding protein, e.g., an antibody or an antibody fragment, to a target antigen are wel known in the art and include, e.g., surface plasmon resonance (e.g., BIACORE® assays), bio-layer interferometry, ligand binding assays (e.g., enzyme-linked immunosorbent assay (ELISA), equilibrium dialysis, fluorescent-activated cel sorting (FACS), or flow cytometry-based binding assays and the like. Specific binding to a particular target antigen from a certain species does not exclude that the antigen-binding protein can also specificaly bind to the analogous target from a diferent species. For example, specific binding to human TRAILR2 does not exclude that the antigen-binding protein can also specificaly bind to TRAILR2 from cynomolgus monkeys (“cyno”) or mice.

[0082] The term "isolated" when used in the context of antigen-binding proteins (e.g., antibodies, such as single-domain antibodies), polypeptides, polynucleotides, and vectors, means the antigen-binding proteins (e.g., antibodies, such as single-domain antibodies), polypeptides, polynucleotides and vectors are at least partialy free of other biological molecules from the cels or cel culture from which they are produced. Such biological molecules include nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, or other material such as celular debris and growth medium. An isolated antigen-binding protein may further be at least partialy free of expression system components such as biological molecules from a host cel or of the growth medium thereof. Generaly, the term "isolated" is not intended to refer to a complete absence of such biological molecules (e.g., minor or insignificant amounts of impurity may remain) or to an absence of water, bufers, or salts or toAttorney Docket No: 260525.000049 components of a pharmaceutical formulation that includes the antigen-binding proteins (e.g., antibodies, such as single-domain antibodies).

[0083] The term “operably linked” as used herein can refer to a functional relationship between two or more regions of a polypeptide chain in which the two or more regions are linked so as to produce a functional polypeptide.

[0084] As used herein, the term “variant”, “derivative” or “derived from” in the context of proteins or polypeptides (e.g., antigen-binding proteins or domains thereof) refer to: (a) a polypeptide that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the polypeptide it is a variant or derivative of; (b) a polypeptide encoded by a nucleotide sequence that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a nucleotide sequence encoding the polypeptide it is a variant or derivative of; (c) a polypeptide that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid mutations (i.e., additions, deletions and / or substitutions) relative to the polypeptide it is a variant or derivative of; (d) a polypeptide encoded by nucleic acids can hybridize under high, moderate or typical stringency hybridization conditions to nucleic acids encoding the polypeptide it is a variant or derivative of; (e) a polypeptide encoded by a nucleotide sequence that can hybridize under high, moderate or typical stringency hybridization conditions to a nucleotide sequence encoding a fragment of the polypeptide, it is a variant or derivative of, of at least 20 contiguous amino acids, at least 30 contiguous amino acids, at least 40 contiguous amino acids, at least 50 contiguous amino acids, at least 75 contiguous amino acids, at least 100 contiguous amino acids, at least 125 contiguous amino acids, or at least 150 contiguous amino acids; or (f) a fragment of the polypeptide it is a variant or derivative of. The terms also encompass a fusion protein or polypeptide comprising the polypeptide it is a variant or derivative of.

[0085] The term "substantial identity" or "substantialy identical," when referring to a nucleic acid or fragment thereof, indicates that, when optimaly aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95%, and more preferably at least about 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any wel-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantialy similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.Attorney Docket No: 260525.000049

[0086] As applied to polypeptides, the term "substantial similarity" or "substantialy similar" means that two peptide sequences, when optimaly aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, residue positions which are not identical difer by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution wil not substantialy change the functional properties of a protein. In cases where two or more amino acid sequences difer from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are wel-known to those of skil in the art. See, e.g., Pearson (1994) Methods Mol. Biol.24: 307-331, herein incorporated by reference. Examples of groups of amino acids that have side chains with 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: aspartate and glutamate, and (7) sulfur- containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate- aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443- 1445, herein incorporated by reference. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[0087] Sequence similarity for polypeptides, which is also referred to as sequence identity, is typicaly measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, 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 polypeptides, such as homologous polypeptides from diferent species of organisms or between a wild-type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (PearsonAttorney Docket No: 260525.000049 (2000) supra). Another preferred algorithm when comparing a sequence of the disclosure to a database containing a large number of sequences from diferent organisms is the computer program BLAST, especialy BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res.25:3389-402, each herein incorporated by reference.

[0088] The terms “enhance” or “promote,” or “increase,” or “expand,” or “improve” refer generaly to the ability of a composition contemplated herein to produce, elicit, or cause a greater physiological response (i.e., downstream efects) compared to the response caused by either vehicle or a control molecule / composition. A measurable physiological response may include an increase in immune cel expansion, activation, efector function, persistence, and / or an increase in tumor cel death kiling ability, among others apparent from the understanding in the art and the description herein. In certain embodiments, an “increased” or “enhanced” amount can be a “statisticaly significant” amount, and may include an increase that is 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 al integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7.1.8, etc.) the response produced by vehicle or a control composition.

[0089] The terms “decrease” or “lower,” or “lessen,” or “reduce,” or “abate” refer generaly to the ability of composition contemplated herein to produce, elicit, or cause a lesser physiological response (i.e., downstream efects) compared to the response caused by either vehicle or a control molecule / composition. In certain embodiments, a “decrease” or “reduced” amount can be a “statisticaly significant” amount, and may include a decrease that is 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 al integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7.1.8, etc.) the response (reference response) produced by vehicle or a control composition.

[0090] The terms “treat” or “treatment” of a state, disorder or condition include: (1) preventing, delaying, or reducing the incidence and / or likelihood of the appearance of at least one clinical or sub- clinical symptom of the state, disorder or condition developing in a subject that may be aflicted with or predisposed to the state, disorder or condition, but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; or (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof or at least one clinical or sub-clinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms. The benefit to a subject to be treated is either statisticaly significant or at least perceptible to the patient or to the physician.Attorney Docket No: 260525.000049

[0091] The terms “efective amount” or “therapeuticaly efective amount” refer to a quantity and / or concentration of a composition containing an active ingredient (e.g., anti-TRAILR2 antigen-binding protein) that when administered into a patient either alone (i.e., as a monotherapy) or in combination with additional therapeutic agents, yields a significant decrease in disease progression as, for example, by ameliorating or eliminating symptoms and / or the cause of the disease. An efective amount may be an amount that relieves, lessens, or aleviates at least one symptom or biological response or efect associated with a disease or disorder, prevents progression of the disease or disorder, or improves physical functioning of the patient. A therapeuticaly efective amount of a composition containing an active agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the active agent to elicit a desired response in the individual. A therapeuticaly efective amount is also one in which any toxic or detrimental efects of the active agent are outweighed by the therapeuticaly beneficial efects. A therapeuticaly efective amount may be delivered in one or more administrations. A therapeuticaly efective amount refers to an amount efective, at dosages and for periods of time necessary, to achieve the desired therapeutic and / or prophylactic result.

[0092] The terms “individual”, “subject” and “patient” are used interchangeably herein to refer to an animal; for example a mammal. The terms include human and veterinary subjects. In some embodiments, methods of treating mammals, including, but not limited to, humans, rodents, simians, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets, are provided. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, a subject can be a subject in need of treatment for a disease or disorder. In particular embodiments, the subject is a human. Anti-TRAILR2 Antigen-binding Proteins

[0093] The present disclosure provides antigen-binding proteins (e.g., antibodies, such as single-domain antibodies) that bind to TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2, also known as Death Receptor 5 or DR5).

[0094] TRAIL receptor 2 (TRAILR2) is a single pass type-1 membrane protein with a molecular weight of ~42 kDa that belongs to the TNFR superfamily. TRAILR2 is comprised of an extracelular domain with three cysteine-rich domains (CRDs) and an intracelular death domain that is involved in apoptotic signaling. The cysteine-rich domains contain a total of 7 disulfide bonds stabilizing the elongatedAttorney Docket No: 260525.000049 structure of the protein. CRD1 is also caled pre-ligand assembly domain (PLAD) and has been found to be involved in low afinity (>1 µM) TRAILR2 dimer formation in the absence of TRAIL. The TRAILR2 death domain is a bundle of six alpha helices that are involved in the binding of intracelular binding partners and formation of the death-inducing signaling complex (DISC) to promote apoptotic signaling. Further, TRAILR2 has been found to be O-glycosylated at two positions within CRD1 and CRD2 altering its ability to induce apoptosis albeit not changing the afinity towards TRAIL. In addition, fucosylation of the O- glycans has been reported to be important for apoptosis induction capabilities of TRAILR2.

[0095] The TRAIL ligand itself consists of a β-sandwich fold and homo-trimerizes in solution. The trimer is non-covalent, and TRAIL dissociates at low concentration leading to the fast degradation of the monomeric units. The TRAIL trimer is stabilized by a zinc ion that is coordinated by 3 free cysteines (one per monomer) at the trimerization interface. The TRAIL ligand exists both in a soluble (sTRAIL) and membrane-anchored (mTRAIL) version.

[0096] The extracelular domain of TRAILR2 has been structuraly characterized as a trimeric complex in presence of the TRAIL ligand. In addition, an NMR structure of the transmembrane domain trimer and hexamer has been reported, while there are no structural data available on the intracelular death domain of TRAILR2. However, homolog structures of death domains and further signaling components provide structural insights into the signaling complex.

[0097] In the absence of the trimeric TRAIL ligand, TRAILR2 has been reported to exist either in monomeric or dimeric state. The dimeric state is mediated via a low afinity (>1 µM) interaction of the PLAD domains. The dimers have been reported to adopt two diferent conformations, an active, paralel conformation in which the TRAIL binding site is accessible or an inactive, anti-paralel conformation, in which the TRAIL binding site is shielded. In the presence of soluble or membrane bound trimeric TRAIL, TRAILR2 can bind to TRAIL trimers with high afinity (<2 nM) and assemble into a hexameric TRAILR23TRAIL3 complex with 3 TRAILR2 molecules bound to trimeric TRAIL on the membrane. The TRAIL binding site on TRAILR2 is located at the concave surface formed by CRD1 and CRD2. Furthermore, it has been reported that in addition to homogenous complexes formed by TRAILR2 bound to trimeric TRAIL, heterogenous complexes with TRAILR1 and TRAILR4 can be assembled exhibiting an overal lower signaling potency when activated. As for other receptors of the TNFR-superfamily it has also been reported for TRAILR2 that dimers and trimers may not be randomly distributed throughout the membrane but may be organized in a higher order hexagonal fashion.

[0098] The actual distribution of TRAILR2 oligomeric states on any given normal or cancer cel remains largely unknown. It has been confirmed that TNFR1 mainly exists in a monomeric and dimeric state inAttorney Docket No: 260525.000049 the absence of the TNF ligand, while in the presence of TNF, most of the receptors assemble into trimers or higher oligomers of the trimers while only few monomers and no dimers were observed. Whether these results can be translated to TRAILR2 remains to be seen.

[0099] In line with these observations, it has been consistently reported that TRAILR2 oligomerization may be needed for eficient signaling. The precise mechanism of signaling complex assembly and protein stoichiometries are not yet fuly understood, but it is assumed that two TRAIL bound TRAILR2 trimers need to come into close proximity to enable eficient apoptotic signaling. In more detail, membrane- bound TRAIL trimers were shown to bind three molecules of TRAILR2 receptors that assemble in a symmetrical trimeric complex, with each TRAILR2 molecule interacting with two TRAIL monomers in the center of the complex. This trimerization has been suggested to induce a rearrangement of the transmembrane helices, resulting in a conformational change of the TRAILR2 death domains into an open conformation. In its open conformation the TRAILR2-DD can recruit an adaptor protein caled 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 now exposed FADD death-efector domain (DED). A single death-inducing signaling complex (DISC) complex was found to be unable to induce downstream signaling as procaspase-8 requires homodimerization to exhibit autocatalytic activity and subsequent activation. Hence, a second TRAILR2 complex with al intracelular components bound may need to be in close proximity to enable procaspase-8 dimerization. DISC complex dimerization was mediated only via membrane bound (mTRAIL) but not soluble TRAIL. Anchoring of mTRAIL in the lipid bilayer restricts difusion of the protein to two dimensions, which would efectively increase the colocalization of mTRAIL molecules, and in turn increase the eficiency of agonist-induced TRAILR2 clustering compared to that promoted by the soluble form.

[0100] A structural model proposed for the entire signaling complex indicates that a distance of >90Å is needed between two TRAILR23TRAIL3 complexes to accommodate the intracelular DISC complex. This distance correlates wel with the distance spanned by monoclonal antibodies (mAbs), which have been shown to induce TRAILR2 clustering and signaling.

[0101] In some embodiments, a human TRAILR2 can be encoded by a TNF receptor superfamily member 10b (TNFRSF10B) gene (NCBI Gene ID 8795) and has the amino acid sequence MEQRGQNAPAASGARKRHGPGPREARGARPGPRVPKTLVLVVAAVLLLVSAESALITQQDLAPQQRAAPQQKRSSPS EGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKC RTGCPRGMVKVGDCTPWSDIECVHKESGTKHSGEVPAVEETVTSSPGTPASPCSLSGIIGVTVAAVVLIVAVFVCKSLL WKKVLPYLKGICSGGGGDPERVDRSSQRPGAEDNVLNEIVSILQPTQVPEQEMEVQEPAEPTGVNMLSPGESEHLLEPAttorney Docket No: 260525.000049 AEAERSQRRRLLVPANEGDPTETLRQCFDDFADLVPFDSWEPLMRKLGLMDNEIKVAKAEAAGHRDTLYTMLIKWVN KTGRDASVHTLLDALETLGERLAKQKIEDHLLSSGKFMYLEGNADSAMS (UniProtKB Accession No. O14763) (SEQ ID NO: 2149).

[0102] In some embodiments, a cyno TRAILR2 can be encoded by a TNF receptor superfamily member 10b (TNFRSF10B) gene (NCBI Gene ID 102133727) and has the amino acid sequence MGQLRQSAPAASGARKGRGPGPREARGARPGLRVLKTLVLVVAAAAVLLSVSADCAPITRQSLDPQRRAAPQQKRSSP TEGLCPPGHHISEDSRECISCKYGQDYSTHWNDFLFCLRCTKCDSGEVEVNSCTTTRNTVCQCEEGTFREEDSPEICRKC RTGCPRGMVKVKDCTPWSDIECVHKESGTKHTGEVPAVEKTVTTSPGTPASPCSLSGIIGVIVLVVIVVVAVIVWKTSL WKKVLPYLKGVCSGGGGDPERVDSSSHSPQRPGAEDNALNEIVSIVQPSQVPEQEMEVQEPAEQTDVNTLSPGESEHL LEPAKAEGPQRRGQLVPVNENDPTETLRQCFDDFAAIVPFDAWEPLVRQLGLTNNEIKVAKAEAASSRDTLYVMLIKW VNKTGRAASVNTLLDALETLEERLAKQKIQDRLLSSGKFMYLEDNADSATS (UniProtKB Accession No. A0A2K5TXK0|A0A2K5TXK0) (SEQ ID NO: 2150).

[0103] In some embodiments, a mouse TRAILR2 can be encoded by a TNF receptor superfamily member 10b (TNFRSF10B) gene (NCBI Gene ID 21933) and has the amino acid sequence MEPPGPSTPTASAAARADHYTPGLRPLPKRRLLYSFALLLAVLQAVFVPVTANPAHNRPAGLQRPEESPSRGPCLAGQY LSEGNCKPCREGIDYTSHSNHSLDSCILCTVCKEDKVVETRCNITTNTVCRCKPGTFEDKDSPEICQSCSNCTDGEEELTSC TPRENRKCVSKTAWASWHKLGLWIGLLVPVVLLIGALLVWKTGAWRQWLLCIKRGCERDPESANSVHSSLLDRQTSST TNDSNHNTEPGKTQKTGKKLLVPVNGNDSADDLKFIFEYCSDIVPFDSWNRLMRQLGLTDNQIQMVKAETLVTREALY QMLLKWRHQTGRSASINHLLDALEAVEERDAMEKIEDYAVKSGRFTYQNAAAQPETGPGGSQCV (UniProtKB Accession No. Q9QZM4) (SEQ ID NO: 2151).

[0104] In various embodiments, antigen-binding proteins of the present disclosure have an agonist efect upon binding to TRAILR2. While not wishing to be bound by theory, an agonistic TRAILR2 binder can promote or increase activation of TRAILR2 and / or potentiate one or more signal transduction pathways mediated by TRAILR2. Agonistic TRAILR2 binders may promote or increase TRAILR2 activation by binding TRAILR2, e.g., to induce TRAILR2 multimerization which can render the receptor biologicaly active. For example, agonistic TRAILR2 binders may nucleate the trimerization of TRAILR2 and may additionaly act to bring a TRAILR2 agonistic binder-associated TRAILR2 trimer into close proximity with one or more additional TRAILR2 agonistic binder-associated TRAILR2 trimer(s) in a manner similar to that which occurs when TRAILR2 interacts with its cognate ligand, TRAIL, thus inducing TRAILR2- mediated signaling. In some embodiments, an agonistic TRAILR2 binder described herein may bind, e.g., three molecules of TRAILR2 which can assemble into a trimeric complex (i.e., a TRAILR2 trimer).Attorney Docket No: 260525.000049

[0105] In some embodiments, agonistic TRAILR2 binding proteins of the present disclosure may be capable of rendering a TRAILR2 described herein capable of binding intracelular binding partners (e.g., adaptor proteins such as but not limited to Fas associated death domain (FADD) and forming a death- inducing signaling complex (DISC) to promote apoptotic signaling.

[0106] In some embodiments, antigen-binding proteins of the present disclosure bind to human TRAILR2. In some embodiments, antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to human TRAILR2 with a K −6 D of less than about 1×10 M, for example, less than about 5×10−7 M, less than about 3×10−7 M, less than about 1×10−7 M, less than about 8×10−8 M, less than about 5×10−8 M, less than about 3×10−8 M, less than about 1×10−8 M, less than about 9×10−9 M, less than about 8×10−9 M, less than about 7×10−9 M, less than about 6×10−9 M, less than about 5×10−9 M, less than about 4×10−9 M, less than about 3×10−9 M, less than about 2×10−9 M, or less than about 1×10−9 M, or about 1×10−10 to 1×10−9 M, 1×10−10 to 5×10−9 M, about 1×10−10 to 1×10−8 M, about 1×10−10 to 5×10−8 M, about 1×10−9 to 5×10−9 M, about 5×10−9 to 1×10−8 M, about 1×10−9 to 1×10−8 M, about 1×10−9 to 5×10−8 M, about 1×10−9 to 1×10−7 M, or about 1×10−8 to 1×10−7 M.

[0107] In some embodiments, antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to human TRAILR2 with a K −7 D of less than about 3×10 M. In some embodiments, antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to human TRAILR2 with a K −10 −8 D of about 1×10 to 5×10 M.

[0108] In some embodiments, antigen-binding proteins of the present disclosure bind to cynomolgus monkey (“cyno”) TRAILR2. In some embodiments, antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to cyno TRAILR2 with a KD of less than about 1×10−6 M, for example, less than about 5×10−7 M, less than about 3×10−7 M, less than about 1×10−7 M, less than about 8×10−8 M, less than about 5×10−8 M, less than about 3×10−8 M, less than about 1×10−8 M, less than about 8×10−9 M, less than about 5×10−9 M, less than about 3×10−9 M, or less than about 1×10−9 M, or about 1×10−10 to 1×10−9 M, 1×10−10 to 5×10−9 M, about 1×10−10 to 1×10−8 M, about 1×10−10 to 5×10−8 M, about 1×10−9 to 1×10−8 M, about 1×10−9 to 5×10−8 M, about 1×10−9 to 1×10−7 M, about 1×10−9 to 2×10−7 M, about 1×10−9 to 5×10−7 M, about 1×10−8 to 1×10−7 M, about 1×10−8 to 2×10−7 M, about 1×10−8 to 5×10−7 M, or about 1×10−8 to 1×10−6 M.

[0109] In some embodiments, antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to cyno TRAILR2 with a K of less than about 3×10−7 D M. In some embodiments, antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to cyno TRAILR2 with a K −9 −7 D of about 1×10 to 1×10 M.Attorney Docket No: 260525.000049

[0110] In some embodiments, antigen-binding proteins of the present disclosure bind to mouse TRAILR2. In some embodiments, antigen-binding proteins of the present disclosure may bind to mouse TRAILR2 with a K of less than about 1×10−6 M, for example −7 D , less than about 5×10 M, less than about 3×10−7 M, less than about 1×10−7 M, less than about 8×10−8 M, less than about 5×10−8 M, less than about 3×10−8 M, less than about 1×10−8 M, less than about 8×10−9 M, less than about 5×10−9 M, less than about 3×10−9 M, or less than about 1×10−9 M, or about 1×10−10 to 1×10−9 M, 1×10−10 to 5×10−9 M, about 1×10−10 to 1×10−8 M, about 1×10−10 to 5×10−8 M, about 1×10−9 to 1×10−8 M, about 1×10−9 to 5×10−8 M, about 1×10−9 to 1×10−7 M, about 1×10−9 to 2×10−7 M, about 1×10−9 to 5×10−7 M, about 1×10−8 to 1×10−7 M, about 1×10−8 to 2×10−7 M, about 1×10−8 to 5×10−7 M, or about 1×10−8 to 1×10−6 M. In some embodiments, antigen-binding proteins of the present disclosure do not bind to mouse TRAILR2.

[0111] In some embodiments, antigen-binding proteins of the present disclosure bind to rat TRAILR2. In some embodiments, antigen-binding proteins of the present disclosure may bind to rat TRAILR2 with a K of less than about 1×10−6 M, for example, les −7 −7 D s than about 5×10 M, less than about 3×10 M, less than about 1×10−7 M, less than about 8×10−8 M, less than about 5×10−8 M, less than about 3×10−8 M, less than about 1×10−8 M, less than about 8×10−9 M, less than about 5×10−9 M, less than about 3×10−9 M, or less than about 1×10−9 M, or about 1×10−10 to 1×10−9 M, 1×10−10 to 5×10−9 M, about 1×10−10 to 1×10−8 M, about 1×10−10 to 5×10−8 M, about 1×10−9 to 1×10−8 M, about 1×10−9 to 5×10−8 M, about 1×10−9 to 1×10−7 M, about 1×10−9 to 2×10−7 M, about 1×10−9 to 5×10−7 M, about 1×10−8 to 1×10−7 M, about 1×10−8 to 2×10−7 M, about 1×10−8 to 5×10−7 M, or about 1×10−8 to 1×10−6 M. In some embodiments, antigen- binding proteins of the present disclosure do not bind to rat TRAILR2.

[0112] In some embodiments, anti-TRAILR2 antigen-binding proteins described herein do not block binding of TNF-related apoptosis-inducing ligand (TRAIL) to TRAILR2.

[0113] In some embodiments, anti-TRAILR2 antigen-binding proteins described herein blocks binding of TNF-related apoptosis-inducing ligand (TRAIL) to TRAILR2. In some embodiments, the anti-TRAILR2 antigen-binding proteins described herein may block binding of TNF-related apoptosis-inducing ligand (TRAIL) to TRAILR2 by from about 10% or more. In some embodiments, the binding of TNF-related apoptosis-inducing ligand (TRAIL) to TRAILR2 can be blocked by the anti-TRAILR2 antigen-binding proteins described herein by from about 10% to about 20%, from about 10% to about 30%, from about 10% to about 40%, from about 10% to about 50%, from about 10% to about 60%, from about 10% to about 70%, from about 10% to about 80%, from about 10% to about 90%, more than 60%, from about 60% to about 70%, from about 60% to about 80%, from about 60% to about 90%, more than about 70%, from about 70% to about 80%, from about 70% to about 90%, more than about 80%, from about 80% toAttorney Docket No: 260525.000049 about 90%, more than 90%, from about 90% to about 95%, from about 90% to about 98%, more than 95%, from about 95% to about 98%, more than about 98%, or more than about 99%. The binding of TNF-related apoptosis-inducing ligand (TRAIL) to TRAILR2 can be blocked by the anti-TRAILR2 antigen- binding proteins described herein by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or even 100%. In some embodiments, the binding of TNF- related apoptosis-inducing ligand (TRAIL) to TRAILR2 can be blocked by the anti-TRAILR2 antigen-binding proteins described herein by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, or more.

[0114] In some embodiments, anti-TRAILR2 antigen-binding proteins of the present disclosure may specificaly bind TRAILR2 without exhibiting specific binding for another receptor of the tumor necrosis factor receptor (TNFR) superfamily.

[0115] In some embodiments, anti-TRAILR2 antigen-binding proteins of the present disclosure do not bind specificaly to TRAILR1, TRAILR3 and / or TRAILR4. In some embodiments, anti-TRAILR2 antigen- binding proteins of the present disclosure do not bind specificaly to TRAILR1. In some embodiments, anti-TRAILR2 antigen-binding proteins of the present disclosure do not bind specificaly to TRAILR3. In some embodiments, anti-TRAILR2 antigen-binding proteins of the present disclosure do not bind specificaly to TRAILR4. In some embodiments, anti-TRAILR2 antigen-binding proteins of the present disclosure do not bind specificaly to TRAILR1, TRAILR3 and TRAILR4.

[0116] In some embodiments, anti-TRAILR2 antigen-binding proteins of the present disclosure bind cysteine-rich domain (CRD) domain 1 (CRD1) and / or CRD2 of TRAILR2. In some embodiments, anti- TRAILR2 antigen-binding proteins of the present disclosure bind CRD1 of TRAILR2. In some embodiments, anti-TRAILR2 antigen-binding proteins of the present disclosure bind CRD2 of TRAILR2. In some embodiments, anti-TRAILR2 antigen-binding proteins of the present disclosure bind CRD1 and CRD2 of TRAILR2.

[0117] In some embodiments, anti-TRAILR2 antigen-binding proteins of the present disclosure bind CRD2 and / or CRD3 of TRAILR2. In some embodiments, anti-TRAILR2 antigen-binding proteins of the present disclosure bind CRD2 of TRAILR2. In some embodiments, anti-TRAILR2 antigen-binding proteins of the present disclosure bind CRD3 of TRAILR2. In some embodiments, anti-TRAILR2 antigen-binding proteins of the present disclosure bind CRD2 and CRD3 of TRAILR2.Attorney Docket No: 260525.000049

[0118] Binding afinity of a molecular interaction between two molecules can be measured via various techniques, such as surface plasmon resonance (SPR), bio-layer interferometry (BLI), enzyme-linked immunosorbent assay (ELISA), equilibrium dialysis, fluorescent-activated cel sorting (FACS), flow cytometry binding assays, or isothermal titration calorimetry (ITC), and the like. Surface plasmon resonance is a biosensor technique that alows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, where one molecule is immobilized on the biosensor chip and the other molecule is passed over the immobilized molecule under flow conditions (see e.g., Ober et al.2001, Intern. Immunology 13: 1551-1559). SPR can for example be performed using the BIACORE® system or Carterra LSA system. Another biosensor technique that can be used to determine afinities of biomolecular interactions is bio-layer interferometry (BLI) (see e.g., Abdiche et al.2008, Anal. Biochem.377: 209-217). Bio-layer Interferometry is a label-free optical technique that analyzes the interference pattern of light reflected from two surfaces: an internal reference layer (reference beam) and a layer of immobilized protein on the biosensor tip (signal beam). A change in the number of molecules bound to the tip of the biosensor causes a shift in the interference pattern, reported as a wavelength shift (nm), the magnitude of which is a direct measure of the number of molecules bound to the biosensor tip surface. Since the interactions can be measured in real-time, association and dissociation rates and afinities can be determined. BLI can for example be performed using the Octet® Systems. Alternatively, afinities can be measured in Kinetic Exclusion Assay (KinExA) (see e.g., Drake et al.2004, Anal. Biochem., 328: 35-43), which is a solution-based method to measure true equilibrium binding afinity and kinetics of unmodified molecules. Equilibrated solutions of an antibody / antigen complex are passed over a column with beads precoated with antigen (or antibody), alowing the free antibody (or antigen) to bind to the coated molecule. Detection of the antibody (or antigen) thus captured is accomplished with a fluorescently labeled protein binding the antibody (or antigen).

[0119] Antigen-binding proteins of the present disclosure can include an antibody or an antigen-binding fragment of an antibody, such as a human antibody, a humanized antibody; a camelid antibody; a chimeric antibody; a recombinant antibody; a heavy chain antibody; a single-domain antibody (e.g., VHH); a single chain antibody (e.g., single chain fragment variable (scFv); a diabody; a triabody; a tetrabody; a Fab fragment; a F(ab′) 2 fragment; an IgD antibody; an IgE antibody; an IgM antibody; an IgG1 antibody; an IgG2 antibody; an IgG3 antibody; or an IgG4 antibody, and fragments thereof.

[0120] In some embodiments, an antigen-binding protein that binds to TRAILR2 is a single-domain antibody (also termed as “sdAb”). The single-domain antibodies of the present disclosure can be derivedAttorney Docket No: 260525.000049 from numerous sources, including but not limited to VHHs, VNARs, or VH domains (naturaly occurring or engineered VH domains). VHHs can be generated from camelid heavy chain only antibodies and libraries (e.g., synthetic libraries) thereof. VNARs can be generated from cartilaginous fish heavy chain only antibodies and libraries (e.g., synthetic libraries) thereof. Various methods have been implemented to generate monomeric sdAbs from conventionaly heterodimeric VH and VL domains, including interface engineering and selection of specific germline families. In some embodiments, the sdAb of the present invention are human or humanized.

[0121] In some embodiments, a single-domain antibody described herein is a VHH fragment (also known as a nanobody). VHH fragments are also referred to as “V-bodies” in the present disclosure. In some embodiments, the VHH is a camelid VHH, a humanized VHH or a camelized VH. In some embodiments, a single-domain antibody described herein is a VH domain. In some embodiments, a single-domain antibody described herein is a naturaly occurring VH domain or engineered VH domain.

[0122] The variable domain of an antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises at least three complementarity determining regions (CDRs) which determine its binding specificity. Preferably, in a variable domain, the CDRs are distributed between framework regions (FRs). The variable domain typicaly contains 4 framework regions interspaced by 3 CDR regions, resulting in the folowing typical antibody variable domain structure: FR1- CDR1-FR2-CDR2-FR3-CDR3-FR4. CDRs and / or FRs of the single domain antibody of the invention may be fragments or derivatives from a naturaly occurring antibody variable domain or may be synthetic.

[0123] Sequence identifiers corresponding to exemplary anti-TRAILR2 VHH antibodies provided herein are listed in Table 1-1. Table 1-1 sets forth the sequence identifiers of amino acid sequences of the complementarity determining regions (CDR1, CDR2 and CDR3), amino acid and DNA sequences of the ful-length camelid VHH antibodies, as wel as amino acid sequences of corresponding humanized VHH antibodies. Amino acid sequences of additional exemplary anti-TRAILR2 VHH antibodies and corresponding humanized VHH antibodies are provided in Table 1-2. Table 1-1. Sequence identifiers for exemplary anti-TRAILR2 VHH antibodies Antibody ID Group CDR1 CDR2 CDR3 Non-humanized VHH Humanized id eAttorney Docket No: 260525.000049 ODY-23A11 C 11 12 16 17 60 18 ODY-24F01 C 11 19 20 21 61 22Attorney Docket No: 260525.000049 ODY- D 28 29 2312 - - 2368 28B01Hu1 Al15Attorney Docket No: 260525.000049 ODY- I 52 53 2325 - - 2381 25C08Hu1 Al11Attorney Docket No: 260525.000049 ODY- 2189 N2280 Ala9 ODY-N 2195 ODY- 2195 N1039 Ala1 ODY- 2195 N1039 Ala2 ODY- 2195 N1039 Ala3 ODY- 2195 N1039 Ala4 ODY- 2195 N1039 Ala5 ODY- 2195 N1039 Ala6 ODY- 2195 N1039 Ala7 ODY-N 2201 ODY-2201 N1047Hu1 Ala1 ODY- R 2201 N1047Hu1 Ala2 ODY- R 2201N1047Hu1 Gly3 ODY- R 2201 2202 2348 - - 2404 N1047Hu1 Ala4 Table 1-2. Sequence identifiers for additional exemplary VHH antibodies and humanized VHH antibodies Non-humanized VHH Humanized VHH Cluster Group Amino Acid Amino Acid Sequence Sequence ODY-N731 A 4 5 ODY-N731 A 146 469Attorney Docket No: 260525.000049 ODY-N742 B 9 10 ODY-N742 B 147 470 DYN742 B 14 471Attorney Docket No: 260525.000049 ODY-23D12-ODY-23A11-ODY- C 170 491 24F01-ODY-N808 ODY23D12ODY23A11ODY C 171 492Attorney Docket No: 260525.000049 ODY-23D12-ODY-23A11-ODY- C 193 513 24F01-ODY-N808 ODY23D12ODY23A11ODY C 194 514Attorney Docket No: 260525.000049 ODY-23D12-ODY-23A11-ODY- C 216 535 24F01-ODY-N808 ODY23D12ODY23A11ODY C 217 536Attorney Docket No: 260525.000049 ODY-23D12-ODY-23A11-ODY- C 239 476 24F01-ODY-N808 ODY23D12ODY23A11ODY C 240 509Attorney Docket No: 260525.000049 ODY-23D12-ODY-23A11-ODY- C 262 578 24F01-ODY-N808 ODY23D12ODY23A11ODY C 263 579Attorney Docket No: 260525.000049 ODY-23D12-ODY-23A11-ODY- C 285 601 24F01-ODY-N808 ODY23D12ODY23A11ODY C 286 602Attorney Docket No: 260525.000049 ODY-23D12-ODY-23A11-ODY- C 308 624 24F01-ODY-N808 ODY23D12ODY23A11ODY C 309 625Attorney Docket No: 260525.000049 ODY-23D12-ODY-23A11-ODY- C 331 647 24F01-ODY-N808 ODY23D12ODY23A11ODY C 332 648Attorney Docket No: 260525.000049 ODY-23D12-ODY-23A11-ODY- C 26 27 24F01-ODY-N808 ODY23D12ODY23A11ODY C 354 670Attorney Docket No: 260525.000049 ODY-28B01 D 385 698 ODY-28B01 D 386 699 DY2B1 D 7 7Attorney Docket No: 260525.000049 ODY-N793 G 428 734 ODY-N793 G 429 735 DYN7 4 7Attorney Docket No: 260525.000049 ODY-N1016 O 463 759 ODY-N1016 O 464 760 DYN11 4 7

[0124] In some embodiments, an anti-TRAILR2 antigen-binding protein (e.g., antibody such as a single- domain antibody) described herein may further comprise a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from (amino acids listed in a pair of brackets represent the possible amino acids at the particular position) 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);Attorney Docket No: 260525.000049 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). GFTLDLGAYA (SEQ ID NO: 81); m). GFTFGALA (SEQ ID NO: 86); n). GFTLS(G / S)YA (SEQ ID NO: 120); o). GSIFGGYN (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)..

[0125] In some embodiments, an anti-TRAILR2 antigen-binding protein (e.g., antibody such as a single- domain antibody) of the present disclosure comprises a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from 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).

[0126] In some embodiments, an anti-TRAILR2 antigen-binding protein (e.g., antibody such as a single- domain antibody) described herein may further comprise a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from (amino acids listed in a pair of brackets represent the possible amino acids at the particular position) 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);Attorney Docket No: 260525.000049 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).

[0127] In some embodiments, an anti-TRAILR2 antigen-binding protein (e.g., antibody such as a single- domain antibody) of the present disclosure comprises a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from VSWNGAST (SEQ ID NO: 2); DHRSGT (SEQ ID NO: 7); ISWTGVDT (SEQ ID NO: 12); ISWTGTDT (SEQ ID NO: 19); ISWSGVDT (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: 91); ITSAGGST (SEQ ID NO: 96); IFISGNN (SEQ ID NO: 2190); ITSDDTT (SEQ ID NO: 2196); and ISSAGGYI (SEQ ID NO: 2202).

[0128] In some embodiments, an anti-TRAILR2 antigen-binding protein (e.g., antibody such as a single- domain antibody) of the present disclosure comprises a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from (amino acids listed in a pair of brackets represent the possible amino acids at the particular position) 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);Attorney Docket No: 260525.000049 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 is optionaly replaced with an alanine, and / or one or more alanine residues in the CDR3 sequence is optionaly replaced with a glycine.

[0129] In some embodiments, one non-alanine residue in the CDR3 sequence is replaced with an alanine. In some embodiments, two non-alanine residues in the CDR3 sequence are replaced with an alanine. In some embodiments, three non-alanine residues in the CDR3 sequence are replaced with an alanine. In some embodiments, four non-alanine residues in the CDR3 sequence are replaced with an alanine. In some embodiments, five or more non-alanine residues in the CDR3 sequence are replaced with an alanine.

[0130] In some embodiments, one alanine residue in the CDR3 sequence is replaced with a glycine. In some embodiments, two alanine residues in the CDR3 sequence are replaced with a glycine. In some embodiments, three alanine residues in the CDR3 sequence are replaced with a glycine. In some embodiments, four alanine residues in the CDR3 sequence are replaced with a glycine. In some embodiments, five alanine or more residues in the CDR3 sequence are replaced with a glycine.

[0131] In some embodiments, any of the above-described non-alanine residue replacement(s) with an alanine in the CDR3 sequence can be combined with any of the above-described alanine residue replacement(s) with a glycine in the CDR3 sequence.

[0132] In some embodiments, the CDR3 comprises an amino acid sequence selected from 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);Attorney Docket No: 260525.000049 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.

[0133] In some embodiments, an anti-TRAILR2 antigen-binding protein (e.g., antibody such as a single- domain antibody) of the present disclosure comprises a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from 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: 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); AAASSWSRGGAPYGMDY (SEQ ID NO: 2308); AAASSWSRGGVAYGMDY (SEQ ID NO: 2309); AAASSWSRGGVPAGMDY (SEQ ID NO: 2310); AAASSWSRGGVPYAMDY (SEQ ID NO: 2311); AAASSWSRGGVPYGADY (SEQ ID NO: 2312); AAASSWSRGGVPYGMAY (SEQ ID NO: 2313); AAASSWSRGGVPYGMDA (SEQ ID NO: 2314); 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); TAGPTMSYSRGAEF (SEQ ID NO: 2326); TAGPTMSYSRGGAF (SEQ ID NO: 2327); TAGPTMSYSRGGEA (SEQ ID NO: 2328); 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); 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); HSRWANLAttorney Docket No: 260525.000049 (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).

[0134] In some embodiments, an anti-TRAILR2 antigen-binding protein (e.g., antibody such as a single- domain antibody) of the present disclosure comprises i). a CDR1 comprising an amino acid sequence of GRTFSSNL (SEQ ID NO: 1), a CDR2 comprising an amino acid sequence of VSWNGAST (SEQ ID NO: 2), and a CDR3 comprising an amino acid sequence of (A / V)ASRL(P / V)FNSRSA(I / V)YTDRIYDS (SEQ ID NO: 100); i). a CDR1 comprising an amino acid sequence of GGT(F / L)(A / S)N(D / N)G (SEQ ID NO: 113), a CDR2 comprising an amino acid sequence of DHR(S / T)GT (SEQ ID NO: 121), a CDR3 comprising an amino acid sequence of AVRRSAWY(S / T)DSIYTVSQYDY (SEQ ID NO: 101); ii). a CDR1 comprising an amino acid sequence of GRTL(D / N / S)(A / D / E)Y(A / G) (SEQ ID NO: 114), a CDR2 comprising an amino acid sequence of I(N / S)W(N / S / T)G(T / V)(D / G)T (SEQ ID NO: 122), a CDR3 comprising an amino acid sequence of NAARSYSR(D / G / N)(G / Y)(E / R)PL(E / K)P(A / D)Y (SEQ ID NO: 102); iv). a CDR1 comprising an amino acid sequence of GRTFS(N / S)YA (SEQ ID NO: 115), a CDR2 comprising an amino acid sequence of LNW(N / S)G(D / E)ST (SEQ ID NO: 123), a CDR3 comprising an amino acid sequence of AAASSWSRGG(A / G / I / V)PYGMDY (SEQ ID NO: 103); v). a CDR1 comprising an amino acid sequence of GRDFSNYV (SEQ ID NO: 33), a CDR2 comprising an amino acid sequence of INWAD(E / T)T (SEQ ID NO: 124), a CDR3 comprising an amino acid sequence of AADS(H / R)FRR(P / Y)(A / T / V)PG(I / Q)QYEY (SEQ ID NO: 104); vi). a CDR1 comprising an amino acid sequence of G(L / R)(I / S)FS(D / S)YA (SEQ ID NO: 116), a CDR2 comprising an amino acid sequence of INWSGG(S / T)T (SEQ ID NO: 125), a CDR3 comprising an amino acid sequence of NAA(K / R)SYHRDY(K / S)PL(K / S)(G / P)DY (SEQ ID NO: 105); vi). a CDR1 comprising an amino acid sequence of GR(A / T)FSTLA (SEQ ID NO: 117), a CDR2 comprising an amino acid sequence of INWSGG(S / T)T (SEQ ID NO: 125), a CDR3 comprising an amino acid sequence of AAAPSFGM(M / R / T)(I / N)PESYVHS (SEQ ID NO: 106); vii). a CDR1 comprising an amino acid sequence of GRTFSSDI (SEQ ID NO: 47), a CDR2 comprising an amino acid sequence of ISWSDMSA (SEQ ID NO: 48), a CDR3 comprising an amino acid sequence of AANRGIMSMRLSRYDD (SEQ ID NO: 49); ix). a CDR1 comprising an amino acid sequence of GRSFGD(D / F / Y)A (SEQ ID NO: 118), a CDR2 comprising an amino acid sequence of I(N / R)W(A / D / T)G(D / N)T (SEQ ID NO: 126), a CDR3 comprising an amino acid sequence of T(A / V)GP(A / T)MSYSRGGEF (SEQ ID NO: 107);Attorney Docket No: 260525.000049 x). a CDR1 comprising an amino acid sequence of G(G / R)TLSNYA (SEQ ID NO: 119), a CDR2 comprising an amino acid sequence of ISQ(S / T)S(D / S)T (SEQ ID NO: 127), a CDR3 comprising an amino acid sequence of (A / V)ADRGAISRSGAGM(D / N)Y (SEQ ID NO: 108); xi). a CDR1 comprising an amino acid sequence of GRSFGAQGMEG (SEQ ID NO: 72), a CDR2 comprising an amino acid sequence of (I / M)KWTGNT (SEQ ID NO: 128), a CDR3 comprising an amino acid sequence of TAGP(A / S)IS(L / Y)SRGGEY (SEQ ID NO: 109); xi). a CDR1 comprising an amino acid sequence of GFTLDLGAYA (SEQ ID NO: 81), a CDR2 comprising an amino acid sequence of ISN(S / T)GTTT (SEQ ID NO: 129), a CDR3 comprising an amino acid sequence of A(A / T)NGWGLDP(S / T)TYH(Y / D) (SEQ ID NO: 110); xii). a CDR1 comprising an amino acid sequence of GFTFGALA (SEQ ID NO: 86), a CDR2 comprising an amino acid sequence of ISNDGEHI (SEQ ID NO: 87), a CDR3 comprising an amino acid sequence of SAGWTRRIFQY (SEQ ID NO: 88); xiv). a CDR1 comprising an amino acid sequence of GRTFS(N / S)YA (SEQ ID NO: 115), a CDR2 comprising an amino acid sequence of ISWNGDIT (SEQ ID NO: 91), a CDR3 comprising an amino acid sequence of TAGQSISLSQGGE(H / Y) (SEQ ID NO: 111); xv). a CDR1 comprising an amino acid sequence of GFTLS(G / S)YA (SEQ ID NO: 120), a CDR2 comprising an amino acid sequence of IT(G / S)(A / S)G(G / S)(N / S)T (SEQ ID NO: 130), a CDR3 comprising an amino acid sequence of KAGIRGE(T / V)Y (SEQ ID NO: 112); xvi). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGN(D / N) (SEQ ID NO: 2207), a CDR3 comprising an amino acid sequence of RAYNDGGEY (SEQ ID NO: 2191); xvi). a CDR1 comprising an amino acid sequence of G(G / S)NFRILS (SEQ ID NO: 2209), a CDR2 comprising an amino acid sequence of (I / L)T(K / M / S)D(D / G)TT (SEQ ID NO: 2210), a CDR3 comprising an amino acid sequence of HS(N / R)WYNL (SEQ ID NO: 2208); xvii). a CDR1 comprising an amino acid sequence of G(F / L)(A / T)F(R / S)(R / S)YA (SEQ ID NO: 2212), a CDR2 comprising an amino acid sequence of ISS(A / G / S)(G / S)G(I / Y)(I / T / V) (SEQ ID NO: 2213), a CDR3 comprising an amino acid sequence of (F / M / N)T(A / S)DY; xix). a CDR1 comprising an amino acid sequence of GRTFS(N / S)YA (SEQ ID NO: 115), a CDR2 comprising an amino acid sequence of LNW(N / S)G(D / E)ST (SEQ ID NO: 123), a CDR3 comprising an amino acid sequence of (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);Attorney Docket No: 260525.000049 xx). a CDR1 comprising an amino acid sequence of GRSFGD(D / F / Y)A (SEQ ID NO: 118), a CDR2 comprising an amino acid sequence of I(N / R)W(A / D / T)G(D / N)T (SEQ ID NO: 126), a CDR3 comprising an amino acid sequence of (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); xxi). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGN(D / N) (SEQ ID NO: 2207), a CDR3 comprising an amino acid sequence of (A / R)(A / G)(A / Y)(A / N)(A / D)(A / G)(A / G)(A / E)(A / Y); xxi). a CDR1 comprising an amino acid sequence of G(G / S)NFRILS (SEQ ID NO: 2209), a CDR2 comprising an amino acid sequence of (I / L)T(K / M / S)D(D / G)TT (SEQ ID NO: 2210), a CDR3 comprising an amino acid sequence of (A / H)(A / S)(A / N / R)(A / W)(A / Y)(A / N)(A / L); or xxii). a CDR1 comprising an amino acid sequence of G(F / L)(A / T)F(R / S)(R / S)YA (SEQ ID NO: 2212), a CDR2 comprising an amino acid sequence of ISS(A / G / S)(G / S)G(I / Y)(I / T / V) (SEQ ID NO: 2213), a CDR3 comprising an amino acid sequence of (A / F / M / N)(A / T)(A / G / S)(A / D)Y.

[0135] Provided herein are anti-TRAILR2 antigen-binding proteins (e.g., antibodies such as single- domain antibodies) comprising a CDR1 (CDR1) comprising an amino acid sequence selected from any of the CDR1 amino acid sequences listed in Table 1-1 or Table 6, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0136] In some embodiments, an anti-TRAILR2 antigen-binding protein (e.g., antibody such as single- domain antibody) comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 1, 6, 11, 23, 28, 33, 38, 43, 47, 52, 62, 67, 72, 81, 86, 95, 762-1084, 2189, 2195, 2201, and 2252-2261, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0137] Provided herein are anti-TRAILR2 antigen-binding proteins (e.g., antibodies such as single- domain antibodies) comprising a CDR2 (CDR2) comprising an amino acid sequence selected from any of the CDR2 amino acid sequences listed in Table 1-1 or Table 6, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0138] In some embodiments, an anti-TRAILR2 antigen-binding protein (e.g., antibody such as single- domain antibody) comprises a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 2, 7, 12, 19, 24, 29, 34, 39, 48, 53, 63, 68, 73, 77, 82, 87, 91, 96, 1085-1407, 2190, 2196, 2202, and 2262-2271, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0139] Provided herein are anti-TRAILR2 antigen-binding proteins (e.g., antibodies such as single- domain antibodies) comprising a CDR3 (CDR3) comprising an amino acid sequence selected from any ofAttorney Docket No: 260525.000049 the CDR3 amino acid sequences listed in Table 1-1 or Table 6, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0140] In some embodiments, an anti-TRAILR2 antigen-binding protein (e.g., antibody such as single- domain antibody) comprises a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 3, 8, 13, 16, 20, 25, 30, 35, 40, 44, 49, 54, 64, 69, 74, 78, 83, 88, 92, 97, 1408-1730, 2191, 2197, 2203, 2272- 2277, and 2298-2348or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0141] Provided herein are anti-TRAILR2 antigen-binding proteins (e.g., antibodies such as single- domain antibodies) 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, Table 1-2, or Table 6. In certain embodiments, an anti-TRAILR2 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises i) a CDR1 comprising an amino acid sequence of GRTFSSNL (SEQ ID NO: 1), a CDR2 comprising an amino acid sequence of VSWNGAST (SEQ ID NO: 2), and a CDR3 comprising an amino acid sequence of VASRLPFNSRSAIYTDRIYDS (SEQ ID NO: 3); i) a CDR1 comprising an amino acid sequence of GGTLANNG (SEQ ID NO: 6), a CDR2 comprising an amino acid sequence of DHRSGT (SEQ ID NO: 7), a CDR3 comprising an amino acid sequence of AVRRSAWYSDSIYTVSQYDY (SEQ ID NO: 8); ii) a CDR1 comprising an amino acid sequence of GRTLDAYG (SEQ ID NO: 11), a CDR2 comprising an amino acid sequence of ISWTGVDT (SEQ ID NO: 12), a CDR3 comprising an amino acid sequence of NAARSYSRDYEPLKPDY (SEQ ID NO: 13); iv) a CDR1 comprising an amino acid sequence of GRTLDAYG (SEQ ID NO: 11), a CDR2 comprising an amino acid sequence of ISWTGVDT (SEQ ID NO: 12), a CDR3 comprising an amino acid sequence of NAARSYSRNYEPLKPDY (SEQ ID NO: 16); v) a CDR1 comprising an amino acid sequence of GRTLDAYG (SEQ ID NO: 11), a CDR2 comprising an amino acid sequence of ISWTGTDT (SEQ ID NO: 19), a CDR3 comprising an amino acid sequence of NAARSYSRGGEPLKPDY (SEQ ID NO: 20); vi) a CDR1 comprising an amino acid sequence of GRTLSDYA (SEQ ID NO: 23) , a CDR2 comprising an amino acid sequence of ISWSGVDT (SEQ ID NO: 24), a CDR3 comprising an amino acid sequence of NAARSYSRGGRPLEPAY (SEQ ID NO: 25);Attorney Docket No: 260525.000049 vi) a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), a CDR3 comprising an amino acid sequence of AAASSWSRGGVPYGMDY (SEQ ID NO: 30); vii) a CDR1 comprising an amino acid sequence of GRDFSNYV (SEQ ID NO: 33), a CDR2 comprising an amino acid sequence of INWADET (SEQ ID NO: 34), a CDR3 comprising an amino acid sequence of AADSHFRRYTPGQQYEY (SEQ ID NO: 35); ix) a CDR1 comprising an amino acid sequence of GRSFSSYA (SEQ ID NO: 38), a CDR2 comprising an amino acid sequence of INWSGGST (SEQ ID NO: 39), a CDR3 comprising an amino acid sequence of NAAKSYHRDYSPLSPDY (SEQ ID NO: 40); x) a CDR1 comprising an amino acid sequence of GRTFSTLA (SEQ ID NO: 43), a CDR2 comprising an amino acid sequence of INWSGGST (SEQ ID NO: 39), a CDR3 comprising an amino acid sequence of AAAPSFGMRNPESYVHS (SEQ ID NO: 44); xi) a CDR1 comprising an amino acid sequence of GRTFSSDI (SEQ ID NO: 47), a CDR2 comprising an amino acid sequence of ISWSDMSA (SEQ ID NO: 48), a CDR3 comprising an amino acid sequence of AANRGIMSMRLSRYDD (SEQ ID NO: 49); xi) a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), a CDR3 comprising an amino acid sequence of TAGPTMSYSRGGEF (SEQ ID NO: 54); xii) a CDR1 comprising an amino acid sequence of GGTLSNYA (SEQ ID NO: 62), a CDR2 comprising an amino acid sequence of ISQTSST (SEQ ID NO: 63), a CDR3 comprising an amino acid sequence of VADRGAISRSGAGMDY (SEQ ID NO: 64); xiv) a CDR1 comprising an amino acid sequence of GRTLSNYA (SEQ ID NO: 67), a CDR2 comprising an amino acid sequence of ISQSSDT (SEQ ID NO: 68), a CDR3 comprising an amino acid sequence of AADRGAISRSGAGMDY (SEQ ID NO: 69); xv) a CDR1 comprising an amino acid sequence of GRSFGAQGMEG (SEQ ID NO: 72), a CDR2 comprising an amino acid sequence of MKWTGNT (SEQ ID NO: 73), a CDR3 comprising an amino acid sequence of TAGPAISLSRGGEY (SEQ ID NO: 74); xvi) a CDR1 comprising an amino acid sequence of GRSFGAQGMEG (SEQ ID NO: 72), a CDR2 comprising an amino acid sequence of IKWTGNT (SEQ ID NO: 77), a CDR3 comprising an amino acid sequence of TAGPSISYSRGGEY (SEQ ID NO: 78);Attorney Docket No: 260525.000049 xvi) a CDR1 comprising an amino acid sequence of GFTLDLGAYA (SEQ ID NO: 81), a CDR2 comprising an amino acid sequence of ISNTGTTT (SEQ ID NO: 82), a CDR3 comprising an amino acid sequence of AANGWGLDPTTYHY (SEQ ID NO: 83); xvii) a CDR1 comprising an amino acid sequence of GFTFGALA (SEQ ID NO: 86), a CDR2 comprising an amino acid sequence of ISNDGEHI (SEQ ID NO: 87), a CDR3 comprising an amino acid sequence of SAGWTRRIFQY (SEQ ID NO: 88); xix) a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of ISWNGDIT (SEQ ID NO: 91), a CDR3 comprising an amino acid sequence of TAGQSISLSQGGEY (SEQ ID NO: 92); xx) a CDR1 comprising an amino acid sequence of GFTLSGYA (SEQ ID NO: 95), a CDR2 comprising an amino acid sequence of ITSAGGST (SEQ ID NO: 96), a CDR3 comprising an amino acid sequence of KAGIRGEVY (SEQ ID NO: 97); xxi). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), a CDR3 comprising an amino acid sequence of RAYNDGGEY (SEQ ID NO: 2191); xxi). a CDR1 comprising an amino acid sequence of GSNFRILS (SEQ ID NO: 2195), a CDR2 comprising an amino acid sequence of ITSDDTT (SEQ ID NO: 2196), a CDR3 comprising an amino acid sequence of HSRWYNL (SEQ ID NO: 2197); xxii). a CDR1 comprising an amino acid sequence of GFTFSRYA (SEQ ID NO: 2201), a CDR2 comprising an amino acid sequence of ISSAGGYI (SEQ ID NO: 2202), a CDR3 comprising an amino acid sequence of FTADY (SEQ ID NO: 2203); xxiv). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of GAASSWSRGGVPYGMDY (SEQ ID NO: 2298); xxv). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AGASSWSRGGVPYGMDY (SEQ ID NO: 2299); xxvi). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAGSSWSRGGVPYGMDY (SEQ ID NO: 2300);Attorney Docket No: 260525.000049 xxvi). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAAASWSRGGVPYGMDY (SEQ ID NO: 2301); xxvii). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASAWSRGGVPYGMDY (SEQ ID NO: 2302); xxix). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSASRGGVPYGMDY (SEQ ID NO: 2303); xxx). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWARGGVPYGMDY (SEQ ID NO: 2304); xxxi). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSAGGVPYGMDY (SEQ ID NO: 2305); xxxi). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRAGVPYGMDY (SEQ ID NO: 2306); xxxii). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRGAVPYGMDY (SEQ ID NO: 2307); xxxiv). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRGGAPYGMDY (SEQ ID NO: 2308); xxxv). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRGGVAYGMDY (SEQ ID NO: 2309); xxxvi). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRGGVPAGMDY (SEQ ID NO: 2310);Attorney Docket No: 260525.000049 xxxvi). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRGGVPYAMDY (SEQ ID NO: 2311); xxxvii). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRGGVPYGADY (SEQ ID NO: 2312); xxxix). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRGGVPYGMAY (SEQ ID NO: 2313); xl). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRGGVPYGMDA (SEQ ID NO: 2314); xli). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of AAGPTMSYSRGGEF (SEQ ID NO: 2315); xli). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TGGPTMSYSRGGEF (SEQ ID NO: 2316); xlii). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAAPTMSYSRGGEF (SEQ ID NO: 2317); xliv). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGATMSYSRGGEF (SEQ ID NO: 2318); xlv). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPAMSYSRGGEF (SEQ ID NO: 2319); xlvi). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTASYSRGGEF (SEQ ID NO: 2320);Attorney Docket No: 260525.000049 xlvi). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTMAYSRGGEF (SEQ ID NO: 2321); xlvii). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTMSASRGGEF (SEQ ID NO: 2322); xlix). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTMSYARGGEF (SEQ ID NO: 2323); l). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTMSYSAGGEF (SEQ ID NO: 2324); li). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTMSYSRAGEF (SEQ ID NO: 2325); li). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTMSYSRGAEF (SEQ ID NO: 2326); lii). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTMSYSRGGAF (SEQ ID NO: 2327); liv). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTMSYSRGGEA (SEQ ID NO: 2328); lv). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of AAYNDGGEY (SEQ ID NO: 2329); lvi). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of RGYNDGGEY (SEQ ID NO: 2330);Attorney Docket No: 260525.000049 lvi). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of RAANDGGEY (SEQ ID NO: 2331); lvii). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of RAYADGGEY (SEQ ID NO: 2332); lix). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of RAYNAGGEY (SEQ ID NO: 2333); lx). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of RAYNDAGEY (SEQ ID NO: 2334); lxi). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of RAYNDGAEY (SEQ ID NO: 2335); lxi). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of RAYNDGGAY (SEQ ID NO: 2336); lxii). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of RAYNDGGEA (SEQ ID NO: 2337); lxiv). a CDR1 comprising an amino acid sequence of GSNFRILS (SEQ ID NO: 2195), a CDR2 comprising an amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and a CDR3 comprising an amino acid sequence of ASRWYNL (SEQ ID NO: 2338); lxv). a CDR1 comprising an amino acid sequence of GSNFRILS (SEQ ID NO: 2195), a CDR2 comprising an amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and a CDR3 comprising an amino acid sequence of HARWYNL (SEQ ID NO: 2339); lxvi). a CDR1 comprising an amino acid sequence of GSNFRILS (SEQ ID NO: 2195), a CDR2 comprising an amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and a CDR3 comprising an amino acid sequence of HSAWYNL (SEQ ID NO: 2340);Attorney Docket No: 260525.000049 lxvi). a CDR1 comprising an amino acid sequence of GSNFRILS (SEQ ID NO: 2195), a CDR2 comprising an amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and a CDR3 comprising an amino acid sequence of HSRAYNL (SEQ ID NO: 2341); lxvii). a CDR1 comprising an amino acid sequence of GSNFRILS (SEQ ID NO: 2195), a CDR2 comprising an amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and a CDR3 comprising an amino acid sequence of HSRWANL (SEQ ID NO: 2342); lxix). a CDR1 comprising an amino acid sequence of GSNFRILS (SEQ ID NO: 2195), a CDR2 comprising an amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and a CDR3 comprising an amino acid sequence of HSRWYAL (SEQ ID NO: 2343); lxx). a CDR1 comprising an amino acid sequence of GSNFRILS (SEQ ID NO: 2195), a CDR2 comprising an amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and a CDR3 comprising an amino acid sequence of HSRWYNA (SEQ ID NO: 2344); lxxi). a CDR1 comprising an amino acid sequence of GFTFSRYA (SEQ ID NO: 2201), a CDR2 comprising an amino acid sequence of ISSAGGYI (SEQ ID NO: 2202), and a CDR3 comprising an amino acid sequence of ATADY (SEQ ID NO: 2345); lxxi). a CDR1 comprising an amino acid sequence of GFTFSRYA (SEQ ID NO: 2201), a CDR2 comprising an amino acid sequence of ISSAGGYI (SEQ ID NO: 2202), and a CDR3 comprising an amino acid sequence of FAADY (SEQ ID NO: 2346); lxxii). a CDR1 comprising an amino acid sequence of GFTFSRYA (SEQ ID NO: 2201), a CDR2 comprising an amino acid sequence of ISSAGGYI (SEQ ID NO: 2202), and a CDR3 comprising an amino acid sequence of FTGDY (SEQ ID NO: 2347); or lxxiv). a CDR1 comprising an amino acid sequence of GFTFSRYA (SEQ ID NO: 2201), a CDR2 comprising an amino acid sequence of ISSAGGYI (SEQ ID NO: 2202), and a CDR3 comprising an amino acid sequence of FTAAY (SEQ ID NO: 2348).

[0142] In certain embodiments, an anti-TRAILR2 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises i). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRGGVPYGMDY (SEQ ID NO: 30); i). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTMSYSRGGEF (SEQ ID NO: 54);Attorney Docket No: 260525.000049 ii). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of RAYNDGGEY (SEQ ID NO: 2191); iv). a CDR1 comprising an amino acid sequence of GSNFRILS (SEQ ID NO: 2195), a CDR2 comprising an amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and a CDR3 comprising an amino acid sequence of HSRWYNL (SEQ ID NO: 2197); or v). a CDR1 comprising an amino acid sequence of GFTFSRYA (SEQ ID NO: 2201), a CDR2 comprising an amino acid sequence of ISSAGGYI (SEQ ID NO: 2202), and a CDR3 comprising an amino acid sequence of FTADY (SEQ ID NO: 2203), wherein one or more non-alanine residues in the CDR3 sequence is optionaly replaced with an alanine, and / or one or more alanine residues in the CDR3 sequence is optionaly replaced with a glycine.

[0143] In a related embodiment, provided herein are anti-TRAILR2 antigen-binding proteins (e.g., antibodies such as single-domain antibodies) comprising a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within a VHH amino acid sequence as defined by any of the exemplary anti-TRAILR2 VHH antibodies listed in Table 1-1, Table 1-2, or Table 6. For example, provided herein are antibodies, or antigen-binding fragments thereof, comprising the set of CDR1-CDR2-CDR3 amino acid sequences contained within a VHH amino acid sequence selected from SEQ ID NOs: 4, 9, 14, 17, 21, 26, 31, 36, 41, 45, 50, 55, 65, 70, 75, 79, 84, 89, 93, 98, 146-468, 2192, 2198, 2204 , 2214-2233, and 2354-2404.

[0144] In some embodiments, an anti-TRAILR2 antigen-binding protein (e.g., antibody such as a single- domain antibody) of the present disclosure can include a) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4; b) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 9; c) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 14; d) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 17; e) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 21; f) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 26;Attorney Docket No: 260525.000049 g) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 31; h) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 36; i) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 41; j) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 45; k) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 50; l) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 55; m) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 65; n) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 70; o) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 75; p) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 79; q) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 84; r) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 89; s) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 93; t) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 98; u) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 2192; v) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 2198; orAttorney Docket No: 260525.000049 w) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 2204.

[0145] In an embodiment provided herein, an anti-TRAILR2 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure can include a VHH amino acid sequence selected from SEQ ID NOs: 4, 9, 14, 17, 21, 26, 31, 36, 41, 45, 50, 55, 65, 70, 75, 79, 84, 89, 93, 98, 146-468, 2192, 2198, 2204 , and 2214-2233, or a similar sequence thereof 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.

[0146] In some embodiments, an anti-TRAILR2 antigen-binding protein (e.g., antibody such as a single- domain antibody) of the present disclosure can include a VHH amino acid sequence of SEQ ID NOs: 4, 9, 14, 17, 21, 26, 31, 36, 41, 45, 50, 55, 65, 70, 75, 79, 84, 89, 93, 98, 146-468, 2192, 2198, 2204 , and 2214- 2233, or a sequence having at least 75% identity thereto.

[0147] In an embodiment provided herein, an anti-TRAILR2 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure can include a VHH amino acid sequence selected from SEQ ID NOs: 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 thereof 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.

[0148] In some embodiments, an anti-TRAILR2 antigen-binding protein (e.g., antibody such as a single- domain antibody) of the present disclosure can 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 thereto.

[0149] In some embodiments, an anti-TRAILR2 antigen-binding protein (e.g., antibody such as a single- domain antibody) of the present disclosure can include a VHH amino acid sequence selected from any one of SEQ ID NOs: 31, 55, 2192, 2198, and 2204, or an amino acid sequence having at least 75% identity thereto.

[0150] In some embodiments of any of the above-described antigen-binding proteins, the VHH may be a humanized VHH.

[0151] In an embodiment provided herein, an anti-TRAILR2 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure can include a humanized VHH amino acid sequence selected from SEQ ID NOs: 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, 2234-2251, and 2354-2404, or a similar sequence thereof 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.Attorney Docket No: 260525.000049

[0152] In some embodiments, an anti-TRAILR2 antigen-binding protein (e.g., antibody such as a single- domain antibody) of the present disclosure can include a humanized VHH amino acid sequence selected from 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, 2234-2251, and 2354-2404, or a sequence having at least 75% identity thereto.

[0153] In an embodiment provided herein, an anti-TRAILR2 antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure can include a humanized VHH amino acid sequence selected from SEQ ID NOs: 5, 10, 15, 18, 22, 27, 32, 37, 42, 46, 51, 56, 66, 71, 76, 80, 85, 90, 94, 99, 2194, 2200, 2206, and 2354-2404, or a similar sequence thereof 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.

[0154] In some embodiments, an anti-TRAILR2 antigen-binding protein (e.g., antibody such as a single- domain antibody) of the present disclosure can include a humanized VHH amino acid sequence selected from 5, 10, 15, 18, 22, 27, 32, 37, 42, 46, 51, 56, 66, 71, 76, 80, 85, 90, 94, 99, 2194, 2200, 2206, and 2354-2404, or a sequence having at least 75% identity thereto.

[0155] In some embodiments, an anti-TRAILR2 antigen-binding protein (e.g., antibody such as a single- domain antibody) of the present disclosure can include a humanized VHH amino acid sequence selected from any one of SEQ ID NOs: 32, 56, 2200, 2206, and 2194, or an amino acid sequence having at least 75% identity thereto.

[0156] In some embodiments, an anti-TRAILR2 antigen-binding protein (e.g., antibody such as a single- domain antibody) of the present disclosure can include a humanized VHH amino acid sequence selected from any one of SEQ ID NOs: 2354-2404, or an amino acid sequence having at least 75% identity thereto.

[0157] In some embodiments, the present disclosure also provides an anti-TRAILR2 antigen-binding protein (e.g., antibody such as a single-domain antibody) that competes for binding to TRAILR2 with any one of the exemplary anti-TRAILR2 VHH antibodies listed in Table 1-1, Table 1-2, or Table 6.

[0158] In some embodiments, the present disclosure also provides an anti-TRAILR2 antigen-binding protein (e.g., antibody such as a single-domain antibody) that binds to the same epitope on TRAILR2 as any one of the exemplary anti-TRAILR2 VHH antibodies listed in Table 1-1, Table 1-2, or Table 6. Single-domain antibodiesantibody (e.g., VHH) can be obtained by immunization of dromedaries, camels, lamas, alpacas, or sharks with the desired antigen and subsequent isolation of the mRNA coding for heavy-chain antibodies. Antigens can be purified from natural sources, or in the course of recombinant production. Immunization and / or screening for immunoglobulin sequences can be performed usingAttorney Docket No: 260525.000049 peptide fragments of such antigens. By reverse transcription and polymerase chain reaction (PCR), a gene library of single-domain antibodies containing several milion clones can be produced. Screening techniques such as phage display, yeast display, and ribosome display help to identify the clones binding the antigen. Methods generation of heavy-chain antibody fragments are described in e.g., WO 94 / 04678; Hamers-Casterman et al.1993; Muyldermans et al.2001; and Arbabi Ghahroudi, M. et al. (1997). FEBS Leters 414 (3): 521-526, each of which is incorporated herein by reference in its entirety.

[0160] A diferent method may use gene libraries from animals that have not been previously immunized. Such naïve libraries usualy contain only antibodies with low afinity to the desired antigen, making it necessary to apply afinity maturation by random mutagenesis as an additional step. See e.g., Saerens, D.; et al. (2008). “Single-domain antibodies as building blocks for novel therapeutics”. Current Opinion in Pharmacology 8 (5): 600-608.

[0161] Afinity maturation strategies can be categorized as either targeted / rational approaches or untargeted / random approaches. For targeted approaches information about the VHH of interest is needed, such as hot spots for afinity maturation or structural information on the VHH:antigen complex, whereas for untargeted approaches no prior information is needed. Targeted approaches that may be applied for afinity maturation of VHHs include site-directed in-vitro mutagenesis and in- silico / computational approaches. Common untargeted approaches used for afinity maturation of VHHs include random in-vitro mutagenesis, CDR swapping and autonomous hypermutation yeast surface display, with the latter two being novel, emerging and very time eficient techniques. Most of these strategies have in common, that after applying a certain randomization strategy to generate a mutational library, the resulting library can be screened by employing standard display techniques such as yeast, phage or ribosome display to select for the best binders. The choice of the display system is often guided by the library size to be displayed, with yeast display being able to handle library sizes of ~107 – 109, phage display ~108-1010 and ribosome display ~1012-1013(Chan and Groves, 2021). Notably, during afinity maturation the number of highly interactive residues such as aromatic amino acids usualy increases in the CDR regions. The selected afinity matured clones may be further evaluated by a developability assessment to test for undesired properties, such as unspecific binding to of-targets or VHH instability.

[0162] For targeted in vitro mutagenesis, a set of selected residues within the CDRs of a VHH may be mutated (Tiler et al., 2017; Yau et al., 2005). Pre-selection of these residues can be either performed using alanine scanning to identify hot spot residues for mutation or by using structural data of the antigen:VHH complex to identify positions to be mutated. These sites can then be either submitted toAttorney Docket No: 260525.000049 saturating mutagenesis to substitute a specific site with al possible amino acids or specific amino acid substitutions yielding several smaler libraries. After mutagenesis binders can be displayed to select the best matured candidate. Usualy, several rounds of targeted mutagenesis are performed with separate sub-libraries to obtain combinations of individual mutations that cooperatively result in increased binding afinity.

[0163] Computer-aided / in silico methods are often used to guide targeted in vitro mutagenesis. Using homology modeling of the target:VHH complex or docking, hotspots for mutations can be identified that are then submited to in vitro mutagenesis (Bert Schepens et al., 2021; Cheng et al., 2019; Inoue et al., 2013; Mahajan et al., 2018). Further, in silico methods can search al designed variants in a virtual library (˜1040 members) in a rather short amount of time to identify a feasible number of promising candidates to be tested experimentaly. These techniques can be especialy valuable if structural data on the drug- target interaction are available.

[0164] Untargeted / random afinity maturation strategies that can be applied to afinity mature VHHs include random in vitro mutagenesis, CDR shufling / swapping and in vivo afinity maturation via yeast display. For random in vitro mutagenesis the sequence of either the entire VHH or only the CDRs are mutated randomly (Chen et al., 2021; Ye et al., 2021; Zupancic et al., 2021). The most commonly used technique is error prone PCR employing a DNA polymerase that lacks proof reading activity and PCR conditions that increase the polymerase error rate even further. This technique can be applied without further structural knowledge or information on the importance of residues that contribute to antigen:VHH interaction. The resulting mutational library can then be displayed to select the best matured candidate. This technique may also be combined with NGS sequencing of the display elutions to get an in-depth readout of al obtained candidates, enabling the identification of low abundant but stil promising clones (Chen et al., 2021).

[0165] In some embodiments, CDR shufling or swapping is applied for VHH afinity maturation, such as described in Zupancic et al., 2021. For CDR swapping, enriched libraries can be used as input material for a PCR reaction to individualy amplify the CDR of the VHHs. The PCR products can then be mixed and reassembled using overlapping PCR to generate the entire plasmid for further rounds of display to select for the best matured binder. One limitation of this approach is that it can only be used for VHHs comprising the same framework as it is the case for synthetic libraries.

[0166] In some embodiments, in vivo afinity maturation via yeast display is applied for VHH afinity maturation, such as described in Welner et al., 2021. The method is based on an autonomous hypermutation yeast surface display (AHEAD), which imitates somatic hypermutation during VHHAttorney Docket No: 260525.000049 selection using engineered yeast strains. The yeast’s error prone orthogonal DNA replication system can generate new variants during plasmid replication by randomly introducing mutations. The new variants can then be displayed and selected using yeast surface display to identify the best binders. This enables the production of high afinity clones in very litle time (about 2 weeks), which is significantly faster than classical afinity maturation procedures. The method can be applied using synthetic or immune libraries using unenriched libraries enriched libraries or a subset of preselected clones.

[0167] In case binders with medium afinity are required, as it is the case for the anti-TRAILR2 V-bodies and the afinity of the identified candidates need to be decreased, very similar techniques can be applied. For example, mutations that are aiming at lowering the afinity can be introduced using the same targeted or untargeted approaches as described for the afinity maturation. The selection afterwards can be adapted accordingly. If larger libraries are generated that need to be screened via a display technique, the selection strategy can be adapted to enrich medium afinity binders while excluding high afinity candidates. This could, for example be a pre-panning in phage display with low antigen concentration to remove al higher afinity candidates, folowed by a selection with high antigen concentration to obtain medium afinity VHHs. For library sizes of up to 1000 candidates a kinetic of- rate characterization can be used to get immediate information about the kinetic behavior of the candidates.

[0168] When the most potent clones have been identified, their DNA sequence can be optimized, for example to improve their stability towards enzymes. Another goal is humanization to prevent immunological reactions of the human organism against the antibody. Humanization can be achieved based on the homology between camelid VHH and human VH fragments, which is described in further detail below. Finaly, the optimized single-domain antibody can be translated and expressed in suitable organisms such as E. coli or Saccharomyces cerevisiae.

[0169] 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, comprising gene libraries from immunized or naïve donors and display techniques for identification of the most specific antigens. However, the binding region of a conventional IgG consists of two domains (VH and VL), which tend to dimerize or aggregate because of their lipophilicity. Monomerization can be accomplished by replacing lipophilic by hydrophilic amino acids. (See e.g., Borrebaeck, C. A. K.; Ohlin, M. (2002). “Antibody evolution beyond Nature”. Nature Biotechnology 20 (12): 1189-90.) If afinity can be retained after monomerization, the single-domain antibodies can likewise be produced in E. coli, S. cerevisiae or other suitable organisms.Attorney Docket No: 260525.000049

[0170] A “humanized antibody” refers to a chimeric, geneticaly engineered, antibody in which the amino acid sequences (typicaly CDRs) from an antibody (donor antibody), e.g., a camelid antibody, are grafted onto a human antibody (acceptor antibody). Thus, a humanized antibody typicaly comprises CDRs from a donor antibody and variable region framework and constant regions, when present, from a human antibody. Accordingly, a “humanized VHH” comprises CDRs that corresponds to the CDRs of a naturaly occurring VHHdomain (e.g., a camelid VHH), but that has been “humanized”. Humanized VHH may be prepared by replacing one or more amino acid residues in the amino acid sequence of the naturaly occurring VHHsequence (particularly in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4- chain human antibody. Such humanized VHHs can be obtained in any suitable manner known to a skiled person in the art and thus not strictly limited to methods described herein.

[0171] Humanization of VHHs can achieved using resurfacing or CDR grafting. Resurfacing strategies have been described in e.g., Conrath et al., 2005 J Mol Biol; Kazemi-Lomedasht et al., 2018; Vincke et al., 2009 J Biol Chem, and CDR grafting strategies have been described in e.g., 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, each of which is incorporated herein by reference in its entirety.

[0172] To humanize a camelid VHH using a resurfacing approach, a human germline reference that is most similar to the camelid germline sequence of the selected VHH may be identified. Most of the isolated camelid VHHs in literature belong to the camelid IGHV3 subfamily 2 (Nguyen et al., 2000, EMBO J) with DP-47 / VH3-23 from the IGHV3 family commonly used as human reference. The framework of the camelid VHH can then be compared to the human reference sequence. Surface exposed residues are substituted to their human counterpart as it is assumed that their contribution to protein stability is rather low. Buried residues however remain of camelid origin, as they likely contribute to the overal VHH stability. Humanization of framework regions 1, 3 and 4 usualy does not impact the physicochemical properties of the VHHs, whereas a general humanization of framework 2 would significantly increase local hydrophobicity. Residues H37, H44, H45 and H47 (Chothia numbering) in framework 2, the so caled tetrade or halmark residues, have a rather hydrophobic nature in human VHs (VGLW) as they are partialy buried and involved in VH / VL paring, while in camelid VHHs these residues are partialy charged (FERG), which significantly increases VHH solubility and inhibits paring of camelid VL (Soler et al., 2021, Biomolecules, Conrath et al., 2005 J Mol Biol). Further, residues H37 and H47 are known to interact with the CDR-H3 loop in many VHHs, stabilizing its conformation and thereby contributing to antigen binding afinity. In addition, a significant number of VHHs use framework 2Attorney Docket No: 260525.000049 residues H44, H45 and H47 for antigen binding (Zavrtanik et al., 2018, J Mol Biol). A ful humanization of these residues hence frequently results in reduced solubility or aggregation of the VHHs and a reduced or complete loss of binding afinity for the target antigen (van Faassen et al., 2020, Vincke et al., 2009). In consequence, al or at least some of these halmark residues in framework 2 remain of camelid origin when humanizing VHHs.

[0173] Another approach that may be applied to humanize VHHs is CDR grafting. CDRs of the selected VHHs can be transplanted onto a universal VHH framework that has been partialy or fuly humanized (Saerens et al., 2009 J Biol Chem, Soler et al., 2021, Vincke et al., 2009 J Biol Chem). CDR grafting has been successfuly used in some cases but failed for several others, with VHHs frequently losing their potential to bind to the desired antigen and / or becoming structuraly instable with a high tendency to aggregate (van Faassen et al., 2020, FASEB). This is mostly atributed to interactions of CDR3 with specific residues in framework 2 that are important for CDR3 conformation, general VHH stability and overal hydrophobicity, which are impaired by this approach. Sometimes camelid backmutations are introduced into the framework to compensate for these efects (van Faassen et al., 2020, FASEB).

[0174] An alternative strategy to mitigate the need of humanizing the selected VHH sequences is to use fuly or partialy humanized synthetic VHH libraries instead of camelid immune libraries for VHH discovery (Moutel et al.2016, eLife; McMahon, 2018, NSMB; Zimmermann et al., 2018, eLife). In many of these libraries the halmark residues are stil of camelid origin for reasons discussed above.

[0175] Other suitable humanizing substitutions are described in WO 09 / 138519 and WO 08 / 020079, as wel as Tables A-3 to A-8 from WO 08 / 020079 (which are lists showing possible humanizing substitutions), each of which is incorporated herein by reference in its entirety. Non-limiting examples of such humanizing substitutions include Q108L and A14P. Such humanizing substitutions may also be suitably combined with one or more other mutations as described herein (such as with one or more mutations that reduce binding by pre-existing antibodies).

[0176] In some embodiments, humanized VHH sequences stil retain the residues that are relevant for protein A binding. In some embodiments, the engineering activities during humanization may be applied to engineer protein A binding properties into a VHH that did previously not interact with protein A (Graile et al., 2000, PNAS).

[0177] Like a “humanized antibody”, a “camelized antibody” refers to an antibody having amino acid sequences (typicaly CDRs) from a donor antibody, e.g., a human antibody, and variable region framework and constant regions, when present, from a camelid antibody. Accordingly, a “camelized VH” comprises an amino acid sequence that corresponds to the amino acid sequence of a naturaly occurringAttorney Docket No: 260525.000049 VHdomain, but that has been “camelized”. Camelized VH may be prepared by replacing one or more amino acid residues in the amino acid sequence of a naturaly occurring VHdomain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a VHHdomain of a heavy chain antibody. This can be performed in a manner, for example as described in WO 2008 / 020079. Such “camelizing” substitutions are usualy inserted at amino acid positions that form and / or are present at the VH—VL interface, and / or at the so-caled Camelidae halmark residues, e.g., F37, E44, R45 and F47 (see for example WO 94 / 04678 and Davies and Riechmann (1994 and 1996). In one embodiment, the VHsequence that is used as a starting material or starting point for generating or designing the camelized VHis a VH sequence from a mammal, or the VH sequence of a human antibody. However, such camelized VH can be obtained in any suitable manner known to a skiled person in the art and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturaly occurring VHdomain as a starting material.

[0178] The amino acid residues of a single-domain antibody can be numbered according to the general numbering for VH domains given by Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, Md., Publication No.91), as applied to VHH domains from Camelids described in Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240 (1-2): 185-195; see for example FIG.2 of this publication). The total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering. For example, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number alowed for by the Kabat numbering. As a result, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. The total number of amino acid residues in a VH domain and a VHH domain is usualy in the range of from 110 to 120, often between 112 and 115. However, smaler and longer sequences may also be suitable for the purposes described herein.

[0179] Determination of CDR regions in a single-domain antibody may be accomplished using diferent methods, including those described by 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); MacCalum 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 M P et al., “IMGT unique numbering for immunoglobulin and T cel receptor variable domains and Ig superfamily V-likeAttorney Docket No: 260525.000049 domains,” Dev Comp Immunol, 2003 January; 27(1):55-77 (“IMGT” numbering scheme); Honegger A and Plückthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun.8; 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 herein is incorporated by reference in its entirety.

[0180] The boundaries of a given CDR or framework (FR) may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at diferent positions, resulting in diferential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. The AbM scheme is a compromise between Kabat and Chothia definitions based on that used by Oxford Molecular's AbM antibody modeling software.

[0181] In some embodiments, CDRs can be defined in accordance with 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 typicaly comprises three CDRs, designated CDR1, CDR2, and CDR3. Table 1-3, below, lists exemplary position boundaries of CDR-H1, CDR-H2, CDR- H3 as identified by Kabat, Chothia, AbM, and Contact schemes, respectively. For CDR-H1, residue numbering is listed using both the Kabat and Chothia numbering schemes. FRs are located between CDRs, for example, with FR-H1 located before CDR-H1, FR-H2 located between CDR-H1 and CDR-H2, FR- H3 located between CDR-H2 and CDR-H3 and so forth. It is noted that because the shown Kabat numbering scheme places insertions at H35A and H35B, the end of the Chothia CDR-H1 loop when numbered using the shown Kabat numbering convention varies between H32 and H34, depending on the length of the loop. Table 1-3. CDRs definitions according to various numbering schemes. CDR Kabat Chothia AbM ContactAttorney Docket No: 260525.000049 (Chothia Numbering2) e,atonansttutes o eat,etesa, ; 2Al-Lazikani et al., (1997) JMB 273, 927-948

[0182] Thus, unless otherwise specified, a “CDR” or “complementary determining region,” or individual specified CDRs (e.g., CDR-H1, CDR-H2, CDR-H3), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) CDR as defined by any of the above-mentioned schemes. For example, where it is stated that a particular CDR (e.g., a CDR-H3) contains the amino acid sequence of a corresponding CDR in a given VHH amino acid sequence, it is understood that such a CDR has a sequence of the corresponding CDR (e.g., CDR-H3) within the VHH, as defined by any of the above-mentioned schemes. In some embodiments, specific CDR sequences are specified. Exemplary CDR sequences of provided antibodies are described using various numbering schemes (see e.g., Table 1-3), although it is understood that a provided antibody can include CDRs as described according to any of the other above-mentioned numbering schemes or other numbering schemes known to a person of ordinary skil in the art.

[0183] In a single-domain antibody sequence of the present disclosure, the framework sequences may be any suitable framework sequences. For example, the framework sequences may be framework sequences derived from a heavy chain variable domain (e.g., a VH sequence or VHH sequence). In some embodiments, the framework sequences are either framework sequences that have been derived from a VHH sequence (in which said framework sequences may optionaly have been partialy or fuly humanized) or are conventional VH sequences (in which said framework sequences may optionaly have been partialy or fuly camelized).

[0184] Antigen-binding fragments (or combinations of fragments) of any of single-domain antibodies described herein, such as fragments that contain one or more CDR sequences, suitably flanked by and / or linked via one or more framework sequences, are also encompassed within the present disclosure.

[0185] It should be noted, however, that the present disclosure is not limited to the origin of the single- domain antibody (or of the nucleotide sequence used to express it), nor to the way that the single- domain antibody or nucleotide sequence is generated or obtained. Thus, an antigen-binding protein of the present disclosure may comprise naturaly occurring sequences (from a suitable species),Attorney Docket No: 260525.000049 recombinant sequences, or synthetic or semi-synthetic sequences. Similarly, nucleotide sequences encoding antigen-binding proteins of the present disclosure may comprise naturaly occurring nucleotide sequences, recombinant sequences, or synthetic or semi-synthetic sequences (for example, sequences that are prepared by PCR or isolated from a library).

[0186] Anti-TRAILR2 antigen-binding proteins (e.g., antibodies such single-domain antibodies) of the present disclosure may comprise one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy chain variable domains as compared to the exemplary antibody sequences provided herein. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The antigen-binding molecules of the present disclosure may comprise antigen-binding domains which are derived from any of the exemplary amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and / or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein colectively as "germline mutations"). A person of ordinary skil in the art, starting with the heavy chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen- binding fragments which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, al of the framework and / or CDR residues within the VHH domains are mutated back to the residues found in the original germline sequence from which the antigen-binding domain was originaly derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) of a diferent germline sequence (i.e., a germline sequence that is diferent from the germline sequence from which the antigen-binding domain was originaly derived).

[0187] Furthermore, the antigen-binding domains may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that difer from the original germline sequence are maintained or are mutated to the corresponding residue of a diferent germline sequence. Once obtained, antigen-binding domains that contain one or more germline mutations can be easily tested for one or more desired property such as, improvedAttorney Docket No: 260525.000049 binding specificity, increased binding afinity, improved or enhanced biological properties (e.g., agonistic efect), reduced immunogenicity, etc. Antigen-binding proteins comprising one or more antigen-binding domains obtained in this general manner are encompassed within the present disclosure.

[0188] Provided herein are 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, the present disclosure includes anti-TRAILR2 antigen-binding proteins having VHH and / or CDR amino acid sequences with, e.g., 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 an antigen-binding protein of interest and the resultant variants can screened for a desired activity, for example, retained / improved antigen binding, decreased immunogenicity, or reduced ADCC or CDC.

[0189] Amino acids may be grouped according to common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. In some embodiments, an amino acid substitution is a conservative substitution, meaning exchanging an amino acid with another amino acid of the same class. In some embodiments, amino acid substitutions may also include a non-conservative substitution, meaning exchanging an amino acid with an amino acid of a diferent class. Other exemplary amino acid substitutions are shown in Table 1-4. Table 1-4. Exemplary amino acid substitutions Original Residue Exemplary Substitutions Ala (A) Val; Leu; IleAttorney Docket No: 260525.000049 Thr (T) Val; Ser Trp (W) Tyr; Phethe present disclosure may comprise one or more mutations to reduce oxidation levels of oxidation-labile residues such as Met (M). In certain embodiments, it may be desirable to address Met (M) oxidation liability by mutation of a Met (M) residue. In some embodiments, the single-domain antibodies (e.g., VHH) of the present disclosure may comprise one or more mutations (e.g., substitution mutations) of a Met residue to reduce oxidation. As a non-limiting example, a Met residue may be substituted in any of the single-domain antibodies described herein with e.g., Ile (I), Ala (A), or Leu (L), to reduce oxidation.

[0191] In some embodiments, single-domain antibodies (e.g., VHH) of the present disclosure comprise one or more modifications that reduce binding of the single-domain antibodies (e.g., VHH) by pre- existing antibodies found in human blood or serum. In some embodiments, single-domain antibodies (e.g., VHHs) of the present disclosure are modified by mutation of amino acid position 11, for example Leu11Glu (L11E), Leu11Lys (L11K), or Leu11Val (L11V). In one embodiment, a single-domain antibody (e.g., VHH) of the present disclosure may comprise a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering). As another example, a single-domain antibody (e.g., VHH) of the present disclosure may comprise an extension of 1 to 5 (naturaly occurring) amino acids, such as a single alanine (A) extension, at the C-terminus of the single-domain antibody (e.g., VHH). The C-terminus of a VHH is normaly VTVSS (SEQ ID NO: 2166). In one embodiment, a single- domain antibody (e.g., VHH) of the present disclosure comprises a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering). In another embodiment, a single-domain antibody (e.g., VHH) of the present disclosure comprises a lysine (K) or glutamine (Q) at position 112 (according to Kabat numbering). Accordingly, the C-terminus of a single-domain antibody (e.g., VHH) can be any one 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, a single-domain antibody (e.g., VHH) of the present disclosure comprises a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering), optionaly a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) and an extension of 1 to 5 (naturaly occurring) amino acids, such as a single alanine (A) extension at the C-terminus of the single-domain antibody (e.g., VHH) (such that the C-terminus of theAttorney Docket No: 260525.000049 single-domain antibody (e.g., VHH) for example has the sequence VTVSSA (SEQ ID NO: 2175), VKVSSA (SEQ ID NO: 2176) or VQVSSA (SEQ ID NO: 2177). In further embodiments, single-domain antibodies (e.g., VHH) of the present disclosure are modified by changes in carboxy-terminal region, for example to a terminal sequence having the sequence GQGTLVTVKPGG (SEQ ID NO: 2178) or GQGTLVTVEPGG (SEQ ID NO: 2179) or modification thereof. Additional modification to reduce binding by pre-existing antibodies in human serum can be found in e.g., WO2012 / 175741, WO2015 / 173325, WO2016 / 150845, WO2011 / 003622, WO2013 / 024059; US 11,426,468, US 10,526,397, which are incorporated herein by reference in their entireties.

[0192] In one embodiment, a single-domain antibody (e.g., VHH) of the present disclosure comprises at the carboxy-terminus starting from position 111 according to Chothia the amino acid sequence VAGG (SEQ ID NO: 2405) or VPAG (SEQ ID NO: 2406). In one embodiment, a single-domain antibody (e.g., VHH) of the present disclosure comprises at the carboxy-terminus starting from position 111 according to Chothia the amino acid sequence VAGG (SEQ ID NO: 2405). In one embodiment, a single-domain antibody (e.g., VHH) of the present disclosure comprises at the carboxy-terminus starting from position 111 according to Chothia the amino acid sequence VPAG (SEQ ID NO: 2406).

[0193] In some embodiments, a single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 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, 146-761, 2192, 2194, 2198, 2200, 2204, 2206, and 2214-2251, 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% sequence identity thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ ID NO: 2405) or VPAG (SEQ ID NO: 2406).

[0194] In some embodiments, a single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 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 thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ ID NO: 2405) or VPAG (SEQ ID NO: 2406).

[0195] In some embodiments, a single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 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 leastAttorney Docket No: 260525.000049 98%, or at least 99% identity thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ ID NO: 2405) or VPAG (SEQ ID NO: 2406).

[0196] In some embodiments, a single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 5, 10, 15, 18, 22, 27, 32, 37, 42, 46, 51, 56, 66, 71, 76, 80, 85, 90, 94, 99, 2194, 2200, 2206, and 2354-2404, 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 thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ ID NO: 2405) or VPAG (SEQ ID NO: 2406).

[0197] In some embodiments, a single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 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 thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ ID NO: 2405) or VPAG (SEQ ID NO: 2406).

[0198] In some embodiments, single-domain antibodies (e.g., VHH) of the present disclosure are modified to enhance binding to staphylococcal protein A (SpA) or streptococcal protein G (SpG). Binding of SpA and SpG to antibodies or antibody fragments can be useful in the manufacturing process of the antibodies or antibody fragments. The high-afinity interaction of the IgG Fc region with SpA and SpG has been extensively exploited and became the gold standard for monoclonal antibody purification (Björck and Kronval, 1984). Other non-Fc containing antibody fragments, such as VHHs and Fabs do not have the capacity to bind to SpA or SpG via their Fc regions. However, sequence-dependent interaction with SpA has been demonstrated for these non-Fc containing antibody fragments(Graile et al., 2000; Henry et al., 2016). This characteristic circumvents potential use of afinity tags fused to the drug candidate for afinity chromatography that have the disadvantage as being regarded as a sequence liability, as it may impact protein immunogenicity as wel as protein structure and stability and could compromise functionality. The interaction of the single-domain antibodies (e.g., VHH) to SpA relies on an alternative binding mode, with a 1-5 µM afinity, which is comparable to the 0.2 -3 µM measured for VH-SpA interactions (To et al., JBC, 2005; Henry et al., Plos One, 2016).

[0199] In some embodiments, single-domain antibodies (e.g., VHH) of the present disclosure have, or are modified to have a SpA-binding motif. For example, The VHH-SpA interface has been mapped to thirteen residues, which cluster within the framework at the back side of the V-body, distant to the CDRs (Graile et al., 2000, Henry et al., 2016). In the absence of a VHH-SpA co-structure, superposition of a SpA-Fab crystal structure and a VHH alows for visualizing the binding mode. Based on a structural andAttorney Docket No: 260525.000049 functional analysis, the thirteen residues of the VHH-SpA interface have been characterized to be intolerant to substitutions (residues Gly15, Arg19, Tyr59, Gly65, and Arg66), tolerant to specific substitutions (residues Thr / Lys / Arg57, Thr68, Gln81, Asn82a, and Ser82b) or generaly tolerant to a variety of substitutions (residues Ser17, Lys64, and Ser70) (al residue positions refer to Kabat numbering) (Henry et al., Plos One, 2016). Thus, a SpA-binding motif included in a single-domain antibody (e.g., VHH) of the present disclosure may include one or more, or al of the thirteen residues.

[0200] In some embodiments, single-domain antibodies (e.g., VHH) of the present disclosure comprise one or more modifications at N-terminus to prevent formation of a pyroglutamate 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 with Asp (E1D).

[0201] In some embodiments, a single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 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, 146-761, 2192, 2194, 2198, 2200, 2204, 2206, and 2214-2251, 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 thereto, wherein the amino acid residue Glu at the first position of the single-domain antibody (e.g., VHH) is replaced with Asp (E1D).

[0202] In some embodiments, a single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 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 thereto, wherein the amino acid residue Glu at the first position of the single-domain antibody (e.g., VHH) is replaced with Asp (E1D).

[0203] In some embodiments, a single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 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 thereto, wherein the amino acid residue Glu at the first position of the single-domain antibody (e.g., VHH) is replaced with Asp (E1D).

[0204] In some embodiments, a single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 5, 10, 15, 18, 22, 27, 32, 37, 42, 46, 51, 56, 66, 71, 76, 80, 85, 90, 94, 99, 2194, 2200, 2206, and 2354-2404, 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% identityAttorney Docket No: 260525.000049 thereto, wherein the amino acid residue Glu at the first position of the single-domain antibody (e.g., VHH) is replaced with Asp.

[0205] In some embodiments, a single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 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 thereto, wherein the amino acid residue Glu at the first position of the single-domain antibody (e.g., VHH) is replaced with Asp.

[0206] In some embodiments, the single-domain antibodies (e.g., VHHs) of the present disclosure are modified by substitution of one or more of any amino acid residue at any position which is not Ala (A) with Ala (A). In some embodiments, the single-domain antibodies (e.g., VHH) of the present disclosure are modified by substitution of one or more of any Ala (A) with Gly (G). In some embodiments, a single- domain antibody (e.g., VHH) described herein is modified by substitution of one or more of any amino acid residue at any position which is not Ala (A) with Ala (A) residue and / or by substitution of one or more of any Ala (A) with a Gly (G).

[0207] In some embodiments, single-domain antibodies (e.g., VHHs) of the present disclosure comprise a single amino acid residue that has been replaced with Ala (A) or, in such instances when the original (i.e., wildtype) residue was Ala (A), a single amino acid residue that has been replaced with Gly (G). Non- limiting examples single-domain antibodies (e.g., VHHs) which have been modified in accordance with the above described amino acid substitution(s) include SEQ ID NO: 2354-2404.

[0208] In some embodiments, a complementarity determining region 3 (CDR3) of a single-domain antibody (e.g., VHH) described herein comprises a single amino acid residue that has been replaced with Ala (A) or, in such instances when the original (i.e., wildtype) residue was Ala (A), a single amino acid residue that has been replace with Gly (G). As an example, a CDR3 comprising the amino acid sequence AAASSWSRGGVPYGMDY (SEQ ID NO: 30) can be modified in accordance with the above-described amino acid substitution(s) to, e.g., an amino acid sequence 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); AAASSWSRGGAPYGMDY (SEQ ID NO: 2308); AAASSWSRGGVAYGMDY (SEQ ID NO: 2309); AAASSWSRGGVPAGMDY (SEQ ID NO: 2310); AAASSWSRGGVPYAMDY (SEQ ID NO: 2311); AAASSWSRGGVPYGADY (SEQ ID NO: 2312);Attorney Docket No: 260525.000049 AAASSWSRGGVPYGMAY (SEQ ID NO: 2313); or AAASSWSRGGVPYGMDA (SEQ ID NO: 2314). As another example, a CDR3 comprising the amino acid sequence TAGPTMSYSRGGEF (SEQ ID NO: 54) can be modified in accordance with the above-described amino acid substitution(s) to, e.g., an amino acid sequence 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); TAGPTMSYSRGAEF (SEQ ID NO: 2326); TAGPTMSYSRGGAF (SEQ ID NO: 2327); or TAGPTMSYSRGGEA (SEQ ID NO: 2328). As another example, a CDR3 comprising the amino acid sequence RAYNDGGEY (SEQ ID NO: 2191) can be modified in accordance with the above-described amino acid substitution(s) to, e.g., an amino acid sequence 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, a CDR3 comprising the amino acid sequence HSRWYNL (SEQ ID NO: 2197) can be modified in accordance with the above-described amino acid substitution(s) to, e.g., an amino acid sequence 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, a CDR3 comprising the amino acid sequence FTADY (SEQ ID NO: 2203) can be modified in accordance with the above-described amino acid substitution(s) to, e.g., an amino acid sequence ATADY (SEQ ID NO: 2345); FAADY (SEQ ID NO: 2346); FTGDY (SEQ ID NO: 2347); or FTAAY (SEQ ID NO: 2348). Alternative protein scafolds

[0209] In some embodiments, anti-TRAILR2 antigen-binding proteins of the present disclosure can adopt an alternative protein scafold. Such alternative protein scafold may be a single chain polypeptidic framework, optionaly with a reduced size (e.g., less than about 200 amino acids), that contains a highly structured core associated with variable domains of high conformational tolerance alowing insertions, deletions, or other substitutions. Such antigen-binding proteins may be generated by grafting CDRs or variable regions described herein onto a suitable protein scafold. The structure of alternative scafolds may vary, but preferably are of human origin for those developed as therapeutics.Attorney Docket No: 260525.000049

[0210] Alternative protein scafolds of the present disclosure can be based either on a conventional immunoglobulin (Ig) backbone, or are derived from a completely unrelated protein. These variable domains can be modified to create novel binding interfaces toward any targeted antigen. In some embodiments, an alternative protein scafold of the present disclosure can be derived from Protein A, e.g., the Z-domain thereof (afibodies), ImmE7 (immunity proteins), BPTI / APPI (Kunitz domains), Ras- binding protein AF-6 (PDZ-domains), charybdotoxin (Scorpion toxin), CTLA-4, Min-23 (knottins), lipocalins (anticalins), neokarzinostatin, a fibronectin domain (used in “adnectin”), an ankyrin repeat (AR) domain (used in “DARPins”), avidity multimers (also known as “avimers”), 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), al of which are hereby incorporated by reference in their entirety).

[0211] Anticalins are a suitable type of non-Ig based alternative scafolds for use in the antigen-binding molecules of the present disclosure. Anticalins are a class of engineered ligand-binding proteins that are based on the lipocalin scafold. Lipocalins are a family of proteins that transport smal hydrophobic molecules such as steroids, bilins, retinoids, and lipids. Lipocalins have limited sequence homology, but share a common tertiary structure architecture based on eight antiparalel β-barrels. Lipocalins contain four exposed loops built on the rigid β-barrel structure. Exemplary anticalin proteins that are commonly used are about a size of about 180 amino acids and a mass of about 20 kDa.

[0212] DARPins are another suitable non-Ig based alternative scafold that can be used in the antigen- binding molecules of the present disclosure. DARPins are geneticaly engineered antibody mimetic proteins typicaly exhibiting highly specific and high-afinity target protein binding. They are derived from natural ankyrin repeat (AR) proteins, which usualy contain a 33 amino acid protein motif consisting of two α-helices separated by loops, which repeats mediate protein—protein interactions. DARPins can be generated using combinatorial AR libraries constructed based on the 33 amino acid AR motif with seven randomized positions. DARPin libraries can be screened using ribosome display, and library members typicaly are wel produced in Escherichia coli, do not aggregate, and display high thermodynamic stability. Preferably, DARPins contain two to four of these motifs flanked by N- and C- terminal capping motifs to shield hydrophobic regions and alow increased solubility.

[0213] The avimer structure can also be used as a protein backbone to generate a suitable non-Ig based alternative scafold. Avimers typicaly consist of two or more peptide sequences of 30 to 35 amino acids each, connected by peptide linker. The individual sequences are derived from A-domains of various membrane receptors and have a rigid structure, stabilized by disulfide bridges and calcium. Each A-Attorney Docket No: 260525.000049 domain can bind to a certain epitope of the target protein. The combination of domains binding to diferent epitopes of the same protein increases afinity to this protein, an efect known as avidity.

[0214] Proteins derived from fibronectin II (FN3) domains can also be used to generate a suitable non- Ig based alternative scafold (also known as “monobody”). For example, the tenth fibronectin type II domain (FN10) of human fibronectin corresponds to a β-sandwich with seven β-strands and three connecting loops showing structural homologies to Ig domains without disulfide bridges. In some cases, the connecting loops of FN10, each about 15 to 21 amino acids in length, can be randomized and the domains displayed on both phage and yeast to select for a scafold with the desirable properties. Adnectins™ is an exemplary scafold generated using 10thFN3 domains randomized and displayed in this way. Another exemplary scafold comprising FN3 domains is a Centyrin™. Centryrins™ contain the consensus sequence of FN3 domains of human Tenascin C (TNC), which is found in the extracelular matrix of various tissues. Centyrin™ scafolds have loops that have structural homology to antibody variable domains (i.e., CDR1, CDR2 and CDR3), and are smal (about 10 kDa), simple, and highly stable single domain proteins that do not contain cysteine, disulfides or glycosylated residues. Centyrin™ possess excelent biophysical properties such as stability to heat, pH, denaturant and organic solvents, reversible unfolding and monodispersity. Another recent exemplary FN3-based scafold that can be used in the present disclosure is fluctuation-regulated afinity proteins (FLAPs), as described in See et al., 2020. Biotechnology Journal 15(12):e2000078, which is incorporated herein by reference in its entirety. Fusion Proteins and Conjugates

[0215] In one aspect, provided herein are fusion proteins and conjugates comprising at least one anti- TRAILR2 antigen-binding protein (e.g., antibody such as a single-domain antibody) linked, directly or indirectly, to one or more additional domains or moieties. The at least one anti-TRAILR2 antigen-binding protein can specificaly bind TRAILR2. In some embodiments, the fusion protein or conjugate of the present disclosure comprises a single polypeptide. In other embodiments, the fusion protein or conjugate of the present disclosure comprises more than one polypeptide. In some embodiments, the fusion protein or conjugate of the present disclosure comprises two polypeptides.

[0216] In some embodiments, the fusion protein or conjugate of the present disclosure comprises at least one anti-TRAILR2 antigen-binding protein (e.g., 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 the present disclosure may be at least bivalent, but can also be e.g.,Attorney Docket No: 260525.000049 trivalent, tetravalent, pentavalent, hexavalent, etc. The terms “bivalent”, “trivalent”, “tetravalent”, “pentavalent”, or “hexavalent” al fal under the term “multivalent” and indicate the presence of two, three, four, five or six binding units (e.g., VHHs), respectively.

[0217] In certain embodiments, the fusion protein or conjugate is multispecific. For example, in some cases, the one or more additional domain or moieties may be one or more additional binding domain that binds to one or more further antigen or protein. The fusion protein or conjugate of the present disclosure may be, for example, bispecific, trispecific, tetraspecific, pentaspecific, etc. The terms “bispecific”, “trispecific”, “tetraspecific”, “pentaspecific”, etc., al fal under the term “multispecific” and refer to binding to two, three, four, five, etc., diferent target molecules, respectively.

[0218] In some embodiments, a fusion protein or conjugate of the present disclosure comprises one or more of an anti-TRAILR2 antigen-binding protein described herein. In some embodiments, a fusion protein or conjugate of the present disclosure comprises two or more of an anti-TRAILR2 antigen- binding protein described herein. In some embodiments, a fusion protein or conjugate of the present disclosure comprises three or more of an anti-TRAILR2 antigen-binding protein described herein. In some embodiments, a fusion protein or conjugate of the present disclosure comprises four or more of an anti-TRAILR2 antigen-binding protein described herein. In some embodiments, a fusion protein or conjugate of the present disclosure comprises one, two, three, four, five, six, seven, eight, nine, or ten, or more of an anti-TRAILR2 antigen-binding protein described herein.

[0219] In some embodiments, the one or more antigen-binding proteins can bind to the same epitope on TRAILR2. In some embodiments, the one or more antigen-binding proteins can bind to diferent epitopes on TRAILR2.

[0220] In various embodiments, the one or more antigen-binding proteins can be one or more single- domain antibodies disclosed herein, for example, one or more VHHs disclosed herein.

[0221] 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 comprise the same sequence or may comprise diferent sequences. In such embodiments, the two or more anti-TRAILR2 antigen-binding proteins may bind to the same epitope on TRAILR2 or diferent epitopes on TRAILR2. For example, a fusion protein or conjugate of the present disclosure may be biparatopic, e.g., if two VHHs bind two diferent epitopes on TRAILR2.

[0222] Exemplary designs of multivalent anti-TRAILR2 fusion constructs comprising two or more anti- TRAILR2 binding units (e.g., VHHs) are shown in Figure 13.Attorney Docket No: 260525.000049 Fusion or Conjugation to Fc regions

[0223] In some embodiments, a fusion protein or conjugate of the present disclosure comprises at least one anti-TRAILR2 antigen-binding protein (e.g., antibody such as a single-domain antibody) provided herein operably linked to a dimerization domain such as an immunoglobulin Fc region. An immunoglobulin Fc region may be linked indirectly or directly to at least one anti-TRAILR2 antigen- binding protein (e.g., antibody such as a single-domain antibody). In some embodiments, a fusion protein or conjugate of the present disclosure comprises one, two, three, four, five, six or more anti- TRAILR2 antigen-binding proteins provided herein operably linked to an Fc region.

[0224] A “Fc region” as used herein refers to a portion of a heavy chain constant region comprising CH2 and CH3. In some embodiments, an Fc region comprises a hinge, CH2, and CH3. In various embodiments, when an Fc region comprises a hinge, the hinge can mediate dimerization between two Fc-containing polypeptides. An Fc region included in a fusion protein or conjugate may be an Fc region from any species, or derived from any species, including, but not limited to, human, mouse, rat, monkey (e.g., cyno), camel, lama, shark, goat, rabbit, and / or bovine. In various embodiments, an Fc region included in a fusion protein or conjugate of the present disclosure is a human immunoglobulin Fc region, or is derived from a human immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region is of 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 subclass. The immunoglobulin Fc region may comprise a variant or fragment of a native IgG Fc region.

[0225] In some embodiments, an Fc region included in a fusion protein or conjugate described herein may be a murine (e.g., a mouse or a rat) immunoglobulin Fc region, or derived from a murine immunoglobulin Fc region. In some embodiments, an Fc region included in a fusion protein or conjugate described herein may be a cyno immunoglobulin Fc region, or derived from a cyno immunoglobulin Fc region.

[0226] A native Fc region typicaly possesses an efector function, including but not limited to, Fc receptor binding; Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cel-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cel surface receptors (for example B-cel receptor); and B-cel activation, etc. Such efector functions generaly require the Fc region to be combined with a binding domain (for example, an antibody variable domain) and can be assessed using various assays.

[0227] In some embodiments, a fusion protein or conjugate of the present disclosure can comprise a dimer of Fc regions. In some embodiments, an Fc region mediates dimerization of the TRAILR2-bindingAttorney Docket No: 260525.000049 units at physiological conditions, such as when expressed from a cel, such that a dimer is formed that doubles the number of TRAILR2 binding units. For example, a fusion polypeptide comprising one VHH domain that binds TRAILR2 and an Fc region is monovalent as a monomer, but the Fc region can mediate dimerization; as a result, the fusion protein is bivalent (i.e., having two anti-TRAILR2 VHH domains per molecule). Similarly, in some embodiments, two anti-TRAILR2 VHH domains (2x) are fused to an IgG Fc region and as a result of dimerization, the fusion protein is tetravalent (i.e., having four anti-TRAILR2 VHH domains per molecule). In some embodiments, three anti-TRAILR2 VHH domain (3×) are fused to an IgG Fc region and as a result of dimerization, the fusion protein is hexavalent (i.e., having six anti- TRAILR2 VHH domains per molecule).

[0228] In some embodiments, a fusion protein or conjugate of the present disclosure may comprise two polypeptide chains, each polypeptide chain having the folowing structure: (anti-TRAILR2 VHH)n- Linker-Fc, wherein n can be any integral number (e.g., 1, 2, 3, 4, 5, etc). When n≥2, each anti-TRAILR2 VHH may be optionaly operably linked to another anti-TRAILR2 VHH via a linker.

[0229] In some embodiments, a fusion protein or conjugate of the present disclosure may comprise two polypeptide chains, each polypeptide chain having the folowing structure: (anti-TRAILR2 VHH)n- Linker-Fc-(anti-TRAILR2 VHH)m, wherein n and m can independently be any integral number (e.g., 1, 2, 3, 4, 5, etc). When n≥2 or m≥2, each anti-TRAILR2 VHH may be optionaly operably linked to another anti-TRAILR2 VHH via a linker.

[0230] In some embodiments, a fusion protein or conjugate of the present disclosure is bivalent. In some embodiments, the bivalent fusion protein or conjugate of the disclosure comprises two polypeptide chains, each polypeptide chain having the folowing structure: (anti-TRAILR2 VHH)-Linker- Fc.

[0231] In some embodiments, a fusion protein or conjugate of the present disclosure is tetravalent. In some embodiments, the tetravalent fusion protein or conjugate of the disclosure comprises two polypeptide chains, each polypeptide chain having the folowing structure: (anti-TRAILR2 VHH)-Linker- (anti-TRAILR2 VHH)-Linker-Fc. In some embodiments, the tetravalent fusion protein or conjugate of the disclosure comprises two polypeptide chains, each polypeptide chain having the folowing structure: (anti-TRAILR2 VHH)-Linker-Fc-Linker-(anti-TRAILR2 VHH). The multiple linkers used in the fusion protein are not necessarily the same.

[0232] In some embodiments, a fusion protein or conjugate of the disclosure is hexavalent. In some embodiments, the hexavalent fusion protein or conjugate of the disclosure comprises two polypeptide chains, each polypeptide chain having the folowing structure: (anti-TRAILR2 VHH)-Linker-(anti-TRAILR2Attorney Docket No: 260525.000049 VHH)-Linker-(anti-TRAILR2 VHH)-Linker-Fc. In some embodiments, the hexavalent fusion protein or conjugate of the disclosure comprises two polypeptide chains, each polypeptide chain having the folowing structure: (anti-TRAILR2 VHH)-Linker-(anti-TRAILR2 VHH)-Linker-Fc-linker-(anti-TRAILR2 VHH). In some embodiments, the hexavalent fusion protein or conjugate of the disclosure comprises two polypeptide chains, each polypeptide chain having the folowing structure: (anti-TRAILR2 VHH)-Linker- Fc-Linker-(anti-TRAILR2 VHH)-Linker-(anti-TRAILR2 VHH). The multiple linkers used in the fusion protein are not necessarily the same.

[0233] In some embodiments, the CH3 domain of the Fc region can be used as homodimerization domain, such that the resulting fusion protein may be formed from two identical polypeptides. In other cases, the CH3 dimer interface region of the Fc region can be mutated to enable heterodimerization. For example, a heterodimerization domain can be incorporated into the fusion protein such that the construct is a heterodimeric fusion protein.

[0234] When a dimer of Fc regions is used in a fusion protein or conjugate of the present disclosure, the first and second Fc regions may be of the same IgG isotype such as, e.g., IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4. Alternatively, the first and second Fc regions may be of diferent IgG isotypes such as, e.g., IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0235] In some embodiments, the Fc region included in a fusion protein or conjugate of the present disclosure can be mutated or modified. In some embodiments, the mutations include one or more amino acid substitutions to reduce an efector function of the Fc region. Various examples of mutations to Fc regions to alter, such as reduce, efector function are known, including any as described below. In general, the numbering of the residues in an immunoglobulin heavy chain or portion thereof, such as an Fc region, is according to the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0236] In some embodiments, the human IgG Fc region is modified to alter antibody-dependent celular 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. Enzyme Regul., 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.Attorney Docket No: 260525.000049

[0237] In some embodiments, an Fc region included in a fusion protein or conjugate of the present disclosure exhibits reduced efector functions (such as CDC and ADCC). Various in vitro and / or in vivo cytotoxicity assays can be conducted to confirm the reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the fusion protein construct and / or cleaved components thereof lack FcγR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cels for mediating ADCC are NK cels which express FcγRII only, whereas monocytes express FcγRI, FcγRI and FcγRII. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in e.g., US 5,500,362; US 5,821,337; Helstrom. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986); and Helstrom 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 assay methods may be employed, such as ACTI™ non-radioactive cytotoxicity assay for flow cytometry or CytoTox96™ non-radioactive cytotoxicity assay. Useful efector cels for such assays include peripheral blood mononuclear cels (PBMC) and Natural Kiler (NK) cels. Alternatively, or additionaly, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be carried out to confirm that the fusion protein construct or cleaved components thereof is unable to bind C1q and hence lacks CDC activity (see, e.g., C1q and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402). To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano- Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S. B. et al., Int'l. Immunol.18(12):1759-1769 (2006).

[0238] Examples of mutations that enhance ADCC include modification at Ser239 and Ile332, for example Ser239Asp and Ile332Glu (S239D, 1332E). Examples of mutations that enhance CDC include modifications at Lys326 and Glu333. In some embodiments, the Fc region is modified at one or both of these positions, for example Lys326Ala and / or Glu333Ala (K326A and E333A) using the Kabat numbering system.

[0239] In some embodiments, the Fc region of the fusion protein is altered at one or more of the folowing positions to reduce Fc receptor binding: Leu234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Ser298 (S298), Asn297 (N297), Asn325 (N325) orAla327 (A327) or Pro329 (P329). For example, Leu234Ala (L234A), Leu235Ala (L235A), Leu235Glu (L235E), Asp265Asn (D265N), Asp265Ala (D265A), Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala (N297A), Pro329Ala (P329A) or Pro239Gly (P329G),Attorney Docket No: 260525.000049 Asn325Glu (N325E) or Ala327Ser (A327S). In some embodiments, modifications within the Fc region reduce binding to Fc-receptor-gamma receptors (FcγRs) while have minimal impact on binding to the neonatal Fc receptor (FcRn).

[0240] In some embodiments, the human IgG1 Fc region is modified at amino acid Asn297 (Kabat Numbering) to prevent glycosylation of the fusion protein, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu235 (Kabat Numbering) to alter Fc receptor interactions, e.g., Leu235Glu (L235E) or Leu235Ala (L235A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu234 (Kabat Numbering) to alter Fc receptor interactions, e.g., Leu234Ala (L234A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu234 (Kabat Numbering) to alter Fc receptor interactions, e.g., Leu235Glu (L235E). In some embodiments, the Fc region of the fusion protein is altered at both amino acids 234 and 235, e.g., 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 at 234, 235, and 297, e.g., Leu234Ala, Leu235Ala, Asn297Ala (L234A / L235A / N297A). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 329, e.g., Leu234Ala, Leu235Ala, Pro239Ala (L234A / L235A / P329A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Asp265 (Kabat Numbering) to alter Fc receptor interactions, e.g Asp265Ala (D265A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Pro329 (Kabat Numbering) to alter Fc receptor interactions, e.g., Pro329Ala (P329A) or Pro329Gly (P329G). In some embodiments, the Fc region of the fusion protein is altered at both amino acids 265 and 329, e.g., Asp265Ala and Pro329Ala (D265A / P329A) or Asp265Ala and Pro329Gly (D265A / P329G). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 265, e.g., Leu234Ala, Leu235Ala, Asp265Ala (L234A / L235A / D265A). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 329, e.g., Leu234Ala, Leu235Ala, Pro329Gly (L234A / L235A / P329G). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, 265 and 329, e.g., Leu234Ala, Leu235Ala, Asp265Ala, Pro329Gly (L234A / L235A / D265A / P329G).

[0241] In some embodiments, the Fc region of the fusion protein is altered at Gly235 to reduce Fc receptor binding. For example, wherein Gly235 is deleted from the fusion protein. In some embodiments, the human IgG1 Fc region is modified at amino acid Gly236 to enhance the interaction with CD32A, e.g., Gly236Ala (G236A). In some embodiments, the human IgG1 Fc region lacks Lys447 (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest).Attorney Docket No: 260525.000049

[0242] In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Gly, Leu235Ser, Gly236Arg (L234G / L235S / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Ser, Leu235Thr, Gly236Arg (L234S / L235T / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Ser, Leu235Val, Gly236Arg (L234S / L235V / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Thr, Leu235Gln, Gly236Arg (L234T / L235Q / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Thr, Leu235Thr, Gly236Arg (L234T / L235T / G236R). In some embodiments, the Fc region of the fusion protein fusion protein is altered at amino acids at 234, 235, and 329, e.g., Leu234Thr, Leu235Thr, Pro329Gly (L234A / L235A / P329G). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 252, 254, and 256, e.g., Met252Tyr, Ser254Thr, Thr256Glu (M252Y / S254T / T256E).

[0243] In some embodiments, the Fc region of the fusion protein is lacking an amino acid at one or more of the folowing positions to reduce Fc receptor binding: Glu233 (E233), Leu234 (L234), or Leu235 (L235). In some embodiments, the Fc region of the fusion protein is lacking an amino acid at one or more of the folowing positions Glu233 (E233), Leu234 (L234), or Leu235 (L235) and is modified at one or more of the Asp265 (D265), Asn297 (N297), or Pro329 (P329) to reduce Fc receptor binding. For example, an Fc region included in a TRAILR2 binding polypeptide is derived from a human Fc domain, and comprises a three amino acid deletion in the lower hinge corresponding to IgG1 E233, L234, and L235. In some embodiments, such Fc polypeptides do not engage FcγRs and thus are referred to as “efector silent” or “efector nul.” For example, Fc deletion of these three amino acids reduces the complement protein C1q binding. In some embodiments, a polypeptide with an Fc region with Fc deletion of these three amino acids retains binding to FcRn and therefore has extended half-life and transcytosis associated with FcRn mediated recycling.

[0244] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human IgG1 Fc region, having an amino acid sequence: IgG1 L234A, L235A (also known as “LALA” variant) (mutations bolded in the sequence below) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2152)

[0245] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human IgG1 Fc region, having an amino acid sequence:Attorney Docket No: 260525.000049 IgG1 L234A, L235A, and P329A (also known as “LALAPA” variant) (mutations bolded in the sequence below)TYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2153)

[0246] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human IgG1 Fc region, having an amino acid sequence: IgG1 D265A, N297A and P329A (also known as “DANAPA” variant) (mutations bolded in the sequence below) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAST YRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2154)

[0247] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human IgG1 Fc region, having an amino acid sequence: IgG1 L234A, L235A, and G237A (also known as “LALAGA” variant) (mutations bolded in the sequence below) DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2155)

[0248] In one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgG1 Fc region, having an amino acid sequence: IgG1 L234G / L235S / G236R (mutations bolded in the sequence below) DKTHTCPPCPAPEGSRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2181)

[0249] In one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgG1 Fc region, having an amino acid sequence: IgG1 L234S / L235T / G236R (mutations bolded in the sequence below) DKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2182)Attorney Docket No: 260525.000049

[0250] In one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgG1 Fc region, having an amino acid sequence: IgG1 L234S / L235V / G236R (mutations bolded in the sequence below) DKTHTCPPCPAPESVRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2183)

[0251] In one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgG1 Fc region, having an amino acid sequence: IgG1 L234T / L235Q / G236R (mutations bolded in the sequence below) DKTHTCPPCPAPETQRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2184)

[0252] In one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgG1 Fc region, having an amino acid sequence: IgG1 L234T / L235T / G236R (mutations bolded in the sequence below) DKTHTCPPCPAPETTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2185)

[0253] In one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgG1 Fc region, having an amino acid sequence: IgG1 L234A / L235A / P329G (mutations bolded in the sequence below) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2186)

[0254] In one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgG1 Fc region, having an amino acid sequence: IgG1 M252Y / S254T / T256E (mutations bolded in the sequence below) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2187)

[0255] In some embodiments, the human IgG Fc region is modified to enhance FcRn binding. Examples of Fc mutations that enhance binding to FcRn are Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S254T,Attorney Docket No: 260525.000049 T256E, respectively) (Kabat numbering, Dal'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) (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest), 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).

[0256] In some embodiments, the Fc region lacks or has reduced fucose attached to the N-linked glycan-chain at N297. There are numerous ways to prevent fucosylation, including but not limited to production in a FUT8 deficient cel line; addition of inhibitors to the mammalian cel culture media, for example Castanospermine; and metabolic engineering of the production cel line.

[0257] In some embodiments, the Fc domain included in a fusion protein or conjugate of the present disclosure is derived from a human Fc domain and comprises mutations M252Y and M428V. In some embodiments, the mutated or modified Fc polypeptide includes the folowing mutations: M252Y and M428L using the Kabat numbering system. In some embodiments, such mutations enhance binding to FcRn at the acidic pH of the endosome (near 6.5), while losing detectable binding at neutral pH (about 7.2), alowing for enhanced FcRn mediated recycling and extended half-life.

[0258] In some embodiments, the Fc domain included in a fusion protein or conjugate is derived from a human Fc domain and comprises mutations to induce heterodimerization. In some embodiments, such mutations include those referred to as “knob” and “hole” mutations. For example, having an amino acid modification within the CH3 domain at Thr366, which when replaced with a bulkier amino acid, e.g., Try (T366W), is able to preferentialy pair with a second CH3 domain having amino acid modifications to less bulky amino acids at positions Thr366, Leu368, and Tyr407, e.g., Ser, Ala, and Val, respectively (T366S / L368A / Y407V). In some embodiments, the “knob” Fc domain comprises the mutation T366W. In some embodiments, the “hole” Fc domain comprises mutations T366S, L368A, and Y407V. Heterodimerization via CH3 modifications can be further stabilized by the introduction of a disulfide bond, for example by changing Ser354 to Cys (S354C) and Y349 to Cys (Y349C) on opposite CH3 domains (Reviewed in Carter, 2001 Journal of Immunological Methods, 248: 7-15). In some embodiments, Fc domains used for heterodimerization comprise additional mutations, such as the mutation S354C on a first member of a heterodimeric Fc pair that forms an asymmetric disulfide with a corresponding mutation Y349C on the second member of a heterodimeric Fc pair. In some embodiments, one memberAttorney Docket No: 260525.000049 of a heterodimeric Fc pair comprises the modification H435R or H435K to prevent protein A binding while maintaining FcRn binding. In some embodiments, one member of a heterodimeric Fc pair comprises the modification H435R or H435K, while the second member of the heterodimeric Fc pair is not modified at H435. In various embodiments, the hole Fc domain comprises the modification H435R or H435K (referred to as “hole-R” in some instances when the modification is H435R), while the knob Fc domain does not. In some instances, the hole-R mutation improves purification of the heterodimer over homodimeric hole Fc domains that may be present.

[0259] In some embodiments, the human IgG Fc region is modified to prevent dimerization. In these embodiments, the fusion proteins of the present disclosure are monomeric. For example, modification at residue Thr366 to a charged residue, e.g. Thr366Lys, Thr366Arg, Thr366Asp, or Thr366Glu (T366K, T366R, T366D, or T366E, respectively), prevents CH3-CH3 dimerization.

[0260] In some embodiments, the immunoglobulin Fc region of the fusion protein is of human IgG3 isotype, or a variant thereof. In one embodiment, the IgG3 Fc region is modified at amino acid Asn297 (Kabat Numbering) to prevent glycosylation of the antibody, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the human IgG3 Fc region is modified at amino acid 435 to extend the half-life, e.g., Arg435His (R435H). In some embodiments, the human IgG3 Fc region lacks Lys447 (EU index of Kabat et al 1991).

[0261] In some embodiments, the immunoglobulin Fc region of the fusion protein is of human IgG4 isotype, or a variant thereof. As a non-limiting example, an immunoglobulin Fc region of human IgG4 isotype may have an amino acid sequence: ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 2091)

[0262] In some embodiments, the immunoglobulin Fc region of human IgG4 isotype may comprise the amino acid sequence set forth in SEQ ID NO: 2091, or a similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0263] In some embodiments, the human IgG4 Fc region comprises one or more mutations selected from, e.g., Ser228Pro (S228P), Leu235Glu (L235E), Leu235Ala (L235A), Phe234Ala (F234A), and / or Pro329Gly (P329G) according to EU numbering.

[0264] In some embodiments, the IgG4 Fc region of the fusion protein is altered at amino acids at 228 and 235, e.g., 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 at 228, 234 and 235,Attorney Docket No: 260525.000049 e.g., 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 at 228, 235, and 329, e.g., Ser228Pro, Leu235Glu and P329G (S228P / L235E / P329G).

[0265] In one embodiment, the human IgG4 Fc region is modified at amino acid 235 to alter Fc receptor interactions, e.g., Leu235Glu (L235E). In some embodiments, the human IgG4 Fc region is modified at amino acid Asn297 (Kabat Numbering) to prevent glycosylation of the antibody, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the human IgG4 Fc region lacks Lys447 (EU index of Kabat et al 1991).

[0266] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human IgG4 Fc region, having an amino acid sequence: IgG4 S228P, L235E (mutations bolded in the sequence below) ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 2156)

[0267] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human IgG4 Fc region, having an amino acid sequence: IgG4 S228P, L235A (mutations bolded in the sequence below) ESKYGPPCPPCPAPEFAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 2157)

[0268] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human IgG4 Fc region, having an amino acid sequence: IgG4 S228P, F234A, L235E (mutations bolded in the sequence below) ESKYGPPCPPCPAPEAEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 2158)

[0269] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human IgG4 Fc region, having an amino acid sequence: IgG4 S228P, F234A, L235A (mutations bolded in the sequence below) ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 2159)Attorney Docket No: 260525.000049

[0270] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human IgG4 Fc region, having an amino acid sequence: IgG4 P329G, S228P, L235E (mutations bolded in the sequence below) ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 2160)

[0271] Additional IgG4 heavy chain modifications suitable for use in the fusion proteins or conjugates of the present disclosure include those described in Tables 1 and 2 of Dumet et al., mAbs, 11:8, 1341-1350, which is incorporated herein by reference in its entirety.

[0272] In some embodiments, the fusion protein or conjugate contains an immunoglobulin hinge region. In some embodiments, the hinge region serves as a linker to connect one or more TRAILR2 binding units (e.g., VHHs) to the Fc region. In other embodiments, the fusion protein can comprise a linker in addition to the hinge region to connect the one or more TRAILR2 binding units (e.g., VHHs) to the Fc region. The hinge region can be selected from any of the human IgG subclasses. For example, the fusion protein may contain a modified IgG1 hinge having the sequence of EPKSSDKTHTCPPC (SEQ ID NO: 2161), wherein the Cys220 that typicaly forms a disulfide bond with the C-terminal cysteine of the light chain is mutated to serine, e.g., Cys220Ser (C220S). In other embodiments, the fusion protein contains a truncated hinge having a sequence DKTHTCPPC (SEQ ID NO: 2162).

[0273] In some embodiments, the fusion protein or conjugate has a modified hinge from IgG4, which is modified to prevent or reduce strand exchange, e.g., Ser228Pro (S228P), having the sequence ESKYGPPCPPC (SEQ ID NO: 2163).

[0274] In alternative embodiments, a fusion protein or conjugate of the present disclosure may comprise sequences other than an Fc region to achieve multimerization (e.g., dimerization). For example, an amino acid sequence containing at least one cysteine residue may be included to facilitate dimerization of two polypeptides by formation of a disulfide bond between the two polypeptides. In some embodiments, such a multimerizing domain may comprise one or more cysteine residues, or a short cysteine-containing peptide. Other multimerizing domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[0275] Fc mutations suitable for use in the fusion proteins disclosed herein are also discussed in, e.g., Wilkinson et al., Fc-engineered antibodies with immune efector 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.Attorney Docket No: 260525.000049 Fusion or Conjugation to Half-Life Extension Moieties

[0276] In some embodiments, a fusion protein or conjugate of the present disclosure may comprise one or more other moieties which provide the fusion protein or conjugate with increased (in vivo) half- life. In vivo half-life extension means that the fusion protein or conjugate has an increased half-life in a mammal, such as a human subject, after administration.

[0277] Non-limiting examples of half-life extension moieties suitable for use in the present disclosure include polyethylene glycol (PEG) molecules, serum proteins or fragments thereof, binding units that can bind to serum proteins, an Fc portion, and smal proteins or peptides that can bind to serum proteins.

[0278] In some embodiments, a fusion protein or conjugate of the present disclosure may comprise a binding moiety that can bind to serum albumin, such as human serum albumin, or a serum immunoglobulin, such as IgG. In one embodiment, a fusion protein or conjugate of the present disclosure may comprise a binding moiety that can bind to human serum albumin. In one embodiment, the binding moiety is a single-domain antibody (e.g., VHH).

[0279] For example and without limitation, albumin binders that are described in, e.g., 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 is its entirety, can be used in the fusion protein or conjugate of the present disclosure. Fusion or Conjugation to Other Moieties

[0280] Anti-TRAILR2 antigen-binding proteins (e.g., antibodies such as single-domain antibodies) provided herein may be operably linked, directly or indirectly, to a second moiety, such as but not limited to, a detectable label, a drug, a toxin, a radionuclide, an enzyme, an immunomodulatory agent, a cytokine, a cytotoxic agent, a smal molecule drug, a chemotherapeutic agent, a therapeutic agent, a diagnostic agent, or a combination thereof.

[0281] In some embodiments, a conjugate of the present disclosure comprises a label, which can generate a detectable signal. Such conjugates can be used for research or diagnostic purposes, such as for the in vivo detection of cancer. Preferably, the label is capable of producing, either directly or indirectly, a detectable signal. For example, the label may be radio-opaque or a radioisotope (such as 3H, 14C, 32P, 35S, 123I, 125I, 131I); a fluorescent (fluorophore) or chemiluminescent (chromophore) compound (such as fluorescein isothiocyanate, rhodamine or luciferin); an enzyme (such as β- galactosidase, alkaline phosphatase, or horseradish peroxidase); an imaging agent; or a metal ion. InAttorney Docket No: 260525.000049 some embodiments, the label is a radioactive atom for scintigraphic studies, for example 99Tc or 123I, or a spin label for nuclear magnetic resonance (NMR) imaging, such as zirconium-89, iodine-123, iodine- 131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron. Zirconium-89 may also be complexed to various metal chelating agents and conjugated to antibodies, e.g., for PET imaging (WO 2011 / 056983).

[0282] Anti-TRAILR2 antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may be conjugated to another moiety, such as an epitope tag, e.g., for the purpose of purification or detection. Examples of such molecules that are useful in protein purification include those that present structural epitopes capable of being recognized by a second molecule. This is commonly employed in protein purification by afinity chromatography, in which a molecule is immobilized on a solid support and exposed to a heterogeneous mixture containing a target protein conjugated to a molecule capable of binding the immobilized compound. Non-limiting examples of epitope tag molecules that can be conjugated to anti-TRAILR2 antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure, e.g., for the purposes of molecular recognition include a poly-histidine tag (His-tag), a myc-tag, human influenza hemagglutinin (HA) tag, a FLAG-tag, maltose-binding protein, glutathione-S-transferase, biotin, and streptavidin. Conjugates containing the epitopes presented by these molecules are capable of being recognized by complementary molecules such as maltose, glutathione, a nickel-containing complex, an anti-FLAG antibody, an anti-myc antibody, an anti-HA antibody, streptavidin, or biotin, respectively. For example, one can purify an anti-TRAILR2 antigen-binding protein of the present disclosure that has been conjugated to an epitope tag from a complex mixture of other proteins and biomolecules (e.g., DNA, RNA, carbohydrates, phospholipids, etc) by treating the mixture with a solid phase resin containing a complementary molecule that can selectively recognize and bind the epitope tag of the TRAILR2 antibody or fragment thereof. Examples of solid phase resins include agarose beads, which are compatible with purifications in aqueous solution.

[0283] In some embodiments, a conjugate of the present disclosure may comprise one or more anti- TRAILR2 VHH domains described herein conjugated to a therapeutic agent, which can be cytotoxic, cytostatic or otherwise provides some therapeutic benefit. In some embodiments, the cytotoxic agent is a drug, a chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g., an enzymaticaly active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (e.g., a radioconjugate). Such conjugates may be applicable to, e.g., the treatment or prevention of a cancer. InAttorney Docket No: 260525.000049 some embodiments, antibody drug conjugates described herein may alow targeted delivery of a drug moiety to a target tissue (e.g., tumors).

[0284] In some embodiments, a conjugate of the present disclosure comprises a toxin. In some embodiments, the toxin includes, for example, bacterial toxins such as diphtheria toxin, plant toxins such as ricin, smal 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 calicheamicin (Lode et al., Cancer Res.58:2928 (1998); Hinman et al., Cancer Res.53:3336-3342 (1993). The toxins may exert their cytotoxic and cytostatic efects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Examples of other therapeutic agents that can be conjugated to an anti-TRAILR2 antigen-binding protein of the present disclosure are described herein (see “Treatment Methods and Other Uses” section).

[0285] In some embodiments, anti-TRAILR2 antigen-binding proteins (e.g., antibodies such as single- domain antibodies) of the present disclosure may be fused or conjugated to one or more moieties that facilitate delivery to the central nervous system (CNS) / brain. The moiety that can facilitate delivery of an anti-TRAILR2 antigen-binding protein to the central nervous system (CNS) / brain can be for example, a peptide, a polypeptide, smal molecule, a lipid, or a synthetic polymer. Various approaches to deliver single-domain antibodies into the brain are described in Pothin et al., Pharmaceutics 2020, 12(10), 937, which is incorporated herein by reference in its entirety.

[0286] As a non-limiting example, an anti-TRAILR2 antigen-binding protein (e.g., antibody such as single-domain antibody) of the present disclosure may be fused or conjugated to a moiety (e.g., an antibody) that binds to the transferrin receptor (TfR) or insulin receptor. The transferrin receptor (TfR) is highly expressed by brain capilary endothelial cels (BCECs) forming the blood-brain barrier (BBB) and has been utilized as a target for brain drug delivery. Monoclonal antibodies binding to the TfR, such as clone Ri7, have been shown to internalize into BCECs in vivo. As another example, an anti-TRAILR2 antigen-binding protein (e.g., antibody such as single-domain antibody) of the present disclosure may be conjugated to hydrophobic fatty acid moieties, such as C18 faty acid (stearic acid), C16 faty acid (palmitic acid) or C8 faty acid (octanoic acid) moieties; or amphiphilic block copolymer moieties, such as poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (pluronics or poloxamers) or poly(2- oxasolines). Various fatty acid moieties and block copolymer moieties that can be utilized for brain delivery of proteins are described in, e.g., Yi and Kabanov, J Drug Target.2013; 21(10): 940–955, which is incorporated herein by reference in its entirety.Attorney Docket No: 260525.000049

[0287] Example methods for ataching a moiety, 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, N.Y. (1983); and Colcher et al., Meth. Enzymol., 121 :802-16 (1986). Additional suitable methods for preparing the conjugates of the present disclosure include those described in, e.g., WO 2009 / 067800, WO 2011 / 133886, and US2014322129, incorporated by reference herein in their entirety.

[0288] In some embodiments, the attachment between an anti-TRAILR2 antigen-binding protein and a second moiety can be covalent or non-covalent, e.g., via a biotin-streptavidin non-covalent interaction. In some embodiments, a second moiety can be atached to an anti-TRAILR2 antigen-binding protein using any of various molecular biological or chemical conjugation and linkage methods known in the art and described below. In some embodiments, linkers such as peptide linkers, cleavable linkers, non- cleavable linkers or linkers that aid in the conjugation reaction, can be used to link or conjugate a second moiety to an anti-TRAILR2 antigen-binding protein described herein.

[0289] In some embodiments, an anti-TRAILR2 antigen-binding protein (e.g., antibody such as single- domain antibody) is conjugated to one or more second moieties, e.g., about 1 to about 20 moieties per molecule, optionaly via a linker. In some embodiments, the one or more second moieties can be the same or diferent. The linker may be composed of one or more linker components. For covalent attachment of an antibody and the second moiety, the linker typicaly has two reactive functional groups, i.e., bivalency in a reactive sense. Bivalent linker reagents which are useful to attach two or more functional or biologicaly active moieties, such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups have been described in, e.g., Hermanson, G. T. (1996) Bioconjugate Techniques; Academic Press: New York, p 234-242.

[0290] In some embodiments, a linker used in a conjugate of the present disclosure may include 6- maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”), valine-citruline (“val-cit”), a alanine- phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-Succinimidyl 4-(2- pyridylthio)pentanoate (“SPP”), N-Succinimidyl 4-(N-maleimidomethyl)cyclohexane-I carboxylate (“SMCC”), or N-Succinimidyl(4-iodo-acetyl)aminobenzoate (“STAB”), or a combination thereof.

[0291] In some embodiments, a linker used in a conjugate of the present disclosure may comprise amino acid residues. Exemplary amino acid linker components include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptides include valine-citruline (vc or val-cit), alanine- phenylalanine (af or ala-phe). Exemplary tripeptides include glycine-valine-citruline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acid residues used in an amino acid linker component mayAttorney Docket No: 260525.000049 include naturaly occurring amino acids, as wel as minor amino acids and non-naturaly occurring amino acid analogs, such as citruline. Amino acid linker components can be designed and optimized in their selectivity for enzymatic cleavage by particular enzymes, for example, a tumor-associated protease, cathepsin B, C and D, or a plasmin protease.

[0292] Conjugates of an anti-TRAILR2 antigen-binding protein (e.g., antibody such as single-domain antibody) and second moiety (e.g., cytotoxic agent) can be made using a variety of bifunctional protein- coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl substrate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p- azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)- ethylenediamine), disocyanates (such as toluene 2,6-disocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

[0293] Conjugates of the present disclosure can be prepared by a variety of methods. For example, the conjugation method may include: (1) reaction of a nucleophilic group of a VHH domain with a bivalent linker reagent, to form VHH-Linker, via a covalent bond, folowed by reaction with a drug moiety; or (2) reaction of a nucleophilic group of a drug moiety with a bivalent linker reagent, to form drug-linker, via a covalent bond, folowed by reaction with the nucleophilic group of a VHH domain.

[0294] Nucleophilic groups on proteins including antibodies (e.g., VHH domains), include, but are not limited to: (i) N-terminal amine groups, (i) side chain amine groups (e.g., lysine), (ii) side chain thiol groups (e.g., cysteine), and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (i) alkyl and benzyl halides such as haloacetamides; (ii) aldehydes, ketones, carboxyl, and maleimide groups. Additional nucleophilic groups can be introduced into proteins (e.g., antibodies such as VHH domains) through the reaction of lysines with 2- iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol. Reactive thiol groups may be introduced into a protein (e.g., antibody such as a VHH domain) by introducing one, two, three, four, or more cysteine residues.

[0295] Conjugates, such as antibody drug conjugates, may also be produced by modification of an antibody, such as a VHH domain, to introduce electrophilic moieties, which can react with nucleophilic substituents on the linker reagent or drug. The sugars of glycosylated antibodies may be oxidized, e.g., with periodate oxidizing reagents, to form aldehyde or ketone groups which may lead with the amineAttorney Docket No: 260525.000049 group of linker reagents or drug moieties. The resulting imine Schif base groups may form a stable linkage, or may be reduced, e.g., by borohydride reagents to form stable amine linkages. In one embodiment, reaction of the carbohydrate portion of a glycosylated antibody with either galactose oxidase or sodium meta-periodate may yield carbonyl (aldehyde and ketone) groups in the protein that can react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, proteins containing N-terminal serine or threonine residues can react with sodium meta- periodate, resulting in production of an aldehyde in place of the first amino acid. Such aldehyde can be reacted with a drug moiety or linker nucleophile.

[0296] Likewise, nucleophilic groups on a drug moiety include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBi esters, haloformates, and acid halides; (i) alkyl and benzyl halides such as haloacetamides; (ii) aldehydes, ketones, carboxyl, and maleimide groups.

[0297] Alternatively, a fusion protein containing a VHH domain and cytotoxic agent may be made, e.g., by recombinant DNA techniques or peptide synthesis. A DNA sequence may be engineered to comprise respective regions encoding the two portions of the fusion protein either adjacent to one another or separated by a region encoding a linker peptide which does not impair the desired properties of the fusion protein. The DNA sequence can be then transfected into a host cel that expresses the fusion protein. The fusion protein can be recovered from the cel culture and purified using techniques known in the art. Linkers

[0298] In some embodiments, the one or more polypeptides of the fusion proteins of the present disclosure are operably linked via peptide linkers. A peptide linker can range from 2 amino acids to 60 or more amino acids, and in certain aspects a peptide linker ranges from 3 amino acids to 50 amino acids, from 4 to 30 amino acids, from 5 to 25 amino acids, from 10 to 25 amino acids, 10 amino acids to 60 amino acids, from 12 amino acids to 20 amino acids, from 20 amino acids to 50 amino acids, or from 25 amino acids to 35 amino acids in length.

[0299] In some embodiments, a peptide linker, e.g., a peptide linker separating two VHH domains or an VHH domain and a heavy chain constant region, is at least 5 amino acids, at least 6 amino acids or atAttorney Docket No: 260525.000049 least 7 amino acids in length and optionaly is up to 30 amino acids, up to 40 amino acids, up to 50 amino acids or up to 60 amino acids in length.

[0300] In some embodiments, the linker ranges from 5 amino acids to 50 amino acids in length, e.g., ranges from 5 to 50, from 5 to 45, from 5 to 40, from 5 to 35, from 5 to 30, from 5 to 25, or from 5 to 20 amino acids in length. In other embodiments of the foregoing, the linker ranges from 6 amino acids to 50 amino acids in length, e.g., ranges from 6 to 50, from 6 to 45, from 6 to 40, from 6 to 35, from 6 to 30, from 6 to 25, or from 6 to 20 amino acids in length. In yet other embodiments of the foregoing, the linker ranges from 7 amino acids to 50 amino acids in length, e.g., ranges from 7 to 50, from 7 to 45, from 7 to 40, from 7 to 35, from 7 to 30, from 7 to 25, or from 7 to 20 amino acids in length.

[0301] 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 the disclosure include those disclosed by Chen et ai, 2013, Adv Drug Deliv Rev.65(10): 1357-1369 and Klein et a / ., 2014, Protein Engineering, Design & Selection 27(10): 325-330. Particularly useful flexible linkers are or comprise repeats of glycines and serines (termed “GS-linker” herein), e.g., a monomer or multimer of GnS (SEQ ID NO: 2136) or SGn(SEQ ID NO: 2137), where n is an integer from 1 to 10, e.g., 12, 3, 4, 5, 6, or 7, 8, 9 or 10. In one embodiment, the linker is or comprises a monomer or multimer of repeat of G4S (SEQ ID NO: 2093), e.g., (GGGGS)n (SEQ ID NO: 2138).

[0302] Polyglycine linkers can suitably be used in the fusion proteins of the disclosure. In some embodiments, a peptide linker used herein comprises two consecutive glycines (2Gly), three consecutive glycines (3Gly), four consecutive glycines (4Gly) (SEQ ID NO: 2090), five consecutive glycines (5Gly) (SEQ ID NO: 2139), six consecutive glycines (6Gly) (SEQ ID NO: 2140), seven consecutive glycines (7Gly) (SEQ ID NO: 2141), eight consecutive glycines (8Gly) (SEQ ID NO: 2142), or nine consecutive glycines (9Gly) (SEQ ID NO: 2143).

[0303] In some embodiments, a GS-linker used herein comprises an amino acid sequence selected from 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 proteins can include a combination of a GS-linker and a glycine linker.

[0304] In one embodiment, two or more VHHs are linked via a GGGGSGGGGSGGGGS (SEQ ID NO: 2094) linker. In one embodiment, two or more VHHs are linked via a GGGGSGGGGS (SEQ ID NO: 2147) linker. In one embodiment, a VHH and an Fc region are linked via a GGGGSESKYGPPCPSCP (SEQ ID NO: 2131) linker. In one embodiment, a VHH and an Fc region are linked via a GGGGS (SEQ ID NO: 2093) linker.Attorney Docket No: 260525.000049

[0305] In some embodiments, the one or more polypeptides of the fusion proteins of the present disclosure are operably linked via a “rigid” peptide linker. Such peptidic linker may comprise a proline- rich peptide. In one embodiment, a rigid peptide linker comprises PAPAPAPAPAPAPAPAP (SEQ ID NO: 2132). In one embodiment, a rigid peptide linker comprises GGGGSPAPAPAPAPAPAPAPAP (SEQ ID NO: 2133). In one embodiment, a rigid peptide linker comprises PAPAPAPAPAPAPAPAPGGGGS (SEQ ID NO: 2134). In one embodiment, a rigid peptide linker comprises GGGGSPAPAPAPAPAPAPAPAPGGGGS (SEQ ID NO: 2135). In one embodiment, a rigid peptide linker comprises GGGGSPAPAPAPAPAPAPAPAPGGGS (SEQ ID NO: 2088).

[0306] In one embodiment, a rigid peptide linker comprises A(EAAAK)nA (SEQ ID NO: 2148), where n is any integer, e.g., 12, 3, 4, 5, 6, or 7, 8, 9 or 10.

[0307] Other exemplary peptide linkers that can be used in the fusion proteins described herein are shown in Table 2. Table 2. Exemplary Peptide Linker Sequences Linker Amino acid sequence SEQ ID NO G4S GGGGS 2093Attorney Docket No: 260525.000049 Linker 24 PRGASKSGSASQTGSAPGS 2118 Linker 25 GTAAAGAGAAGGAAAGAAG 2119 Lik 2 T 212Signal Sequences

[0308] In some embodiments, the fusion protein described herein may further comprise a signal sequence at its N-terminus. Signal sequences may be present in the precursor molecule of the fusion protein and may be removed after the protein is secreted from the host cel during production. 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. Exemplary fusion proteins of the present disclosure

[0309] Non-limiting examples of fusion proteins (e.g., bivalent, trivalent or tetravalent constructs with / without Fc regions) are disclosed in the “Examples” and “List of Sequences” sections below.Attorney Docket No: 260525.000049

[0310] Non-limiting examples of amino acid sequences of fusion proteins are described in Table 3. The VHH amino acid sequence(s) are in bolded text; the linker sequence(s) are underlined with a single line; and the Fc region is in italicized text. Table 3. Non-limiting examples of amino acid sequences of fusion proteins Fusion Amino acid sequence SEQ ID NO proteinAttorney Docket No: 260525.000049 ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAASSWSRGGVPYGM DYWGKGTLVTVSS 23D12 EVQLVESGGGLVQAGGSLRLSCAASGRTLDAYGMGWFRQAPGKEREPVGVI 2059Attorney Docket No: 260525.000049 HYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCKAGIRGEVYWGQG TQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESG GGLVQPGGSLRLSCAASGFTLSGYAMSWYRQAPGKERELVALITSAGGSTHYAttorney Docket No: 260525.000049 VSFISNTGTTTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAANG WGLDPTTYHYWGQGTQVTVSS N742h 3 EVQLLESGGGLVQPGGSLRVSCAASGGTLANNGMAWFRQAPGKEREFVAA 2068, ID NOs: 2054-2070, or a similar sequence thereof 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.

[0312] In various embodiments, a fusion protein of the present disclosure comprises any one of SEQ ID NOs: 2054-2070, or a sequence having at least 75% identity thereto.

[0313] It is to be understood that although the exemplary fusion proteins described herein contain non-humanized VHH amino acid sequences, such non-humanized VHH amino acid sequences can beAttorney Docket No: 260525.000049 replaced with any of the humanized VHH amino acid sequences described herein (e.g., in Tables 1-1 and 1-2).

[0314] Any of various fusion proteins described herein may have an agonistic efect upon binding to TRAILR2. Polynucleotide Molecules

[0315] In another aspect, provided herein are polynucleotide molecules encoding the anti-TRAILR2 antigen-binding proteins (e.g., antibodies including single-domain antibodies) or fusion proteins described herein. Polynucleotide molecules encoding polypeptide portion(s) of a conjugate of the present disclosure are also encompassed within the present disclosure.

[0316] In some embodiments, a polynucleotide molecule of the present disclosure encodes an anti- TRAILR2 VHH amino acid sequence selected from SEQ ID NOs: 4, 9, 14, 17, 21, 26, 31, 36, 41, 45, 50, 55, 65, 70, 75, 79, 84, 89, 93, 98, 146-468, 2192, 2198, 2204, and 2214-2233, or a similar sequence thereof 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.

[0317] In some embodiments, a polynucleotide molecule of the present disclosure encoding an anti- TRAILR2 VHH comprises the nucleotide sequence of any one of SEQ ID NOs: 57-61, 131-145, 1731-2053, 2193, 2199, 2205, and 2278-2297, or a similar sequence thereof 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.

[0318] In some embodiments, a polynucleotide molecule of the present disclosure encoding an anti- TRAILR2 VHH comprises the nucleotide sequence of any one of SEQ ID NOs: 57-61, 131-145, 2071-2087, 2193, 2199, and 2205, or a similar sequence thereof 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.

[0319] In some embodiments, a polynucleotide molecule of the present disclosure encoding an anti- TRAILR2 VHH comprises the nucleotide sequence of any one of SEQ ID NOs: 132, 137, 2193, 2199, and 2205, or a similar sequence thereof 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.

[0320] In an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence selected from SEQ ID NOs: 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, 2234-2251, and 2354-2404, or aAttorney Docket No: 260525.000049 similar sequence thereof 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.

[0321] In an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a fusion protein comprising an amino acid sequence selected from SEQ ID NOs: 2054-2070, or a similar sequence thereof 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.

[0322] In some embodiments, a polynucleotide molecule of the present disclosure encoding fusion protein comprises the nucleotide sequence of any one of SEQ ID NOs: 2071-2087, or a similar sequence thereof 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.

[0323] A polynucleotide molecule may be used to transform / transfect a host cel or host organism, e.g., for expression and / or production of a polypeptide. Suitable hosts or host cels for production of an anti- TRAILR2 polypeptides described herein include any suitable fungal, prokaryotic or eukaryotic cel or cel line or any suitable fungal, prokaryotic or eukaryotic organism. A host or host cel comprising a polynucleotide molecule encoding an anti-TRAILR2 antigen-binding protein polypeptide or fusion protein described herein is also encompassed by the present disclosure.

[0324] A polynucleotide molecule may be for example DNA, RNA, or a hybrid thereof, and may also comprise (e.g., chemicaly) modified nucleotides, like locked nucleic acids (LNA) or peptide nucleic acids (PNA). 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., plasmid). In some embodiments, the polynucleotide is in the form of a single- stranded RNA (e.g., mRNA).

[0325] Techniques for generating polynucleotides may include, for example but not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more naturaly occurring and / or synthetic sequences (or two or more parts thereof), introduction of mutations that lead to the expression of a truncated expression product; introduction of one or more restriction sites (e.g. to create cassettes and / or regions that may easily be digested and / or ligated using suitable restriction enzymes), and / or the introduction of mutations by means of a PCR reaction using one or more “mismatched” primers. Alternatively, polynucleotides of the present disclosure may be isolated from a suitable natural source. Polynucleotide sequences encoding naturaly occurring (poly)peptides can for example be subjected to site-directed mutagenesis, to generate a polynucleotide molecule encoding polypeptide with sequence variation.Attorney Docket No: 260525.000049 Vectors

[0326] Also provided herein are vectors comprising the polynucleotide molecules encoding the anti- TRAILR2 antigen-binding proteins (e.g., antibodies including single-domain antibodies), fusion proteins, or other relevant polypeptides of the present disclosure. A “vector” as used herein is a vehicle suitable for carrying genetic material into a host cel. A vector can include a nucleic acid vector, such as a plasmid or mRNA, or nucleic acids embedded into a bigger structure, such as a liposome or viral vector.

[0327] A vector can include one or more of the folowing elements: an origin of replication, one or more regulatory sequences (e.g., promoters, enhancers, terminators) that regulate the expression of a polypeptide of interest, and / or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that can be used in colorimetric assays, for example, β-galactosidase). For DNA-based vectors, this usualy includes the presence of elements for transcription (e.g., a promoter and a polyA signal) and translation (e.g., Kozak sequence). In some embodiments, the vector is an expression vector, i.e. a vector suitable for expressing an encoded polypeptide or construct under suitable conditions in a host cel.

[0328] To express an anti-TRAILR2 antigen-binding protein or fusion protein (or fragments thereof) of the present disclosure, polynucleotides encoding partial or ful-length polypeptide chains, e.g., obtained as described above (e.g., VHH, VHH-Fc), can be inserted into expression vectors such that the genes are operatively linked to one or more transcriptional and translational control sequences. The expression vector and expression control sequences are chosen to be compatible with the expression host cel used. Polynucleotides encoding the two or more polypeptide chains (when present and difer from one another) of an anti-TRAILR2 antigen-binding protein or fusion protein of the present disclosure can be inserted into separate vectors, or, optionaly, incorporated into the same expression vector.

[0329] In addition to polynucleotides encoding the polypeptide chain(s) of an anti-TRAILR2 antigen- binding protein or fusion protein, the recombinant expression vectors of the invention may include regulatory sequences that control the expression of genes encoding the polypeptide chain(s) in a host cel. The design of the expression vector, including the selection of regulatory sequences, may depend on the choice of the host cel to be transformed and / or the desired level of protein expression. For example, suitable regulatory sequences for mammalian host cel expression include viral elements that direct high levels of protein expression in mammalian cels, such as promoters and / or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter (AdMLP) and polyoma. Additional examples of viral regulatory elements, and sequencesAttorney Docket No: 260525.000049 thereof, include those described in, e.g., U.S. Pat. Nos.5, 168,062; 4,510,245; and 4,968,615; the disclosures of each of which are incorporated herein by reference.

[0330] Recombinant expression vectors of the present disclosure may carry additional sequences, such as sequences that regulate replication of the vector in host cels (e.g., origins of replication) and selectable marker genes. A selectable marker gene facilitates selection of host cels into which the vector has been introduced (see e.g., US4,399,216; US 4,634,665; and US 5,179,017; the disclosure of each of which is incorporated herein by reference in its entirety). For example, typicaly the selectable marker gene confers resistance to antibiotics, such as ampicilin, chloramphenicol, kanamycin, or nourseothricin, or cytotoxic drugs, such as G418, puromycin, blasticidin, hygromycin or methotrexate, to a host cel into which the vector has been introduced. Suitable selectable marker genes can include the dihydrofolate reductase (DHFR) gene (for use in DHFR deficient host cels with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0331] Vectors of the present disclosure may further include sequence elements that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements include, e.g., 5′ and 3′ untranslated regions, an internal ribosomal entry site (IRES), and polyadenylation signal site in order to direct eficient transcription of the gene carried on the expression vector.

[0332] Viral vectors can be used for the eficient delivery of exogenous genes into the genome of a cel (e.g., a eukaryotic or prokaryotic cel). Viral vectors are particularly useful for gene delivery because the polynucleotides contained within such genomes are typicaly incorporated into the genome of a target cel by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents to induce gene integration. Examples of suitable viral vectors include a retrovirus, adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses (AAV) such as AAV2, AAV8, AAV9), negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpes virus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), baculovirus, coronavirus, and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses useful for delivering polynucleotides encoding polypeptides of the present disclosure include, for example Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include, but are not limited to, avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-typeAttorney Docket No: 260525.000049 viruses, HTLV-BLV group, lentivirus, spumavirus (Cofin, J. M.1996. Fundamental Virology, DMKDN Fields, PM Howley, ed. (Philadelphia, Lippincot-Raven Publishers): 763-843., the disclosure of which is incorporated herein by reference). Other examples of viral genomes useful in the compositions and methods of the present disclosure include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline sarcoma virus, feline leukemia virus, avian leukemia virus, human T-cel leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentiviruses. Host Cels

[0333] In one aspect, the present disclosure also provides host cels or host organisms that comprise the polynucleotides or vectors encoding the anti-TRAILR2 antigen-binding proteins (e.g., antibodies including single-domain antibodies), fusion proteins, or other relevant polypeptides described herein. Suitable host cels or host organisms can be any suitable fungal, prokaryotic or eukaryotic cel or cel line or any suitable fungal, prokaryotic or eukaryotic organism. Host cels include progeny of a single host cel, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cel due to natural, accidental, or deliberate mutation. Host cels can also include cels transfected in vivo with a polynucleotide(s) or vector provided herein.

[0334] Exemplary eukaryotic cels include mammalian cels, such as primate or non-primate animal cels; fungal cels, such as yeast (e.g., Saccharomyces cerevisiae or Pichia pastoris); plant cels; and insect cels. Non-limiting exemplary mammalian cels include, but are not limited to, NSO cels, PER.C6® cels (Crucel), COS cels, SP2 / 0 cels, and 293 and CHO cels, and their derivatives, such as 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cels. Exemplary prokaryotic cels include bacterial cels such as Escherichia coli. Preparation Methods

[0335] The present disclosure also provides methods of producing the anti-TRAILR2 antigen-binding proteins (e.g., antibodies including single-domain antibodies), fusion proteins, or conjugates described herein.

[0336] In some embodiments, a method may comprise transforming / transfecting a host cel or host organism with a polynucleotide encoding an anti-TRAILR2 antigen-binding protein (e.g., antibody such as single-domain antibody), fusion protein, or other relevant polypeptide(s) described herein, expressingAttorney Docket No: 260525.000049 the anti-TRAILR2 antigen-binding protein (e.g., antibody such as single-domain antibody), fusion protein, or other relevant polypeptide(s) in the host, optionaly folowed by one or more isolation and / or purification steps.

[0337] When recombinant expression vectors encoding one or more polypeptide(s) of an anti-TRAILR2 antigen-binding protein (e.g., antibody such as single-domain antibody), fusion protein, or conjugate of the present disclosure are introduced into mammalian host cels, the host cels are cultured for a period of time suficient to alow for expression of the protein(s) or polypeptide(s) in the host cels or secretion of the protein(s) or polypeptide(s) into the culture medium in which the host cels are grown. Protein(s) or polypeptide(s) can be recovered from the culture medium using standard protein purification methods. Host cels can also be used to produce portions of intact antibodies, such as VHH domains.

[0338] Once a protein or polypeptide of the present disclosure has been produced by recombinant expression, it can be purified by any method known in the art for purification of a protein or polypeptide, for example, by chromatography (e.g., ion exchange, afinity, particularly by afinity for TRAILR2 after Protein A or Protein G selection, and sizing column chromatography), centrifugation, diferential solubility, or by any other standard technique for the purification of proteins. Further, the proteins or polypeptides of the present disclosure can be fused to heterologous polypeptide sequences described herein (e.g., His-tag) or otherwise known in the art to facilitate purification or to produce therapeutic conjugates below). Once isolated, a protein or polypeptide of the present disclosure can, if desired, be further purified, e.g., by high performance liquid chromatography, or by gel filtration chromatography, such as on a Superdex™ column. Pharmaceutical Compositions and Formulations

[0339] The present disclosure also provides a composition comprising anti-TRAILR2 antigen-binding protein (e.g., antibody such as single-domain antibody), fusion protein, or conjugate of the present technology, at least one polynucleotide molecule encoding the same, at least one vector comprising such a polynucleotide molecule, or at least one host cel comprising the polynucleotide molecule or vector. The composition may be a pharmaceutical composition. The composition may further comprise at least one pharmaceuticaly acceptable carrier, diluent or excipient and / or adjuvant, and optionaly comprise one or more further pharmaceuticaly active polypeptides and / or compounds.

[0340] As used herein, the term “pharmaceuticaly acceptable carrier” is intended to include any and al solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers areAttorney Docket No: 260525.000049 described in the most recent 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, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. Supplementary active compounds can also be incorporated into the compositions.

[0341] Examples of suitable formulations include, but are not limited to, solutions, suspensions, powders, pastes, ointments, jelies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in- water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powel et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Phdomain Sci Technol 52:238- 311.

[0342] A pharmaceutical composition of the present disclosure may be formulated according to its intended route of administration. Examples of suitable routes of administration include, e.g., intravenous, subcutaneous, intratumoral, oral (e.g., buccal, sublingual), intranasal, inhalation, intraocular, intramuscular, intradermal, transdermal (i.e., topical), intraperitoneal, transmucosal, vaginal, and rectal administration, or injection to the CNS / brain (e.g., intraspinal, intracerebral, or intrathecal administration). Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the folowing components: a sterile diluent such as water for injection, saline solution, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; fixed oils; chelating agents such as ethylenediaminetetraacetic acid (EDTA); bufers such as phosphates, acetates, or citrates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of plastic or glass.

[0343] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include, for example, physiological saline, bacteriostatic water, Cremophor EL®, or phosphate bufered saline (PBS). The composition is preferably sterile and has a proper fluidity. In most embodiments, the composition is stable under the conditions of manufacture and storage and can be preserved against the contaminatingAttorney Docket No: 260525.000049 action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, e.g., water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the contamination by microorganisms can be achieved by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0344] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients described above, as required, folowed by filtered sterilization. Generaly, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and / or freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0345] Oral compositions may include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, capsules, or liquid forms. Formulation in tablet and liquid forms may be used for protease insensitive VHHs. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied oraly and swished and expectorated or swalowed. Pharmaceuticaly compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pils, capsules, troches and the like can contain any of the folowing ingredients, or compounds of a similar nature: a binder such as microcrystaline celulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as coloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.Attorney Docket No: 260525.000049

[0346] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propelant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0347] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generaly known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generaly known in the art.

[0348] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0349] For brain delivery, compounds of the present disclosure may be formulated to facilitate crossing of the blood-brain barrier. For example, anti-TRAILR2 antigen-binding proteins (e.g., antibody such as single-domain antibody), fusion proteins, or conjugates of the present disclosure may be encapsulated into brain targeted liposomes, lipid nanoparticles, lipid microparticles, or lipid microcapsules for brain delivery. Example liposomes delivery systems are described in Pothin et al., Pharmaceutics 2020, 12(10), 937, which is incorporated herein by reference in its entirety.

[0350] In some embodiments, the active compounds are prepared with carriers that can protect the compound against rapid elimination from the body, such as a controled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, colagen, polyorthoesters, and polylactic acid. Liposomal suspensions can also be used as pharmaceuticaly acceptable carriers. These can be prepared according to methods known to those skiled in the art, for example, as described in US 4,522,811, which is incorporated herein by reference in its entirety.

[0351] It is especialy advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physicaly discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic efect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure is dependent on the unique characteristics of the active compound and the particular therapeutic efect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.Attorney Docket No: 260525.000049

[0352] The pharmaceutical compositions (or components thereof) can be included in a kit, container, pack, or dispenser together with instructions for administration. These pharmaceutical compositions can be included in diagnostic kits with instructions for use.

[0353] Pharmaceutical compositions are administered in an amount efective for treatment or prophylaxis of the specific indication. The therapeuticaly efective amount is typicaly dependent on the weight of the subject being treated, the physical or health condition of the subject, the extensiveness of the condition to be treated, or the age of the subject being treated. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of 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 an amount in the range of 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 an amount in the range of 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 an amount in the range of about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted. Efective dosages and schedules for administering a pharmaceutical composition of the present disclosure may be determined empiricaly; for example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly. Moreover, interspecies scaling of dosages can be performed using wel- known methods in the art (e.g., Mordenti et al., 1991, Phdomainaceut. Res.8:1351).

[0354] In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 10 mg to about 1,000 mg per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 20 mg to about 500 mg per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 20 mg to about 300 mg per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 20 mg to about 200 mg per dose.

[0355] In some embodiments wherein the antigen-binding proteins of the present disclosure are administered as a viral vector (e.g., an AAV), dose ranges and frequency of administration of the viral vector described herein can vary depending on the nature of the viral vector, and the medical condition, as wel as parameters of a specific patient and the route of administration used. In some embodiments, viral vector compositions can be administered to a subject at a dose ranging from about 1×105 plaque forming units (pfu) to about 1×1015 pfu, depending on mode of administration, the route of administration, the nature of the disease and condition of the subject. In some cases, the viral vectorAttorney Docket No: 260525.000049 compositions can be administered at a dose ranging from about 1×108 pfu to about 1×1015 pfu, or from about 1×1010 pfu to about 1×1015 pfu, or from about 1×108 pfu to about 1×1012 pfu. A more accurate dose can also depend on the subject in which it is being administered. For example, a lower dose may be required if the subject is juvenile, and a higher dose may be required if the subject is an adult human subject. In certain embodiments, a more accurate dose can depend on the weight of the subject. In certain embodiments, for example, a juvenile human subject can receive from about 1×108 pfu to about 1×1010 pfu, while an adult human subject can receive a dose from about 1×1010 pfu to about 1×1012 pfu.

[0356] Various delivery systems are known and can be used to administer the pharmaceutical composition of the disclosure, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cels capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem.262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, intraocular, epidural, intraspinal, intracerebral, intrathecal and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologicaly active agents. Administration can be systemic or local.

[0357] A pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present disclosure. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generaly utilizes a replaceable cartridge that contains a pharmaceutical composition. Once al of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefiled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0358] In certain situations, the pharmaceutical composition can be delivered in a controled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng.14:201). In another embodiment, polymeric materials can be used; see, Medical Applications of Controled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a controled release system can be placed in proximity of the composition’s target,Attorney Docket No: 260525.000049 thus requiring only a fraction of the...

Claims

Attorney Docket No: 260525.000049 Claims 1. An antigen-binding protein that specificaly binds TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2), comprising a complementarity determining region 3 (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, wherein one or more non-alanine residues in the CDR3 sequence is optionaly replaced with an alanine, and / or one or more alanine residues in the CDR3 sequence is optionaly replaced with a glycine.

2. The antigen-binding protein of claim 1, wherein the CDR3 comprises an amino acid sequence selected from 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); andAttorney Docket No: 260525.000049 w). (A / F / M / N)(A / T)(A / G / S)(A / D)Y.

3. The antigen-binding protein of claim 1, wherein the CDR3 comprises an amino acid sequence selected from 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: 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); AAASSWSRGGAPYGMDY (SEQ ID NO: 2308); AAASSWSRGGVAYGMDY (SEQ ID NO: 2309); AAASSWSRGGVPAGMDY (SEQ ID NO: 2310); AAASSWSRGGVPYAMDY (SEQ ID NO: 2311); AAASSWSRGGVPYGADY (SEQ ID NO: 2312); AAASSWSRGGVPYGMAY (SEQ ID NO: 2313); AAASSWSRGGVPYGMDA (SEQ ID NO: 2314); 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); TAGPTMSYSRGAEF (SEQ ID NO: 2326); TAGPTMSYSRGGAF (SEQ ID NO: 2327); TAGPTMSYSRGGEA (SEQ ID NO: 2328); 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); 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); HSRWANLAttorney Docket No: 260525.000049 (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).

4. The antigen-binding protein of any one of claims 1-3, further comprising a CDR1 comprising an amino acid sequence selected from 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). GFTLDLGAYA (SEQ ID NO: 81); m). GFTFGALA (SEQ ID NO: 86); n). GFTLS(G / S)YA (SEQ ID NO: 120); o). GSIFGGYN (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).

5. The antigen-binding protein of claim 4, wherein the CDR1 comprises an amino acid sequence selected from 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).Attorney Docket No: 260525.000049 6. The antigen-binding protein of any one of claims 1-5, further comprising a CDR2 comprising an amino acid sequence selected from 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 of claim 6, wherein the CDR2 comprises an amino acid sequence selected from VSWNGAST (SEQ ID NO: 2); DHRSGT (SEQ ID NO: 7); ISWTGVDT (SEQ ID NO: 12); ISWTGTDT (SEQ ID NO: 19); ISWSGVDT (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: 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 specificaly binds TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2), comprising a CDR1 comprising an amino acid sequence selected from SEQ ID Nos: 1, 6, 11, 23, 28, 33, 38, 43, 47, 52, 62, 67, 72, 81, 86, 95, 762-1084, 2189, 2195, 2201, and 2252-2261; aAttorney Docket No: 260525.000049 CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 2, 7, 12, 19, 24, 29, 34, 39, 48, 53, 63, 68, 73, 77, 82, 87, 91, 96, 1085-1407, 2190, 2196, 2202, and 2262-2271; and / or a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 3, 8, 13, 16, 20, 25, 30, 35, 40, 44, 49, 54, 64, 69, 74, 78, 83, 88, 92, 97, 1408-1730, 2191, 2197, 2203, 2272-2277, and 2298-2348.

9. The antigen-binding protein of any one of claims 1, 3-8, wherein the antigen-binding protein comprises i). a CDR1 comprising an amino acid sequence of GRTFSSNL (SEQ ID NO: 1), a CDR2 comprising an amino acid sequence of VSWNGAST (SEQ ID NO: 2), and a CDR3 comprising an amino acid sequence of VASRLPFNSRSAIYTDRIYDS (SEQ ID NO: 3); i). a CDR1 comprising an amino acid sequence of GGTLANNG (SEQ ID NO: 6), a CDR2 comprising an amino acid sequence of DHRSGT (SEQ ID NO: 7), and a CDR3 comprising an amino acid sequence of AVRRSAWYSDSIYTVSQYDY (SEQ ID NO: 8); ii). a CDR1 comprising an amino acid sequence of GRTLDAYG (SEQ ID NO: 11), a CDR2 comprising an amino acid sequence of ISWTGVDT (SEQ ID NO: 12), and a CDR3 comprising an amino acid sequence of NAARSYSRDYEPLKPDY (SEQ ID NO: 13); iv). a CDR1 comprising an amino acid sequence of GRTLDAYG (SEQ ID NO: 11), a CDR2 comprising an amino acid sequence of ISWTGVDT (SEQ ID NO: 12), and a CDR3 comprising an amino acid sequence of NAARSYSRNYEPLKPDY (SEQ ID NO: 16); v). a CDR1 comprising an amino acid sequence of GRTLDAYG (SEQ ID NO: 11), a CDR2 comprising an amino acid sequence of ISWTGTDT (SEQ ID NO: 19), and a CDR3 comprising an amino acid sequence of NAARSYSRGGEPLKPDY (SEQ ID NO: 20); vi). a CDR1 comprising an amino acid sequence of GRTLSDYA (SEQ ID NO: 23) , a CDR2 comprising an amino acid sequence of ISWSGVDT (SEQ ID NO: 24), and a CDR3 comprising an amino acid sequence of NAARSYSRGGRPLEPAY (SEQ ID NO: 25); vi). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRGGVPYGMDY (SEQ ID NO: 30); vii). a CDR1 comprising an amino acid sequence of GRDFSNYV (SEQ ID NO: 33), a CDR2 comprising an amino acid sequence of INWADET (SEQ ID NO: 34), and a CDR3 comprising an amino acid sequence of AADSHFRRYTPGQQYEY (SEQ ID NO: 35);Attorney Docket No: 260525.000049 ix). a CDR1 comprising an amino acid sequence of GRSFSSYA (SEQ ID NO: 38), a CDR2 comprising an amino acid sequence of INWSGGST (SEQ ID NO: 39), and a CDR3 comprising an amino acid sequence of NAAKSYHRDYSPLSPDY (SEQ ID NO: 40); x). a CDR1 comprising an amino acid sequence of GRTFSTLA (SEQ ID NO: 43), a CDR2 comprising an amino acid sequence of INWSGGST (SEQ ID NO: 39), and a CDR3 comprising an amino acid sequence of AAAPSFGMRNPESYVHS (SEQ ID NO: 44); xi). a CDR1 comprising an amino acid sequence of GRTFSSDI (SEQ ID NO: 47), a CDR2 comprising an amino acid sequence of ISWSDMSA (SEQ ID NO: 48), and a CDR3 comprising an amino acid sequence of AANRGIMSMRLSRYDD (SEQ ID NO: 49); xi). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTMSYSRGGEF (SEQ ID NO: 54); xii). a CDR1 comprising an amino acid sequence of GGTLSNYA (SEQ ID NO: 62), a CDR2 comprising an amino acid sequence of ISQTSST (SEQ ID NO: 63), and a CDR3 comprising an amino acid sequence of VADRGAISRSGAGMDY (SEQ ID NO: 64); xiv). a CDR1 comprising an amino acid sequence of GRTLSNYA (SEQ ID NO: 67), a CDR2 comprising an amino acid sequence of ISQSSDT (SEQ ID NO: 68), and a CDR3 comprising an amino acid sequence of AADRGAISRSGAGMDY (SEQ ID NO: 69); xv). a CDR1 comprising an amino acid sequence of GRSFGAQGMEG (SEQ ID NO: 72), a CDR2 comprising an amino acid sequence of MKWTGNT (SEQ ID NO: 73), and a CDR3 comprising an amino acid sequence of TAGPAISLSRGGEY (SEQ ID NO: 74); xvi). a CDR1 comprising an amino acid sequence of GRSFGAQGMEG (SEQ ID NO: 72), a CDR2 comprising an amino acid sequence of IKWTGNT (SEQ ID NO: 77), and a CDR3 comprising an amino acid sequence of TAGPSISYSRGGEY (SEQ ID NO: 78); xvi). a CDR1 comprising an amino acid sequence of GFTLDLGAYA (SEQ ID NO: 81), a CDR2 comprising an amino acid sequence of ISNTGTTT (SEQ ID NO: 82), and a CDR3 comprising an amino acid sequence of AANGWGLDPTTYHY (SEQ ID NO: 83); xvii). a CDR1 comprising an amino acid sequence of GFTFGALA (SEQ ID NO: 86), a CDR2 comprising an amino acid sequence of ISNDGEHI (SEQ ID NO: 87), and a CDR3 comprising an amino acid sequence of SAGWTRRIFQY (SEQ ID NO: 88);Attorney Docket No: 260525.000049 xix). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of ISWNGDIT (SEQ ID NO: 91), and a CDR3 comprising an amino acid sequence of TAGQSISLSQGGEY (SEQ ID NO: 92); xx). a CDR1 comprising an amino acid sequence of GFTLSGYA (SEQ ID NO: 95), a CDR2 comprising an amino acid sequence of ITSAGGST (SEQ ID NO: 96), and a CDR3 comprising an amino acid sequence of KAGIRGEVY (SEQ ID NO: 97); xxi). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of RAYNDGGEY (SEQ ID NO: 2191); xxi). a CDR1 comprising an amino acid sequence of GSNFRILS (SEQ ID NO: 2195), a CDR2 comprising an amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and a CDR3 comprising an amino acid sequence of HSRWYNL (SEQ ID NO: 2197); xxii). a CDR1 comprising an amino acid sequence of GFTFSRYA (SEQ ID NO: 2201), a CDR2 comprising an amino acid sequence of ISSAGGYI (SEQ ID NO: 2202), and a CDR3 comprising an amino acid sequence of FTADY (SEQ ID NO: 2203); xxiv). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of GAASSWSRGGVPYGMDY (SEQ ID NO: 2298); xxv). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AGASSWSRGGVPYGMDY (SEQ ID NO: 2299); xxvi). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAGSSWSRGGVPYGMDY (SEQ ID NO: 2300); xxvi). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAAASWSRGGVPYGMDY (SEQ ID NO: 2301); xxvii). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASAWSRGGVPYGMDY (SEQ ID NO: 2302);Attorney Docket No: 260525.000049 xxix). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSASRGGVPYGMDY (SEQ ID NO: 2303); xxx). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWARGGVPYGMDY (SEQ ID NO: 2304); xxxi). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSAGGVPYGMDY (SEQ ID NO: 2305); xxxi). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRAGVPYGMDY (SEQ ID NO: 2306); xxxii). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRGAVPYGMDY (SEQ ID NO: 2307); xxxiv). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRGGAPYGMDY (SEQ ID NO: 2308); xxxv). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRGGVAYGMDY (SEQ ID NO: 2309); xxxvi). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRGGVPAGMDY (SEQ ID NO: 2310); xxxvi). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRGGVPYAMDY (SEQ ID NO: 2311); xxxvii). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRGGVPYGADY (SEQ ID NO: 2312);Attorney Docket No: 260525.000049 xxxix). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRGGVPYGMAY (SEQ ID NO: 2313); xl). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRGGVPYGMDA (SEQ ID NO: 2314); xli). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of AAGPTMSYSRGGEF (SEQ ID NO: 2315); xli). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TGGPTMSYSRGGEF (SEQ ID NO: 2316); xlii). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAAPTMSYSRGGEF (SEQ ID NO: 2317); xliv). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGATMSYSRGGEF (SEQ ID NO: 2318); xlv). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPAMSYSRGGEF (SEQ ID NO: 2319); xlvi). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTASYSRGGEF (SEQ ID NO: 2320); xlvi). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTMAYSRGGEF (SEQ ID NO: 2321); xlvii). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTMSASRGGEF (SEQ ID NO: 2322);Attorney Docket No: 260525.000049 xlix). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTMSYARGGEF (SEQ ID NO: 2323); l). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTMSYSAGGEF (SEQ ID NO: 2324); li). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTMSYSRAGEF (SEQ ID NO: 2325); li). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTMSYSRGAEF (SEQ ID NO: 2326); lii). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTMSYSRGGAF (SEQ ID NO: 2327); liv). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTMSYSRGGEA (SEQ ID NO: 2328); lv). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of AAYNDGGEY (SEQ ID NO: 2329); lvi). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of RGYNDGGEY (SEQ ID NO: 2330); lvi). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of RAANDGGEY (SEQ ID NO: 2331); lvii). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of RAYADGGEY (SEQ ID NO: 2332);Attorney Docket No: 260525.000049 lix). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of RAYNAGGEY (SEQ ID NO: 2333); lx). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of RAYNDAGEY (SEQ ID NO: 2334); lxi). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of RAYNDGAEY (SEQ ID NO: 2335); lxi). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of RAYNDGGAY (SEQ ID NO: 2336); lxii). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of RAYNDGGEA (SEQ ID NO: 2337); lxiv). a CDR1 comprising an amino acid sequence of GSNFRILS (SEQ ID NO: 2195), a CDR2 comprising an amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and a CDR3 comprising an amino acid sequence of ASRWYNL (SEQ ID NO: 2338); lxv). a CDR1 comprising an amino acid sequence of GSNFRILS (SEQ ID NO: 2195), a CDR2 comprising an amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and a CDR3 comprising an amino acid sequence of HARWYNL (SEQ ID NO: 2339); lxvi). a CDR1 comprising an amino acid sequence of GSNFRILS (SEQ ID NO: 2195), a CDR2 comprising an amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and a CDR3 comprising an amino acid sequence of HSAWYNL (SEQ ID NO: 2340); lxvi). a CDR1 comprising an amino acid sequence of GSNFRILS (SEQ ID NO: 2195), a CDR2 comprising an amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and a CDR3 comprising an amino acid sequence of HSRAYNL (SEQ ID NO: 2341); lxvii). a CDR1 comprising an amino acid sequence of GSNFRILS (SEQ ID NO: 2195), a CDR2 comprising an amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and a CDR3 comprising an amino acid sequence of HSRWANL (SEQ ID NO: 2342);Attorney Docket No: 260525.000049 lxix). a CDR1 comprising an amino acid sequence of GSNFRILS (SEQ ID NO: 2195), a CDR2 comprising an amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and a CDR3 comprising an amino acid sequence of HSRWYAL (SEQ ID NO: 2343); lxx). a CDR1 comprising an amino acid sequence of GSNFRILS (SEQ ID NO: 2195), a CDR2 comprising an amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and a CDR3 comprising an amino acid sequence of HSRWYNA (SEQ ID NO: 2344); lxxi). a CDR1 comprising an amino acid sequence of GFTFSRYA (SEQ ID NO: 2201), a CDR2 comprising an amino acid sequence of ISSAGGYI (SEQ ID NO: 2202), and a CDR3 comprising an amino acid sequence of ATADY (SEQ ID NO: 2345); lxxi). a CDR1 comprising an amino acid sequence of GFTFSRYA (SEQ ID NO: 2201), a CDR2 comprising an amino acid sequence of ISSAGGYI (SEQ ID NO: 2202), and a CDR3 comprising an amino acid sequence of FAADY (SEQ ID NO: 2346); lxxii). a CDR1 comprising an amino acid sequence of GFTFSRYA (SEQ ID NO: 2201), a CDR2 comprising an amino acid sequence of ISSAGGYI (SEQ ID NO: 2202), and a CDR3 comprising an amino acid sequence of FTGDY (SEQ ID NO: 2347); or lxxiv). a CDR1 comprising an amino acid sequence of GFTFSRYA (SEQ ID NO: 2201), a CDR2 comprising an amino acid sequence of ISSAGGYI (SEQ ID NO: 2202), and a CDR3 comprising an amino acid sequence of FTAAY (SEQ ID NO: 2348).

10. The antigen-binding protein of any one of claims 1-9, wherein the antigen-binding protein comprises i). a CDR1 comprising an amino acid sequence of GRTFSSYA (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LNWSGEST (SEQ ID NO: 29), and a CDR3 comprising an amino acid sequence of AAASSWSRGGVPYGMDY (SEQ ID NO: 30); i). a CDR1 comprising an amino acid sequence of GRSFGDFA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IRWTGDT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of TAGPTMSYSRGGEF (SEQ ID NO: 54); ii). a CDR1 comprising an amino acid sequence of GSIFGGYN (SEQ ID NO: 2189), a CDR2 comprising an amino acid sequence of IFISGNN (SEQ ID NO: 2190), and a CDR3 comprising an amino acid sequence of RAYNDGGEY (SEQ ID NO: 2191); iv). a CDR1 comprising an amino acid sequence of GSNFRILS (SEQ ID NO: 2195), a CDR2 comprising an amino acid sequence of ITSDDTT (SEQ ID NO: 2196), and a CDR3 comprising an amino acid sequence of HSRWYNL (SEQ ID NO: 2197); orAttorney Docket No: 260525.000049 v). a CDR1 comprising an amino acid sequence of GFTFSRYA (SEQ ID NO: 2201), a CDR2 comprising an amino acid sequence of ISSAGGYI (SEQ ID NO: 2202), and a CDR3 comprising an amino acid sequence of FTADY (SEQ ID NO: 2203).

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

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

13. The antigen-binding protein of claim 12, wherein the VHH is a camelid VHH.

14. The antigen-binding protein of claim 13, wherein the VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 4, 9, 14, 17, 21, 26, 31, 36, 41, 45, 50, 55, 65, 70, 75, 79, 84, 89, 93, 98, 146-468, 2192, 2198, 2204, and 2214-2233, or an amino acid sequence having at least 75% identity thereto.

15. The antigen-binding protein of claim 13 or 14, wherein the VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 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 thereto.

16. The antigen-binding protein of any one of claims 13-15, wherein the VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 31, 55, 2192, 2198, and 2204, or an amino acid sequence having at least 75% identity thereto.

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 NOs: 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, 2234-2251, and 2354-2404, or an amino acid sequence having at least 75% identity thereto.Attorney Docket No: 260525.000049 19. The antigen-binding protein of claim 17 or 18, wherein the humanized VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 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 2354-2404, or an amino acid sequence having at least 75% identity thereto.

20. The antigen-binding protein of any one of claims 17-19, wherein the humanized VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 32, 56, 2200, 2206, 2194, and 2354- 2404, or an amino acid sequence having at least 75% identity thereto.

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

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

23. The antigen-binding protein of claim 22, wherein the antigen-binding protein binds to human TRAILR2 with a K of about −10 −8 D 1×10 to 5×10 M.

24. The antigen-binding protein of any one of claims 1-23, wherein the antigen-binding protein binds to cyno TRAILR2.

25. The antigen-binding protein of claim 24, wherein the antigen-binding protein binds to cyno TRAILR2 with a K −7 D of less than about 3×10 M.

26. The antigen-binding protein of claim 25, wherein the antigen-binding protein binds to cyno TRAILR2 with a K of about 1×10−9 t −7 D o 1×10 M.

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

28. The antigen-binding protein of any one of claims 1-26, wherein the antigen-binding protein blocks binding of TRAIL to TRAILR2.Attorney Docket No: 260525.000049 29. The antigen-binding protein of any one of claims 1-28, wherein the antigen-binding protein does not bind specificaly to TRAILR1, TRAILR3 and / or TRAILR4.

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

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

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

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

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

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

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

37. The fusion protein of claim 36, which comprises three or more said antigen-binding proteins.

38. The fusion protein of claim 37, which comprises four said antigen-binding proteins.

39. The fusion protein of any one of claims 36-38, wherein the one or more antigen-binding proteins bind to the same epitope on TRAILR2.Attorney Docket No: 260525.000049 40. The fusion protein of any one of claims 36-38, wherein the one or more antigen-binding proteins bind to diferent epitopes on TRAILR2.

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

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

43. The fusion protein of any one of claims 36-42, which further comprises an immunoglobulin Fc region.

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

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

47. The fusion protein of claim 46, wherein the Fc region of human IgG1 comprises one or more mutations selected from 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) according to EU numbering.

48. The fusion protein of claim 47, wherein the Fc region of human IgG1 comprises a set of mutations selected from 1). L234A and L235A;Attorney Docket No: 260525.000049 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 an Fc region of human IgG4, or a variant thereof.

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

51. The fusion protein of claim 50, wherein the Fc region of human IgG4 comprises a set of mutations selected from 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 of any one of claims 36-51, which further comprises a moiety that binds to serum albumin.

53. The fusion protein of claim 36, which comprises the amino acid sequence of any one of SEQ ID Nos: 2054-2070, or a sequence having at least 75% identity thereto.Attorney Docket No: 260525.000049 54. The fusion protein of any one of claims 36-53, wherein the fusion protein has an agonist efect upon binding to TRAILR2.

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

56. The conjugate of claim 55, wherein the second moiety is selected from a detectable label, a drug, a toxin, a radionuclide, an enzyme, an immunomodulatory agent, a cytokine, a cytotoxic agent, a chemotherapeutic agent, a diagnostic agent, or a combination thereof.

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

58. The polynucleotide molecule of claim 57, which comprises the nucleotide sequence of any one of SEQ ID NOs: 57-61, 131-145, 1731-2053, 2071-2087, 2193, 2199, 2205, and 2278-2297, or a nucleotide sequence having at least 70% identity thereto.

59. The polynucleotide molecule of claim 57 or 58, which comprises the nucleotide sequence of any one of SEQ ID NOs: 57-61, 131-145, 2071-2087, 2193, 2199, and 2205, or a nucleotide sequence having at least 70% identity thereto.

60. The polynucleotide molecule of any one of claims 57-59, which comprises the nucleotide sequence of any one of SEQ ID NOs: 132, 137, 2193, 2199, and 2205, or a nucleotide sequence having at least 70% identity thereto.

61. A recombinant vector comprising the polynucleotide molecule of any one of claims 57-60.

62. A host cel comprising polynucleotide molecule of any one of claims 57-60, or the recombinant vector of claim 61.Attorney Docket No: 260525.000049 63. A kit comprising the antigen-binding protein of any one of claims 1-35, the fusion protein of any one of claims 36-54, or the conjugate of any one of claims 55 -56, the polynucleotide molecule of any one of claims 57-60, or the recombinant vector of claim 61, and optionaly, instructions and / or packaging for the same.

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

65. A method for preparing an antigen-binding protein or a fusion protein that specificaly binds TNF- related apoptosis-inducing ligand receptor 2 (TRAILR2), comprising the steps of: (a) culturing the host cel of claim 62 in a culture medium under conditions suitable for expression of the antigen-binding protein or fusion protein, and (b) isolating the antigen-binding protein or fusion protein from the host cel and / or culture medium.

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

67. The method of claim 66, wherein said contacting occurs in vitro.

68. The method of claim 66, wherein said contacting occurs in vivo.

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

70. The method of any one of claims 66-69, wherein the cel expressing TRAILR2 is a cel of a cancer.

71. The method of claim 70, wherein the cancer is a solid tumor.Attorney Docket No: 260525.000049 72. The method of claim 70, wherein the cancer is selected from adenoid cystic carcinoma, adrenal gland tumor, amyloidosis, anal cancer, appendix cancer, astrocytoma, ataxia-telangiectasia, Beckwith-Wiedemann syndrome, bile duct cancer (cholangiocarcinoma), Birt-Hogg-Dubé syndrome, bladder cancer, bone cancer (sarcoma of bone), brain stem glioma, brain tumor, breast cancer, inflammatory breast cancer, metastatic breast cancer, male breast cancer, Carney complex, central nervous system tumors (brain and spinal cord), cervical cancer, childhood cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, desmoid tumor, desmoplastic infantile ganglioglioma, childhood tumor, ependymoma, esophageal cancer, Ewing sarcoma, eye cancer, eyelid cancer, familial adenomatous polyposis, familial GIST, familial malignant melanoma, familial pancreatic cancer, galbladder cancer, gastrointestinal stromal tumor (GIST), germ cel tumor, gestational trophoblastic disease, head and neck cancer, hereditary breast and ovarian cancer, hereditary difuse gastric cancer, hereditary leiomyomatosis and renal cel cancer, hereditary mixed polyposis syndrome, hereditary pancreatitis, hereditary papilary renal carcinoma, HIV / AIDS-related cancer, juvenile polyposis syndrome, kidney cancer, lacrimal gland tumor, laryngeal and hypopharyngeal cancer, Li-Fraumeni syndrome, liver cancer, lung cancer, non-smal cel lung cancer, smal cel lung cancer, lynch syndrome, mastocytosis, meduloblastoma, melanoma, meningioma, mesothelioma, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, MUTYH (or MYH)-associated polyposis, myelodysplastic syndromes (MDS), nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine tumor of the gastrointestinal tract, neuroendocrine tumor of the lung, neuroendocrine tumor of the pancreas, neuroendocrine tumors, neurofibromatosis type 1, neurofibromatosis type 2, nevoid basal cel carcinoma syndrome, oral and oropharyngeal cancer, osteosarcoma, ovarian, falopian tube, and peritoneal cancer, pancreatic cancer, parathyroid cancer, penile cancer, Peutz-Jeghers syndrome, pheochromocytoma and paraganglioma, pituitary gland tumor, pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Kaposi sarcoma, soft tissue sarcomas, skin cancer (non-melanoma), smal bowel cancer, stomach cancer, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis complex, uterine cancer, vaginal cancer, Von Hippel-Lindau syndrome, vulvar cancer, Werner syndrome, Wilms tumor, or xeroderma pigmentosum.

73. The method of claim 70, wherein the cancer is gastrointestinal cancer, breast cancer, or lung cancer.Attorney Docket No: 260525.000049 74. The method of claim 73, wherein the gastrointestinal cancer is colorectal cancer, gastric cancer, esophageal cancer, pancreatic cancer, or cholangiocarcinoma cancer.

75. The method of any one of claims 66-74, wherein the method further comprises contacting the cel with one or more additional therapeutic agents.

76. A method of treating or preventing a cancer in a subject in need thereof, said method comprising administering to the subject the antigen-binding protein of claim 1-35, the fusion protein of any one of claims 36-54, or the conjugate of any one of claims 55 -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 gland tumor, amyloidosis, anal cancer, appendix cancer, astrocytoma, ataxia-telangiectasia, Beckwith-Wiedemann syndrome, bile duct cancer (cholangiocarcinoma), Birt-Hogg-Dubé syndrome, bladder cancer, bone cancer (sarcoma of bone), brain stem glioma, brain tumor, breast cancer, inflammatory breast cancer, metastatic breast cancer, male breast cancer, Carney complex, central nervous system tumors (brain and spinal cord), cervical cancer, childhood cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, desmoid tumor, desmoplastic infantile ganglioglioma, childhood tumor, ependymoma, esophageal cancer, Ewing sarcoma, eye cancer, eyelid cancer, familial adenomatous polyposis, familial GIST, familial malignant melanoma, familial pancreatic cancer, galbladder cancer, gastrointestinal stromal tumor (GIST), germ cel tumor, gestational trophoblastic disease, head and neck cancer, hereditary breast and ovarian cancer, hereditary difuse gastric cancer, hereditary leiomyomatosis and renal cel cancer, hereditary mixed polyposis syndrome, hereditary pancreatitis, hereditary papilary renal carcinoma, HIV / AIDS-related cancer, juvenile polyposis syndrome, kidney cancer, lacrimal gland tumor, laryngeal and hypopharyngeal cancer, Li-Fraumeni syndrome, liver cancer, lung cancer, non-smal cel lung cancer, smal cel lung cancer, lynch syndrome, mastocytosis, meduloblastoma, melanoma, meningioma, mesothelioma, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, MUTYH (or MYH)-associated polyposis, myelodysplastic syndromes (MDS), nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine tumor of the gastrointestinal tract, neuroendocrine tumor of the lung, neuroendocrine tumor of the pancreas,Attorney Docket No: 260525.000049 neuroendocrine tumors, neurofibromatosis type 1, neurofibromatosis type 2, nevoid basal cel carcinoma syndrome, oral and oropharyngeal cancer, osteosarcoma, ovarian, falopian tube, and peritoneal cancer, pancreatic cancer, parathyroid cancer, penile cancer, Peutz-Jeghers syndrome, pheochromocytoma and paraganglioma, pituitary gland tumor, pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Kaposi sarcoma, soft tissue sarcomas, skin cancer (non-melanoma), smal bowel cancer, stomach cancer, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis complex, uterine cancer, vaginal cancer, Von Hippel-Lindau syndrome, vulvar cancer, Werner syndrome, Wilms tumor, 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 cancer.

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

82. The method of claim 75 or 81, wherein the one or more additional therapeutic agents are selected from a chemotherapeutic agent, a vascular endothelial growth factor (VEGF) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, or an apoptosis-inducing agent, an immunotherapeutic agent, or a combination thereof.

83. The method of claim 82, wherein the chemotherapeutic agent is selected from bleomycin, carboplatin, chlorambucil, cisplatin, colchicine, cyclophosphamide, daunorubicin, doxorubicin or liposomal doxorubicin, mitomycin C, actinomycin, diethylstilbestrol, etoposide, 5-fluorouracil, floxuridine, melphalan, methotrexate, mitomycin, 6-mercaptopurine, teniposide, 6-thioguanine, vincristine and vinblastine, leflunomide, tamoxifen, interferon α-2b, glutamic acid, plicamycin, mercaptopurine, 6-thioguanine, carmustine, BCNU, limousine, CCNU, cytosine arabinose, estramustine, hydroxyurea, procarbazine, busulfan, medroxyprogesterone, estramustine phosphate sodium, ethenyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, testolactone, melphalan, chlorambucil, mechlorethamine, thiourea,Attorney Docket No: 260525.000049 bethamethasone sodium phosphate, dicarbazine, asparagine, mitotane, vincristine sulfate, vinblastine sulfate, FOLFOX (folinic acid, 5-fluorouracil and oxaliplatin) or FOLFIRI (folinic acid, 5- fluorouracil, and irinotecan), and a combination thereof.

84. The method of claim 82, wherein the VEGF inhibitor is bevacizumab, ramucirumab, regorafenib, ziv- 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-inducing agent is selected from a B-cel lymphoma 2 (BCL2) inhibitor, a BCL-extra large (BCL-XL) inhibitor, or an inhibitor of apoptosis proteins (IAP) inhibitor, or a combination thereof.

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

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

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

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