Trispecific and / or trivalent binding proteins using the crossover dual variable domain (CODV) format for the treatment of HIV infection - Patent Application 20070123633

Abstract

Using the crossover dual variable domain (CODv) format, the present disclosure relates to compositions comprising trispecific and / or trivalent binding proteins comprising four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first pair of polypeptides forming the binding protein have dual variable domains with a crossover orientation and a second pair of polypeptides have a single variable domain. Also provided herein are methods of making trispecific and / or trivalent binding proteins and uses of such binding proteins for the treatment and / or prevention of HIV / AIDS.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 831,415, filed April 9, 2019, and European Patent Application No. EP19306312.0, filed October 8, 2019, the disclosures of each of which are incorporated herein by reference in their entireties.

[0002] Submitting a sequence listing as an ASCII text file The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 183952031940SEQLIST.TXT, Recorded: March 25, 2020, Size: 580KB).

[0003] Field The present disclosure relates to trispecific and / or trivalent binding proteins comprising four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first pair of polypeptides forming the binding protein has dual variable domains with a crossover orientation. The present disclosure also relates to methods of making trispecific and / or trivalent binding proteins and to the use of such binding proteins for treating and / or preventing HIV / AIDS. [Background technology]

[0004] Antiretroviral therapy (ART) has been the standard treatment for HIV / AIDS patients in the past decades. ART drugs target internal proteins, such as reverse transcriptase (RT), integrase (IN), and viral protease (PI), by inhibiting the proteolytic cleavage of protein precursors required for reverse transcription of the HIV-1 genome, integration of the HIV-1 genome, and production of infectious viral particles. Treatment with ART or a combination of different classes of ART results in the inhibition of HIV-1 replication and a subsequent reduction of viremia, often to undetectable levels (non-viremic state). Although ART greatly aids HIV patients in controlling disease progression and containing the global HIV spread, it requires patients to take drugs on a daily basis, often according to a strict regimen. Approximately 10% of patients fail therapy each year due to drug toxicity, suboptimal adherence, and emerging drug resistance. As more HIV patients are able to live normal life spans (>80 years), chronic complications such as aging and drug-drug interactions, as well as cardiovascular / renal / bone toxicity, become particularly problematic. The economic burden of treating HIV / AIDS has not subsided.

[0005] HIV latently infects long-lived resting memory CD4+ T cells and others in the form of proviral DNA integrated into the host genome. Latently infected cells survive for decades, self-renew like stem cells through homeostatic proliferation, and are considered HIV-1 reservoirs. HIV-1 reservoirs do not express viral proteins and are therefore unaffected by ART or the host immune system. However, a low percentage of cells in the reservoir randomly reactivate by unknown mechanisms, which is responsible for recurrent viremia when ART is stopped.

[0006] Thus, there is a need to develop HIV / AIDS treatments to target HIV-1 reservoirs and ultimately eliminate them completely to achieve a cure for HIV, or long-term remission without any further treatment. Any therapeutic strategy to eliminate the HIV-1 reservoir requires first activating the reservoir and then eliminating the activated HIV-1 reservoir cells.

[0007] All references referred to in this specification, including patent applications, patent publications, and UniProtKB / Swiss-Prot accession numbers, are incorporated herein by reference in their entirety to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference. Summary of the Invention [Problem to be solved by the invention]

[0008] To meet these and other needs, multispecific binding proteins (e.g., antibodies) that form three antigen-binding sites are provided herein. In some embodiments, the binding proteins bind one or more HIV target proteins and CD3 polypeptides. In some embodiments, the binding proteins bind HIV target proteins, CD28 polypeptides, and CD3 polypeptides. The concept of trispecific anti-HIV / CD28×CD3 T cell engagers (TCEs) disclosed herein is believed to be an effective eliminator of the HIV-1 reservoir through activation by anti-CD3, co-activation by anti-CD28, and subsequent killing of activated HIV-1 reservoir cells through anti-HIV / anti-CD28 by engaging activated CD8 T cells, providing a potential strategy to attack the HIV-1 reservoir. Additionally, anti-CD3 binding sites are described with high affinity binding to human CD3 polypeptides and potential manufacturing disadvantages removed (e.g., deamidation sites).

[0009] In some embodiments, a binding protein comprises four polypeptide chains that form three antigen binding sites that specifically bind to one or more HIV target proteins, wherein a first polypeptide chain has the formula: V L2 -L 1 -V L1 -L 2 -C L [I] The structure includes: The second polypeptide chain has the formula: V H1 -L 3 -V H2 -L 4 -C H1 - Hinge-C H2 -C H3 [II] The structure includes: The third polypeptide chain has the formula: V H3 -C H1 - Hinge-C H2 -C H3 [III] The structure includes: The fourth polypeptide chain has the formula: V L3 -C L [IV] The structure includes: Where: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V L3 is a third immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; V H3 is a third immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; CH1 is immunoglobulin C H1 a heavy chain constant domain; C H2 is immunoglobulin C H2 a heavy chain constant domain; C H3 is immunoglobulin C H3 a heavy chain constant domain; The hinge is C H1 and C H2 an immunoglobulin hinge region connecting the domains; L 1 , L 2 , L 3 and L 4 is an amino acid linker; a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair; V H1 and V L1 forms a first antigen-binding site; V H2 and V L2 forms a second antigen-binding site that binds to the CD3 polypeptide, and V H2 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 22), L2 The domain is QSLVHX 1 NX 2 X 3 TY where X 1 is E or Q, and X 2 is A or L, and X 3 QSLVHX is Q, R, or F 1 NX 2 X 3 comprising a CDR-L1 sequence comprising the amino acid sequence of TY (SEQ ID NO: 293), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 30); V H3 and V L3forms a third antigen-binding site that binds to the HIV target protein, Said binding proteins are provided herein.

[0010] In some embodiments, the first binding site binds to a CD28 polypeptide (e.g., a human CD28 polypeptide). H1 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO: 33), L1 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of KAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO: 36). H1 The domain is and / or comprising the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWGKGTTVTVSS (SEQ ID NO: 59); L1 The domain is It comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO: 60).

[0011] In some embodiments, V L2 The CDR-L1 sequence of the domain comprises an amino acid sequence selected from the group consisting of QSLVHQNAQTY (SEQ ID NO: 24), QSLVHENLQTY (SEQ ID NO: 25), QSLVHENLFTY (SEQ ID NO: 26), and QSLVHENLRTY (SEQ ID NO: 27). H2The domain comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 22); L2 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHQNAQTY (SEQ ID NO: 24), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 30). H2 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 22); L2 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLQTY (SEQ ID NO: 25), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 30). H2 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and L2 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLFTY (SEQ ID NO: 26), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 30). H2 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 21), and an amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 22); and L2The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLRTY (SEQ ID NO: 27), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 29), and an amino acid sequence of GQGTQYPFT (SEQ ID NO: 30). H2 The domain is and / or comprising the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO:52); L2 The domain is DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 54), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 55), DIVMTQTPLSLS VTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:56), and DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:57). In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:54. In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 52, and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 55. In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 52, and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 56. In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 52, and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 57.

[0012] In some embodiments, the third antigen-binding site binds to an HIV target protein selected from the group consisting of glycoprotein 120, glycoprotein 41, and glycoprotein 160. H3 The domain includes a CDR-H1 sequence including the amino acid sequence of NCPIN (SEQ ID NO: 1), a CDR-H2 sequence including the amino acid sequence of WMKPRHGAVSYARQLQG (SEQ ID NO: 2), and a CDR-H3 sequence including the amino acid sequence of GKYCTARDYYNWDFEH (SEQ ID NO: 3), L3 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of RTSQYGSLA (SEQ ID NO: 4), a CDR-L2 sequence comprising the amino acid sequence of SGSTRAA (SEQ ID NO: 5), and a CDR-L3 sequence comprising the amino acid sequence of QQYEF (SEQ ID NO: 6). H3 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTAHI (SEQ ID NO: 7), a CDR-H2 sequence comprising the amino acid sequence of IKPQYGAV (SEQ ID NO: 8) or IKPQYGAT (SEQ ID NO: 9), and a CDR-H3 sequence comprising the amino acid sequence of DRSYGDSSWALDA (SEQ ID NO: 10), L3 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGVGSD (SEQ ID NO: 11), a CDR-L2 sequence comprising the amino acid sequence of HTS (SEQ ID NO: 12), and a CDR-L3 sequence comprising the amino acid sequence of CQVLQF (SEQ ID NO: 13). H3 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of DCTLN (SEQ ID NO: 14), a CDR-H2 sequence comprising the amino acid sequence of WLKPRWGAVNYARPLQG (SEQ ID NO: 15), and a CDR-H3 sequence comprising the amino acid sequence of GKNCDYNWDFEH (SEQ ID NO: 16), L3 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of RTSQYGSLA (SEQ ID NO: 17), a CDR-L2 sequence comprising the amino acid sequence of SGSTRAA (SEQ ID NO: 18), and a CDR-L3 sequence comprising the amino acid sequence of QQYEF (SEQ ID NO: 19). H3 The domain is and / or comprising the amino acid sequence of QVRLSQSGGQMKKPGDSMRISCRASGYEFINCPINWIRLAPGKRPEWMGWMKPRHGAVSYARQLQGRVTMTRDMYSETAFLELRSLTSDDTAVYFCTRGKYCTARDYYNWDFEHWGQGTPVTVSS (SEQ ID NO: 43); L3 The domain comprises the amino acid sequence of SLTQSPGTLSLSPGETAIISCRTSQYGSLAWYQQRPGQAPRLVIYSGSTRAAGIPDRFSGSRWGPDYNLTISNLESGDFGVYYCQQYEFFGQGTKVQVDIK (SEQ ID NO: 45). H3 The domain is and / or comprising the amino acid sequence of QVRLSQSGGQMKKPGDSMRISCRASGYEFINCPINWIRLAPGKRPEWMGWMKPRHGAVSYARQLQGRVTMTRQLSQDPDDPDWGTAFLELRSLTSDDTAVYFCTRGKYCTARDYYNWDFEHWGQGTPVTVSS (SEQ ID NO: 44); L3 The domain comprises the amino acid sequence of SLTQSPGTLSLSPGETAIISCRTSQYGSLAWYQQRPGQAPRLVIYSGSTRAAGIPDRFSGSRWGPDYNLTISNLESGDFGVYYCQQYEFFGQGTKVQVDIK (SEQ ID NO: 45). H3 The domain is and / or comprising the amino acid sequence of: RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQYGAVNFGGGFRDRVTLTRDVYREIAYMDIRGLKPDDTAVYYCARDRSYGDSSWALDAWGQGTTVVVSA (SEQ ID NO: 46); L3 The domain comprises the amino acid sequence of YIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGVPSRFSGSGFHTSFNLTISDLQADDIATYYCQVLQFFGRGSRLHIK (SEQ ID NO: 49). H3 The domain is and / or comprising the amino acid sequence of: RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQYGATNFGGGFRDRVTLTRDVYREIAYMDIRGLKPDDTAVYYCARDRSYGDSSWALDAWGQGTTVVVSA (SEQ ID NO: 47); L3 The domain comprises the amino acid sequence of YIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGVPSRFSGSGFHTSFNLTISDLQADDIATYYCQVLQFFGRGSRLHIK (SEQ ID NO: 49). H3 The domain is and / or comprising the amino acid sequence of: RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQYGAVNFGGGFRDRVTLTRQLSQDPDDPDWGIAYMDIRGLKPDDTAVYYCARDRSYGDSSWALDAWGQGTTVVVSA (SEQ ID NO: 48); L3 The domain comprises the amino acid sequence of YIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGVPSRFSGSGFHTSFNLTISDLQADDIATYYCQVLQFFGRGSRLHIK (SEQ ID NO: 49). H3 The domain is and / or comprising the amino acid sequence of QVQLVQSGGQMKKPGESMRISCRASGYEFIDCTLNWIRLAPGKRPEWMGWLKPRWGAVNYARPLQGRVTMTRQLSQDPDDPDWGTAFLELRSLTVDDTAVYFCTRGKNCDYNWDFEHWGRGTPVIVSS (SEQ ID NO:50); L3 The domain comprises the amino acid sequence LTQSPGTLSLSPGETAIISCRTSQYGSLAWYQQRPGQAPRLVIYSGSTRAAGIPDRFSGSRWGPDYNLTISNLESGDFGVYYCQQYEFFGQGTKVQVDIK (SEQ ID NO:51).

[0013] In some embodiments that may be combined with any other embodiments described herein, L 1 , L 2 , L 3 or L 4 In some embodiments, at least one of L 1 , L 2 , L 3 and L 4 are each independently 0 amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 40), GGGSGGGGSGGGGS (SEQ ID NO: 41), S, RT, TKGPS (SEQ ID NO: 39), GQPKAAP (SEQ ID NO: 38), and GGSGSSGSGG (SEQ ID NO: 42). 1 , L 2 , L 3 and L 4 each independently comprises a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 40), GGGSGGGGSGGGGS (SEQ ID NO: 41), S, RT, TKGPS (SEQ ID NO: 39), GQPKAAP (SEQ ID NO: 38), and GGSGSSGSGG (SEQ ID NO: 42). 1 contains the sequence GQPKAAP (SEQ ID NO: 38), 2 contains the sequence TKGPS (SEQ ID NO:39), 3 contains a sequence S, and L 4 In some embodiments, L comprises the sequence RT. 1 , L 2 , L 3 or L 4 At least one of the L 1 , L 2 , L 3 and L 4 contains the sequence DKTHT (SEQ ID NO:37).

[0014] In some embodiments that may be combined with any other embodiments described herein, the hinge-C of the second and third polypeptide chains H2 -C H3Domain is human IgG4 hinge-C H2 -C H3 domain, hinge-C H2 -C H3 In some embodiments, the domains each comprise an amino acid substitution at a position corresponding to positions 234 and 235 of human IgG4 according to the EU index, the amino acid substitutions being F234A and L235A. H2 -C H3 Domain is human IgG4 hinge-C H2 -C H3 domain, hinge-C H2 -C H3 Each domain comprises an amino acid substitution at a position corresponding to 233-236 of human IgG4 according to the EU index, the amino acid substitutions being E233P, F234V, L235A, and a deletion at 236. In some embodiments, the hinge-C of the second and third polypeptide chains H2 -C H3 Domain is human IgG4 hinge-C H2 -C H3 domain, hinge-C H2 -C H3 In some embodiments, the domains each comprise an amino acid substitution at a position corresponding to positions 228 and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P and R409K. H2 -C H3 Domain is human IgG1 hinge-C H2 -C H3 domain, hinge-C H2 -C H3 In some embodiments, the domains comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, and the amino acid substitutions are L234A, L235A, and P329A. H2 -C H3 Domain is human IgG1 hinge-C H2 -C H3domain, hinge-C H2 -C H3 The domains each comprise an amino acid substitution at a position corresponding to positions 298, 299, and 300 of human IgG1 according to the EU index, the amino acid substitutions being S298N, T299A, and Y300S. H2 -C H3 The domain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being Y349C, T366S, L368A, and Y407V; the hinge-C of the third polypeptide chain H2 -C H3 In some embodiments, the domain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being S354C and T366W. H2 -C H3 The domain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being S354C and T366W; the hinge-C of the third polypeptide chain H2 -C H3 The domain contains amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being Y349C, T366S, L368A, and Y407V.

[0015] In some embodiments, the first polypeptide chain comprises an amino acid sequence of SEQ ID NO:61 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:61; the second polypeptide chain comprises an amino acid sequence of SEQ ID NO:62 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:62; the third polypeptide chain comprises an amino acid sequence of SEQ ID NO:63 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:63; the fourth polypeptide chain comprises an amino acid sequence of SEQ ID NO:64 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:64. In some embodiments, the first polypeptide chain comprises an amino acid sequence of SEQ ID NO:65 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:65; the second polypeptide chain comprises an amino acid sequence of SEQ ID NO:66 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:66; the third polypeptide chain comprises an amino acid sequence of SEQ ID NO:67 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:67; the fourth polypeptide chain comprises an amino acid sequence of SEQ ID NO:68 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:68. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:69 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:69; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:70 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:70; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:71 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:71; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:72 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:72.In some embodiments, the first polypeptide chain comprises an amino acid sequence of SEQ ID NO:73 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:73; the second polypeptide chain comprises an amino acid sequence of SEQ ID NO:74 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:74; the third polypeptide chain comprises an amino acid sequence of SEQ ID NO:75 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:75; the fourth polypeptide chain comprises an amino acid sequence of SEQ ID NO:76 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:76. In some embodiments, the first polypeptide chain comprises an amino acid sequence of SEQ ID NO:77 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:77; the second polypeptide chain comprises an amino acid sequence of SEQ ID NO:78 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:78; the third polypeptide chain comprises an amino acid sequence of SEQ ID NO:79 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:79; the fourth polypeptide chain comprises an amino acid sequence of SEQ ID NO:80 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:80. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:81 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:81; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:82 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:82; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:83 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:83; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:84 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:84.In some embodiments, the first polypeptide chain comprises an amino acid sequence of SEQ ID NO:85 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:85; the second polypeptide chain comprises an amino acid sequence of SEQ ID NO:86 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:86; the third polypeptide chain comprises an amino acid sequence of SEQ ID NO:87 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:87; the fourth polypeptide chain comprises an amino acid sequence of SEQ ID NO:88 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:88. In some embodiments, the first polypeptide chain comprises an amino acid sequence of SEQ ID NO:89 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:89; the second polypeptide chain comprises an amino acid sequence of SEQ ID NO:90 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:90; the third polypeptide chain comprises an amino acid sequence of SEQ ID NO:91 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:91; the fourth polypeptide chain comprises an amino acid sequence of SEQ ID NO:92 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:92. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:93 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:93; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:94 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:94; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:95 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:95; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:96 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:96.In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:97 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:97; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:98 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:98; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:99 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:99; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:100 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:100. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:101 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:101; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:102 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:102; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:103 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:103; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:104 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:104. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:105 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:105; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:106 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:106; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:107 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:107; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:108 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:108. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:109 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:109; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:110 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:110; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:111 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:111; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:112 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:112.In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:113 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:113; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:114 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:114; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:115 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:115; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:116 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:116. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:117 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:117; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:118 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:118; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:119 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:119; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:120 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:120. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:121 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:121; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:122 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:122; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:123 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:123; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:124 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:124.In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:129 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:129; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:130 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:130; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:131 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:131; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:132 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:132. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:133 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:133; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:134 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:134; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:135 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:135; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:136 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:136. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:137 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:137; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:138 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:138; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:139 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:139; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:140 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:140. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:141 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:141; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:142 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:142; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:143 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:143; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:144 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:144.In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:145 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:145; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:146 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:146; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:147 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:147; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:148 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:148. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:149 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:149; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:150 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:150; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:151 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:151; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:152 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:152. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 153 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 153; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 154 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 154; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 155 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 155; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 156 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 156.In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:157 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:157; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:158 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:158; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:159 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:159; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:160 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:160. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:161 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:161; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:162 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:162; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:163 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:163; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:164 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:164. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:165 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:165; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:166 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:166; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:167 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:167; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:168 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:168. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 169 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 169; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 170 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 170; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 171 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 171; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 172 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 172. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 173 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 173; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 174 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 174; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 175 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 175; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 176 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 176.

[0016] In some embodiments, provided herein is an isolated nucleic acid molecule comprising the nucleotide sequence encoding the binding protein of any one of the above embodiments.In some embodiments, provided herein is an expression vector comprising the nucleic acid molecule of any one of the above embodiments.In some embodiments, provided herein is an isolated host cell comprising the nucleic acid molecule of any one of the above embodiments or the expression vector of any one of the above embodiments.In some embodiments, the host cell is a mammalian or insect cell.

[0017] In some embodiments, provided herein is a pharmaceutical composition comprising a binding protein of any one of the above embodiments and a pharma- ceutically acceptable carrier.

[0018] In some embodiments, provided herein is a method for preventing and / or treating HIV infection in a patient, comprising administering to the patient a therapeutically effective amount of at least one of the binding proteins of any one of the above embodiments or the pharmaceutical composition of any one of the above embodiments. In some embodiments, the binding protein is administered in combination with standard antiretroviral therapy. In some embodiments, administration of at least one binding protein results in the elimination of one or more latent and / or chronically HIV-infected cells in the patient. In some embodiments, the patient is a human.

[0019] In some embodiments, the binding protein or pharmaceutical composition of any one of the above embodiments is provided for the prevention and / or treatment of HIV infection in a patient. In some embodiments, the binding protein is administered in combination with standard antiretroviral therapy. In some embodiments, the binding protein causes elimination of one or more latent and / or chronically HIV-infected cells in the patient. In some embodiments, the patient is a human.

[0020] In some embodiments, the binding protein or pharmaceutical composition of any one of the above embodiments is provided for use in the manufacture of a medicament for the prevention and / or treatment of HIV infection in a patient. In some embodiments, the binding protein is administered in combination with standard antiretroviral therapy. In some embodiments, the binding protein causes elimination of one or more latent and / or chronically HIV-infected cells in the patient. In some embodiments, the patient is a human.

[0021] In some embodiments, a vector system is provided herein, comprising one or more vectors encoding the first, second, third and fourth polypeptide chains of the binding protein of any one of the above embodiments. In some embodiments, the vector system comprises a first vector encoding the first polypeptide chain of the binding protein, a second vector encoding the second polypeptide chain of the binding protein, a third vector encoding the third polypeptide chain of the binding protein, and a fourth vector encoding the fourth polypeptide chain of the binding protein.

[0022] In some embodiments, a kit is provided herein, comprising one, two, three, or four polypeptide chains of a binding protein according to any one of the above embodiments. In some embodiments, the kit further comprises instructions for using the polypeptide chains or binding protein according to any of the methods or uses described herein, e.g., above.

[0023] In some embodiments, a kit is provided herein, comprising one, two, three or four polynucleotides according to any one of the above embodiments.In some embodiments, a kit of polynucleotides, comprising: (a) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 177, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 178, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 179, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 180; (b) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 181, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 182, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 183, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 184; (c) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 185, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 186, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 187, and a polynucleotide sequence of SEQ ID NO: 188. (d) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:189, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:190, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:191, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:192; (e) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:193, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:194, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:195, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:196; (f) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:197, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:198, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:199, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:200;(g) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:201, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:202, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:203, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:204; (h) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:205, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:206, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:207, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:208; (i) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:209, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:210, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:211, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:212; (j) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:213, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:214, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:215, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:216; (k) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:217, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:218, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:219, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:220; (l) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:221, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:222, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:223, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:224; (m) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:225, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:226, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:227, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:228;(n) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:229, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:230, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:231, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:232; (o) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:233, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:234, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:235, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:236; (p) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:237, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:238, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:239, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:240; (q) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:241, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:242, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:243, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:244; (r) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:245, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:246, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:247, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:248; (s) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:249, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:250, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:251, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:252; (t) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:253, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:254, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:255, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:256;(u) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:257, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:258, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:259, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:260; (v) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:261, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:262, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:263, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:264; (w) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:265, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:266, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:267, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:268; (x) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:269, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:270, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:271, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:272; (y) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:273, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:274, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:275, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:276; (z) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:277, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:278, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:279, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:280; (aa) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:281, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:282, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:283, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:284;Provided herein is a kit comprising one, two, three, or four of the following polynucleotides: (bb) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:285, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:286, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:287, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:288; or (cc) a kit of polynucleotides comprising a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:289, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:290, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:291, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:292. In some embodiments, the first, second, third, and fourth polynucleotides are present on one or more expression vectors, e.g., one, two, three, or four expression vectors.;

[0024] It should be understood that one, some, or all of the characteristics of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the present invention will be apparent to those skilled in the art. [Brief description of the drawings]

[0025] [Figure 1] FIG. 1 provides a schematic diagram of a trispecific binding protein that includes four polypeptide chains that form three antigen-binding sites that bind to three target proteins: CD28, CD3, and HIV Env. The first pair of polypeptides has dual variable domains (VH1-VH2 and VL2-VL1) with a crossover orientation that forms two antigen-binding sites (VH1 and VL1; VH2 and VL2) that recognize CD28 and CD3, respectively, and the second pair of polypeptides has a single variable domain (VH3 and VL3) that forms a single antigen-binding site that recognizes HIV Env. The trispecific binding protein shown in FIG. 1 uses a constant region with a "knob-into-hole" mutation, with the knob on the second pair of polypeptides having a single variable domain. [Diagram 2] FIG. 2 shows a schematic representation of the trispecific T cell engager (TCE) strategy for targeting and eliminating the HIV reservoir using the anti-HIV trispecific binding protein shown in FIG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present disclosure provides trispecific and / or trivalent binding proteins comprising four polypeptide chains forming three antigen-binding sites that specifically bind to one or more human immunodeficiency virus (HIV) target proteins and / or one or more T cell receptor target proteins, wherein a first pair of polypeptides forming the binding protein have dual variable domains with a crossover orientation and a second pair of polypeptides have a single variable domain.

[0027] The following description sets forth example methods, parameters, etc. However, it should be recognized that such description is not intended as a limitation on the scope of the present disclosure, but is provided as a description of example embodiments.

[0028] general definition As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings: Unless otherwise required by context, singular terms include pluralities and plural terms include the singular.

[0029] It is understood that the aspects and embodiments of the disclosure described herein include aspects and embodiments that "comprise," "consist," and "consist essentially of."

[0030] The term "polynucleotide" as used herein refers to a single-stranded or double-stranded nucleic acid polymer of at least 10 nucleotides in length. In certain embodiments, the nucleotides that make up a polynucleotide can be ribonucleotides or deoxyribonucleotides or modified forms of any type of nucleotide. Such modifications can include base modifications such as bromouridine, ribose modifications such as arabinoside and 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoroaniladate, and phosphoroamidate. The term "polynucleotide" specifically includes single-stranded and double-stranded forms of DNA.

[0031] An "isolated polynucleotide" is a polynucleotide of genomic, cDNA, or synthetic origin, or any combination thereof, which (1) is not associated with all or a portion of a polynucleotide with which the isolated polynucleotide is found in nature, (2) is linked to a polynucleotide with which it is not linked in nature, or (3) is not found in nature as part of a larger sequence.

[0032] An "isolated polypeptide" is a polypeptide that is (1) free from at least some other polypeptides with which it is normally found, (2) essentially free from other polypeptides from the same source, e.g., the same species, (3) expressed by cells from a different species, (4) separated from at least about 50 percent of the polynucleotides, lipids, carbohydrates, or other materials with which it is naturally associated, (5) not associated (by covalent or non-covalent interactions) with portions of the polypeptide with which it is naturally associated, (6) operably associated (by covalent or non-covalent interactions) with polypeptides with which it is not naturally associated, or (7) not naturally occurring. Such isolated polypeptides can be encoded by genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination of these. Preferably, an isolated polypeptide is substantially free from polypeptides or other contaminants found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research, or otherwise).

[0033] Naturally occurring antibodies typically comprise tetramers. Each such tetramer is typically composed of two identical pairs of polypeptide chains, with each pair having one full-length "light" chain (typically having a molecular weight of about 25 kDa) and one full-length "heavy" chain (typically having a molecular weight of about 50-70 kDa). The terms "heavy chain" and "light chain", as used herein, refer to any immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen. The amino-terminal portion of each light and heavy chain typically contains a variable domain of about 100-110 or more amino acids that are typically responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant domain that is responsible for effector function. Thus, in naturally occurring antibodies, the full-length heavy chain immunoglobulin polypeptide contains a variable domain (V H ) and three constant domains (C H1 , C H2 , and C H3), including V H The domain is located at the amino terminus of the polypeptide and is C H3 The domains are located at the carboxyl terminus, and the full-length light chain immunoglobulin polypeptide contains the variable domain (V L ) and the constant domain (C L ), including V L The domain is located at the amino terminus of the polypeptide and is C L The domain is at the carboxyl terminus.

[0034] Human light chains are typically classified as kappa and lambda light chains, and human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, defining the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including, but not limited to, IgM1 and IgM2. IgA is similarly further divided into subclasses, including, but not limited to, IgA1 and IgA2. Within full-length light and heavy chains, the variable and constant domains are typically joined by a "J" region of about 12 or more amino acids, and heavy chains also include a "D" region of about 10 additional amino acids. See, for example, FUNDAMENTAL IMMUNOLOGY (Paul, W., ed., Raven Press, 2nd ed., 1989) (incorporated in its entirety by reference for all purposes). The variable regions of each light / heavy chain pair typically form an antigen-binding site. The variable domains of naturally occurring antibodies typically exhibit the same overall structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are typically aligned by the framework regions, which may allow binding to a specific epitope. From the amino terminus to the carboxyl terminus, the variable domains of both the light and heavy chains typically include the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0035] The term "CDR set" refers to a group of three CDRs present in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries that define the three CDRs. These CDRs can be referred to as Kabat CDRs. Chothia and coworkers (Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17; Chothia et al., 1989, Nature 342:877-83) have described the Kabat CDRs as It has been found that certain sub-portions within the CDRs adopt nearly identical peptide backbone conformations despite the large diversity at the amino acid sequence level. These sub-portions are designated L1, L2, and L3 or H1, H2, and H3, with "L" and "H" referring to the light and heavy chain regions, respectively. These regions can be referred to as Chothia CDRs, and have boundaries that overlap with the Kabat CDRs. Other boundaries that define CDRs that overlap with the Kabat CDRs are described by Padlan, 1995, FASEB J. 9:133-39; MacCallum, 1996, J. Mol. Biol. 262(5):732-45; and Lefranc, 2003, Dev. Comp. Immunol. 27:55-77. Still other CDR boundary definitions may not strictly follow one of the systems herein, but nevertheless follow the Kabat CDRs. They overlap the CDRs, but may be shortened or lengthened in light of prediction or experimental findings that certain residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding.The methods used herein can utilize CDRs defined according to any of these systems, although certain embodiments use Kabat or Chothia-defined CDRs. Identification of predicted CDRs using amino acid sequences is well known in the art, such as in Martin, AC, "Protein sequence and structure analysis of antibody variable domains," Antibody Engineering, Vol. 2. Kontermann R., Dubel S., eds., Springer-Verlag, Berlin, pp. 33-51 (2010). Other conventional methods can be used to inspect the amino acid sequences of heavy and / or light chain variable domains to identify sequences of CDRs, for example, by comparing known amino acid sequences of other heavy and light chain variable regions to determine regions of sequence hypervariability. Numbered sequences can be aligned by eye or by using an alignment program, such as one of the programs in the CLUSTAL suite, as described in Thompson, 1994, Nucleic Acids Res. 22:4673-80. Molecular models are conventionally used to accurately delineate framework and CDR regions and therefore accurate sequence-based assignments.

[0036] The term "Fc" as used herein refers to a molecule comprising a sequence of non-antigen-binding fragments resulting from the digestion of an antibody or produced by other means, whether in monomeric or multimeric form, and may contain a hinge region. The original immunoglobulin source of native Fc is preferably of human origin and may be any of the immunoglobulins, with IgG1 and IgG2 being preferred. Fc molecules are composed of monomeric polypeptides that can be linked by covalent (i.e., disulfide bonds) and non-covalent associations into dimeric or multimeric forms. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4, depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). One example of an Fc is a disulfide-bonded dimer resulting from papain digestion of IgG. The term "Fc" as used herein is inclusive to monomeric, dimeric and multimeric forms.

[0037] F(ab) fragments typically contain one light chain and one heavy chain V H and C H1 domain, and the V of the F(ab) fragment H -C H1 The heavy chain portion cannot form disulfide bonds with another heavy chain polypeptide. As used herein, a F(ab) fragment also refers to one light chain containing two variable domains separated by an amino acid linker, and one light chain containing two variable domains and a C separated by an amino acid linker. H1 The antibody may comprise one heavy chain containing the domain.

[0038] F(ab') fragments typically contain one light chain and one H1 and C H2 The F(ab') domain comprises a portion of one heavy chain that contains an additional constant region, such that interchain disulfide bonds are formed between the two heavy chains to form the F(ab') domain. 2 Molecules can be formed.

[0039] The term "binding protein," as used herein, refers to a non-naturally occurring (or recombinant or engineered) molecule that specifically binds to at least one target antigen. Trispecific binding proteins of the present disclosure, unless otherwise specified, typically comprise four polypeptide chains that form at least three antigen-binding sites, with the first polypeptide chain having the formula: V L2 -L 1 -V L1 -L 2 -C L [I] having a structure represented by The second polypeptide chain has the formula: V H1 -L 3 -V H2 -L 4 -C H1 - Hinge-C H2 -C H3 [II] having a structure represented by The third polypeptide chain has the formula: V H3 -C H1 [III] having a structure represented by The fourth polypeptide chain has the formula: V L3 -C L [IV] having a structure represented by Where: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V L3 is a third immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; V H3is a third immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1 a heavy chain constant domain; The hinge is C H1 and C H2 an immunoglobulin hinge region connecting the domains; L 1 , L 2 , L 3 and L 4 is an amino acid linker; The polypeptide of formula I and the polypeptide of formula II form a crossover light chain-heavy chain pair.

[0040] A "recombinant" molecule is one that is produced, expressed, created, or isolated by recombinant means.

[0041] One embodiment of the disclosure provides a binding protein having biological and immunological specificity for one to three target antigens. Another embodiment of the disclosure provides a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide chain forming such a binding protein. Another embodiment of the disclosure provides an expression vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide chain forming such a binding protein. Yet another embodiment of the disclosure provides a host cell that expresses such a binding protein (i.e., comprises a nucleic acid molecule or vector encoding a polypeptide chain forming such a binding protein).

[0042] The term "swappability" as used herein refers to the interchangeability of variable domains within a binding protein format and with retention of folding and ultimately binding affinity. "Full swappability" refers to the interchangeability of variable domains in a polypeptide chain of formula I or a polypeptide chain of formula II while maintaining full functionality of the binding protein as evidenced by retention of binding affinity. H1 and V H2The order of both domains, and therefore V L1 and V L2 It refers to the ability to swap (i.e. reverse) the order of domains. H and V L It should be noted that the designation simply refers to the location of the domain on a particular protein chain in the final format. For example, V H1 and V H2 is the V in the parent antibody L1 and V L2 domains, V in binding proteins H1 and V H2 Similarly, V L1 and V L2 is the V in the parent antibody H1 and V H2 domains, V in binding proteins H1 and V H2 Therefore, V H and V L The designation refers to the current location, not the original location in the parent antibody. H and V L Domains are therefore "swappable".

[0043] The term "antigen" or "target antigen" or "antigen target" as used herein refers to a molecule or a portion of a molecule to which a binding protein can bind and which can additionally be used in an animal to produce an antibody capable of binding to an epitope of that antigen. A target antigen may have one or more epitopes. For each target antigen recognized by a binding protein, the binding protein can compete with an intact antibody that recognizes the target antigen.

[0044] The term "HIV" as used herein means human immunodeficiency virus. As used herein, the term "HIV infection" generally encompasses infection of a host, particularly a human host, with a retrovirus of the human immunodeficiency virus (HIV) family, including but not limited to HIV I, HIV II, HIV III (also known as HTLV-II, LAV-1, LAV-2). HIV can be used herein to refer to any strain, form, subtype, clade and variation in the HIV family. Thus, treatment of HIV infection encompasses treatment of a person who is a carrier of any retrovirus of the HIV family or who has been diagnosed with active AIDS, as well as treatment or prevention of AIDS-related conditions in such a person.

[0045] The term "AIDS" as used herein means Acquired Immune Deficiency Syndrome. AIDS is caused by HIV.

[0046] The term "CD4bs" or "CD4 binding site" refers to a binding site for CD4 (cluster of differentiation 4), a glycoprotein found on the surface of immune cells, such as T helper cells, monocytes, macrophages, and dendritic cells.

[0047] The term "CD3" refers to cluster of differentiation factor 3 polypeptide, which is typically a T-cell surface protein that is part of the T-cell receptor (TCR) complex.

[0048] "CD28" is cluster of differentiation 28 polypeptide, a T cell surface protein that provides costimulatory signals for T cell activation and survival.

[0049] The term "glycoprotein 160" or "gp160 protein" refers to the envelope glycoprotein complex of HIV, which is a homotrimer that is cleaved into gp120 and gp41 subunits.

[0050] The term "MPER" refers to the membrane-proximal external region of glycoprotein 41 (gp41), a subunit of the envelope protein complex of retroviruses, including HIV.

[0051] The term "glycan" refers to the carbohydrate moiety of a glycoconjugate, such as a glycoprotein, glycolipid, or proteoglycan. In the disclosed binding proteins, the glycan refers to the HIV-1 envelope glycoprotein gp120.

[0052] The term "T cell engager" refers to a binding protein directed against an HIV target protein in addition to being directed against the host's immune system, and more specifically, the cytotoxic activity of T cells.

[0053] The term "trimeric apex" refers to the apex of the HIV-1 envelope glycoprotein gp120.

[0054] The term "monospecific binding protein" refers to a binding protein that specifically binds to one antigen target.

[0055] The term "monovalent binding protein" refers to a binding protein that has one antigen-binding site.

[0056] The term "bispecific binding protein" refers to a binding protein that specifically binds to two different antigen targets.

[0057] The term "bivalent binding protein" refers to a binding protein that has two binding sites.

[0058] The term "trispecific binding protein" refers to a binding protein that specifically binds to three different antigen targets.

[0059] The term "trivalent binding protein" refers to a binding protein having three binding sites. In certain embodiments, the trivalent binding protein can bind to one antigen target. In other embodiments, the trivalent binding protein can bind to two antigen targets. In other embodiments, the trivalent binding protein can bind to three antigen targets.

[0060] An "isolated" binding protein is a binding protein that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with the diagnostic or therapeutic use of the binding protein, and can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the binding protein is purified (1) to greater than 95% by weight, most preferably greater than 99% by weight, as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver stain. Since at least one component of the binding protein's natural environment is absent, an isolated binding protein includes the binding protein in situ in a recombinant cell.

[0061] The terms "substantially pure" or "substantially purified," as used herein, refer to a compound or species that is the predominant species present (i.e., it is more abundant than any other individual species in the composition on a molar basis). In some embodiments, a substantially purified fraction is a composition in which the species constitutes at least about 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition is a composition in which all macromolecular species present in the composition constitute greater than about 80%, 85%, 90%, 95%, or 99%. In still other embodiments, the species is purified to an essential homogeneity state in which the composition consists essentially of a single macromolecular species (contaminant species cannot be detected in the composition by conventional detection methods).

[0062] A "neutralizing" binding protein, as used herein, refers to a molecule that can block or substantially reduce the effector function of a target antigen to which it is bound. As used herein, "substantially reduce" means a reduction in the effector function of the target antigen by at least about 60%, preferably at least about 70%, more preferably at least about 75%, even more preferably at least about 80%, even more preferably at least about 85%, and most preferably at least about 90%.

[0063] The term "epitope" includes any determinant, preferably a polypeptide determinant, capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitopic determinants include chemically active surface groupings of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments may have distinctive three-dimensional structural features and / or distinctive charge characteristics. An epitope is the region of an antigen that is bound by an antibody or binding protein. In certain embodiments, a binding protein is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. In some embodiments, the equilibrium dissociation constant is ≦10-8 M, more preferably the equilibrium dissociation constant is ≦10 -9 M, most preferably the dissociation constant is ≦10 -10 When it is M, the binding protein is said to specifically bind to the antigen.

[0064] Dissociation constant of the binding protein (K D ) can be determined, for example, by surface plasmon resonance. Generally, surface plasmon resonance analysis measures real-time binding interactions between a ligand (target antigen on a biosensor matrix) and an analyte (binding protein in solution) by surface plasmon resonance (SPR) using a BIAcore system (Pharmacia Biosensor; Piscataway, NJ). Surface plasmon analysis can also be performed by immobilizing the analyte (binding protein on a biosensor matrix) and presenting the ligand (target antigen). "K D The term "dissociation constant" as used herein refers to the dissociation constant of the interaction between a particular binding protein and a target antigen.

[0065] The term "specifically binds" as used herein means a concentration of about 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M, 1×10 -11 M, 1×10 -12 It refers to the ability of a binding protein or antigen-binding fragment thereof to bind to an antigen containing the epitope with a Kd of M or less and / or to bind to the epitope with an affinity at least two-fold higher than its affinity for a nonspecific antigen.

[0066] The term "linker" as used herein refers to one or more amino acid residues that are inserted between immunoglobulin domains to provide the light and heavy chain domains with sufficient mobility to fold into a crossover dual variable region immunoglobulin. The linker is inserted at the transition between the variable domains or between the variable and constant domains at the sequence level. The approximate sizes of immunoglobulin domains are well understood, so that transitions between domains can be identified. The precise location of the domain transition can be determined by locating secondary structure elements, such as beta-sheets or peptide stretches that do not form alpha-helices, as demonstrated by experimental data or as can be predicted by modeling or secondary structure prediction techniques. The linkers described herein are V L2 C-terminus and V-terminus of the domain L1 L located on the light chain between the N-terminus of the domain 1 ; and V L1 The C-terminus and C L L located on the light chain between the N-terminus of the domain 2 The heavy chain linker is called V H1 C-terminus and V-terminus of the domain H2 L located between the N-terminus of the domain 3 ; and V H2 The C-terminus and C H1 L located between the N-terminus of the domain 4 It is known as.

[0067] The term "vector" as used herein refers to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coding information to a host cell. The term "vector" includes a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA molecule into which additional DNA segments can be inserted. Another type of vector is a viral vector, in which additional DNA segments can be inserted into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Additionally, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. The terms "plasmid" and "vector" are sometimes used interchangeably herein, as plasmids are the most commonly used form of vector. However, the present disclosure is intended to include other forms of expression vectors that serve equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0068] The phrase "recombinant host cell" (or "host cell"), as used herein, refers to a cell into which a recombinant expression vector has been introduced. A recombinant host cell or host cell is intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Such progeny may not actually be identical to the parent cell, since certain modifications may occur in successive generations, either due to mutations or environmental influences, but such cells are still included within the scope of the term "host cell" as used herein. A variety of host cell expression systems can be used to express the binding protein, including bacteria, yeast, baculovirus, and mammalian expression systems (as well as phage display expression systems). An example of a suitable bacterial expression vector is pUC19. To recombinantly express the binding protein, the host cell is transformed or transfected with one or more recombinant expression vectors carrying DNA fragments encoding the polypeptide chain of the binding protein, so that the polypeptide chain is expressed in the host cell and is preferably secreted into the medium in which the host cell is cultured, from which the binding protein can be recovered.

[0069] The term "transformation" as used herein refers to a change in the genetic characteristics of a cell, and a cell is transformed when it is modified to contain new DNA. For example, a cell is transformed and genetically modified from its native state. After transformation, the transforming DNA can recombine with the cell's DNA by being physically integrated into the cell's chromosome, or can be maintained transiently as an episomal element without being replicated, or can be independently replicated as a plasmid. A cell is considered stably transformed when the DNA is replicated with the division of the cell. The term "transfection" as used herein refers to the uptake of foreign or exogenous DNA by a cell, and a cell is "transfected" when the exogenous DNA is introduced inside the cell membrane. Numerous transfection techniques are well known in the art. Using such techniques, one or more exogenous DNA molecules can be introduced into a suitable host cell.

[0070] The term "naturally occurring" as used herein, when applied to an object, refers to the fact that the object can be found in nature and has not been manipulated by humans. For example, a polynucleotide or polypeptide that can be isolated from a source in nature and exists in an organism (including viruses) that has not been intentionally modified by humans is naturally occurring. Similarly, "non-naturally occurring" as used herein refers to an object that is not found in nature or that has been structurally modified or synthesized by humans.

[0071] As used herein, the 20 conventional amino acids and their abbreviations follow conventional usage. Stereoisomers of the 20 conventional amino acids (e.g., D-amino acids); non-natural amino acids and analogs, such as α-, α-disubstituted amino acids, N-alkylamino acids, lactic acid, and other non-conventional amino acids, may also be suitable components for the polypeptide chain of binding proteins. Examples of non-conventional amino acids include 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxyl terminal direction, in accordance with standard usage and convention.

[0072] Naturally occurring residues can be divided into classes based on common side chain properties: (1) Hydrophobicity: Met, Ala, Val, Leu, Ile, Phe, Trp, Tyr, Pro; (2) Polar hydrophilic: Arg, Asn, Asp, Gln, Glu, His, Lys, Ser, Thr; (3) Aliphatic: Ala, Gly, Ile, Leu, Val, Pro; (4) Aliphatic hydrophobicity: Ala, Ile, Leu, Val, Pro; (5) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (6) Acidic: Asp, Glu; (7) Basic: His, Lys, Arg; (8) Residues affecting chain orientation: Gly, Pro; (9) Aromatic: His, Trp, Tyr, Phe; and (10) Aromatic hydrophobic: Phe, Trp, Tyr.

[0073] Conservative amino acid substitutions may involve the exchange of a member of one of these classes for another member of the same class. Non-conservative substitutions may involve the exchange of a member of one of these classes for a member of another class.

[0074] Those skilled in the art can use well-known techniques to determine suitable variants of the polypeptide chain of binding protein.For example, those skilled in the art can identify suitable sections of the polypeptide chain that can be changed without destroying activity by targeting regions that do not seem to be important for activity.Alternatively, those skilled in the art can identify residues and parts of molecules that are conserved between similar polypeptides.In addition, even sections that may be important for biological activity or structure can be subjected to conservative amino acid substitutions without destroying biological activity or adversely affecting polypeptide structure.

[0075] The term "patient", as used herein, includes human and animal subjects.

[0076] The term "treatment" or "treating" as used herein refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those with the disorder as well as those predisposed to having the disorder or those in which the disorder is to be prevented. In certain embodiments, the binding proteins can be used to treat humans infected with HIV or susceptible to HIV infection, or to ameliorate HIV infection in HIV-infected human subjects. The binding proteins can also be used to prevent HIV in human patients.

[0077] Humans infected with HIV may present with, for example, an acute primary infection syndrome (which may be asymptomatic or may involve a flu-like illness with fever, malaise, diarrhea, and neurological symptoms, e.g., headache), asymptomatic infection (high levels of circulating CD4 +It should be understood that the term includes subjects at any one of several stages in the progression of HIV infection, including HIV infection (a long latency period with a gradual decline in the number of T cells), and AIDS (defined by more severe AIDS-defining illnesses and / or a decline in circulating CD4 cell counts below levels compatible with effective immune function). Additionally, treatment or prevention of HIV infection also encompasses treating suspected infection with HIV following suspected past exposure to HIV, such as contact with HIV-contaminated blood, blood transfusions, exchange of bodily fluids, "unsafe" sexual intercourse with an infected person, accidental punctures, receiving a tattoo or acupuncture with a contaminated instrument, or transmission of the virus from mother to infant during pregnancy, childbirth or shortly thereafter.

[0078] The term "pharmaceutical composition" or "therapeutic composition," as used herein, refers to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

[0079] The term "pharmacologically acceptable carrier" or "physiologically acceptable carrier" as used herein refers to one or more formulation materials suitable for achieving or enhancing delivery of a binding protein.

[0080] The terms "effective amount" and "therapeutically effective amount", when used in reference to a pharmaceutical composition comprising one or more binding proteins, refer to an amount or dosage sufficient to produce a desired therapeutic result. More specifically, a therapeutically effective amount is an amount of binding protein sufficient to inhibit one or more clinically defined pathological processes associated with the condition being treated over a period of time. The effective amount may vary depending on the specific binding protein being used, as well as on various factors and conditions related to the patient being treated and the severity of the disorder. For example, when the binding protein is administered in vivo, factors such as the age, weight, and health of the patient, as well as dose-response curves and toxicity data obtained in preclinical animal studies, are among the factors to be considered. The determination of the effective amount or therapeutically effective amount of a given pharmaceutical composition is well within the capabilities of one of ordinary skill in the art.

[0081] One embodiment of the disclosure provides a pharmaceutical composition comprising a pharma- ceutically acceptable carrier and a therapeutically effective amount of a binding protein.

[0082] Trispecific and / or trivalent binding proteins Certain aspects of the present disclosure relate to trispecific and / or trivalent binding proteins. Any CDR or variable domain of any antigen binding protein described herein may have application in the trispecific binding protein of the present disclosure. Various formats of trispecific binding proteins are contemplated. In some embodiments, the binding protein of the present disclosure is a trispecific and / or trivalent binding protein, comprising four polypeptide chains that form three antigen binding sites that specifically bind to one or more HIV target proteins, wherein the first polypeptide chain is represented by the formula: V L2 -L 1 -V L1 -L 2 -C L [I] The structure includes: The second polypeptide chain has the formula: V H1 -L3 -V H2 -L 4 -C H1 - Hinge-C H2 -C H3 [II] The structure includes: The third polypeptide chain has the formula: V H3 -C H1 - Hinge-C H2 -C H3 [III] The structure includes: The fourth polypeptide chain has the formula: V L3 -C L [IV] The structure includes: Where: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V L3 is a third immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; V H3 is a third immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1 a heavy chain constant domain; C H2 is immunoglobulin C H2 a heavy chain constant domain; C H3 is immunoglobulin C H3 a heavy chain constant domain; The hinge is C H1 and C H2 an immunoglobulin hinge region connecting the domains; L 1, L 2 , L 3 and L 4 is an amino acid linker; The polypeptide of formula I and the polypeptide of formula II form a crossover light chain-heavy chain pair, said binding protein.

[0083] In some embodiments, the first polypeptide chain and the second polypeptide chain have a crossover orientation forming two separate antigen binding sites. H1 and V L1 form a binding pair to form a first antigen-binding site. H2 and V L2 In some embodiments, the V polypeptide forms a binding pair and forms a second antigen-binding site. In some embodiments, the third polypeptide and the fourth polypeptide form a third antigen-binding site. H3 and V L3 form a binding pair to form a third antigen-binding site.

[0084] In some embodiments, one or more of the antigen binding sites bind to an HIV target protein. H3 and V L3 forms a third antigen-binding site that binds to the HIV target protein. H1 and V L1 forms the first antigen-binding site that binds to the T cell target protein, and V H2 and V L2 forms a second antigen-binding site that binds to T cell target proteins, and V H3 and V L3 forms a third antigen-binding site that binds to the HIV target protein. H1 and V L1 forms the first antigen-binding site that binds to the T cell target protein, and V H2 and V L2forms a second antigen-binding site that binds to the CD3 polypeptide, and V H3 and V L3 forms a third antigen-binding site that binds to the HIV target protein. H1 and V L1 forms a first antigen-binding site that binds to the CD28 polypeptide, and V H2 and V L2 forms a second antigen-binding site that binds to the CD3 polypeptide, and V H3 and V L3 forms the third antigen-binding site that binds to the HIV target protein.

[0085] In some embodiments, the binding proteins specifically bind to one or more target proteins on T cells, including one or more HIV target proteins (e.g., those described below) and the T cell receptor complex. These T cell engager binding proteins can transiently recruit T cells to target cells and simultaneously activate the cytolytic activity of the T cells. T cell engager trispecific antibodies can activate the HIV-1 reservoir and redirect / activate T cells to target latently infected HIV-1. + It can be used to lyse T cells. Examples of target proteins on T cells include, but are not limited to, CD3 and CD28, among others. In some embodiments, trispecific binding proteins can be generated by combining the antigen-binding domains of two or more monospecific antibodies (parent antibodies) into one antibody. See International Publications WO2011 / 038290A2, WO2013 / 086533A1, WO2013 / 070776A1, WO2012 / 154312A1, and WO2013 / 163427A1. The binding proteins of the present disclosure can be produced using domains or sequences obtained or derived from any human or non-human antibody, including, for example, human, murine, or humanized antibodies.

[0086] In some embodiments of the present disclosure, the trivalent binding protein is capable of binding to three different antigen targets, hi one embodiment, the binding protein is trispecific, with one light chain-heavy chain pair capable of binding to two different antigen targets or epitopes and one light chain-heavy chain pair capable of binding to one antigen target or epitope.

[0087] In some embodiments, the binding proteins of the present disclosure bind to one or more HIV target proteins and one or more T cell target proteins. In some embodiments, the binding proteins can specifically bind to one HIV target protein and two different epitopes on a single T cell target protein. In some embodiments, the binding proteins can specifically bind to one HIV target protein and two different T cell target proteins (e.g., CD28 and CD3). In some embodiments, the first and second polypeptide chains of the binding protein form two antigen binding sites that specifically target two T cell target proteins, and the third and fourth polypeptide chains of the binding protein form an antigen binding site that specifically binds to an HIV target protein. In some embodiments, the one or more HIV target proteins are one or more of glycoprotein 120, glycoprotein 41, and glycoprotein 160. In some embodiments, the one or more T cell target proteins are one or more of CD3 and CD28.

[0088] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first polypeptide chain comprises the amino acid sequence of SEQ ID NO:61 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:61; a second polypeptide chain comprises the amino acid sequence of SEQ ID NO:62 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:62; a third polypeptide chain comprises the amino acid sequence of SEQ ID NO:63 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:63; and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:64 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:64.

[0089] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first polypeptide chain comprises the amino acid sequence of SEQ ID NO:65 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:65; a second polypeptide chain comprises the amino acid sequence of SEQ ID NO:66 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:66; a third polypeptide chain comprises the amino acid sequence of SEQ ID NO:67 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:67; and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:68 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:68.

[0090] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first polypeptide chain comprises the amino acid sequence of SEQ ID NO:69 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:69; a second polypeptide chain comprises the amino acid sequence of SEQ ID NO:70 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:70; a third polypeptide chain comprises the amino acid sequence of SEQ ID NO:71 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:71; and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:72 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:72.

[0091] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first polypeptide chain comprises the amino acid sequence of SEQ ID NO:73 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:73; a second polypeptide chain comprises the amino acid sequence of SEQ ID NO:74 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:74; a third polypeptide chain comprises the amino acid sequence of SEQ ID NO:75 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:75; and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:76 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:76.

[0092] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first polypeptide chain comprises the amino acid sequence of SEQ ID NO:77 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:77; a second polypeptide chain comprises the amino acid sequence of SEQ ID NO:78 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:78; a third polypeptide chain comprises the amino acid sequence of SEQ ID NO:79 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:79; and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:80 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:80.

[0093] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first polypeptide chain comprises the amino acid sequence of SEQ ID NO:81 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:81; a second polypeptide chain comprises the amino acid sequence of SEQ ID NO:82 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:82; a third polypeptide chain comprises the amino acid sequence of SEQ ID NO:83 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:83; and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:84 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:84.

[0094] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first polypeptide chain comprises the amino acid sequence of SEQ ID NO:85 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:85; a second polypeptide chain comprises the amino acid sequence of SEQ ID NO:86 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:86; a third polypeptide chain comprises the amino acid sequence of SEQ ID NO:87 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:87; and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:88 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:88.

[0095] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first polypeptide chain comprises the amino acid sequence of SEQ ID NO:89 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:89; a second polypeptide chain comprises the amino acid sequence of SEQ ID NO:90 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:90; a third polypeptide chain comprises the amino acid sequence of SEQ ID NO:91 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:91; and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:92 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:92.

[0096] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first polypeptide chain comprises the amino acid sequence of SEQ ID NO:93 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:93; a second polypeptide chain comprises the amino acid sequence of SEQ ID NO:94 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:94; a third polypeptide chain comprises the amino acid sequence of SEQ ID NO:95 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:95; and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:96 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:96.

[0097] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first polypeptide chain comprises the amino acid sequence of SEQ ID NO:97 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:97; a second polypeptide chain comprises the amino acid sequence of SEQ ID NO:98 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:98; a third polypeptide chain comprises the amino acid sequence of SEQ ID NO:99 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:99; and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:100 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:100.

[0098] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first polypeptide chain comprises the amino acid sequence of SEQ ID NO:101 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:101; a second polypeptide chain comprises the amino acid sequence of SEQ ID NO:102 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:102; a third polypeptide chain comprises the amino acid sequence of SEQ ID NO:103 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:103; and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:104 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:104.

[0099] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first polypeptide chain comprises the amino acid sequence of SEQ ID NO:105 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:105; a second polypeptide chain comprises the amino acid sequence of SEQ ID NO:106 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:106; a third polypeptide chain comprises the amino acid sequence of SEQ ID NO:107 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:107; and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:108 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:108.

[0100] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first polypeptide chain comprises the amino acid sequence of SEQ ID NO:109 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:109; a second polypeptide chain comprises the amino acid sequence of SEQ ID NO:110 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:110; a third polypeptide chain comprises the amino acid sequence of SEQ ID NO:111 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:111; and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:112 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:112.

[0101] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first polypeptide chain comprises the amino acid sequence of SEQ ID NO:113 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:113; a second polypeptide chain comprises the amino acid sequence of SEQ ID NO:114 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:114; a third polypeptide chain comprises the amino acid sequence of SEQ ID NO:115 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:115; and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:116 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:116.

[0102] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first polypeptide chain comprises the amino acid sequence of SEQ ID NO:117 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:117; a second polypeptide chain comprises the amino acid sequence of SEQ ID NO:118 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:118; a third polypeptide chain comprises the amino acid sequence of SEQ ID NO:119 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:119; and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:120 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:120.

[0103] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first polypeptide chain comprises the amino acid sequence of SEQ ID NO:121 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:121; a second polypeptide chain comprises the amino acid sequence of SEQ ID NO:122 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:122; a third polypeptide chain comprises the amino acid sequence of SEQ ID NO:123 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:123; and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:124 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:124.

[0104] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first polypeptide chain comprises the amino acid sequence of SEQ ID NO:129 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:129; a second polypeptide chain comprises the amino acid sequence of SEQ ID NO:130 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:130; a third polypeptide chain comprises the amino acid sequence of SEQ ID NO:131 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:131; and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:132 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:132.

[0105] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first polypeptide chain comprises the amino acid sequence of SEQ ID NO:133 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:133; a second polypeptide chain comprises the amino acid sequence of SEQ ID NO:134 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:134; a third polypeptide chain comprises the amino acid sequence of SEQ ID NO:135 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:135; and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:136 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:136.

[0106] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first polypeptide chain comprises the amino acid sequence of SEQ ID NO:137 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:137; a second polypeptide chain comprises the amino acid sequence of SEQ ID NO:138 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:138; a third polypeptide chain comprises the amino acid sequence of SEQ ID NO:139 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:139; and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:140 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:140.

[0107] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first polypeptide chain comprises the amino acid sequence of SEQ ID NO:141 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:141; a second polypeptide chain comprises the amino acid sequence of SEQ ID NO:142 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:142; a third polypeptide chain comprises the amino acid sequence of SEQ ID NO:143 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:143; and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:144 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:144.

[0108] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first polypeptide chain comprises the amino acid sequence of SEQ ID NO:145 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:145; a second polypeptide chain comprises the amino acid sequence of SEQ ID NO:146 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:146; a third polypeptide chain comprises the amino acid sequence of SEQ ID NO:147 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:147; and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:148 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:148.

[0109] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first polypeptide chain comprises the amino acid sequence of SEQ ID NO:149 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:149; a second polypeptide chain comprises the amino acid sequence of SEQ ID NO:150 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:150; a third polypeptide chain comprises the amino acid sequence of SEQ ID NO:151 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:151; and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:152 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:152.

[0110] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first polypeptide chain comprises the amino acid sequence of SEQ ID NO:153 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:153; a second polypeptide chain comprises the amino acid sequence of SEQ ID NO:154 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:154; a third polypeptide chain comprises the amino acid sequence of SEQ ID NO:155 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:155; and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:156 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:156.

[0111] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first polypeptide chain comprises the amino acid sequence of SEQ ID NO:157 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:157; a second polypeptide chain comprises the amino acid sequence of SEQ ID NO:158 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:158; a third polypeptide chain comprises the amino acid sequence of SEQ ID NO:159 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:159; and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:160 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:160.

[0112] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first polypeptide chain comprises the amino acid sequence of SEQ ID NO:161 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:161; a second polypeptide chain comprises the amino acid sequence of SEQ ID NO:162 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:162; a third polypeptide chain comprises the amino acid sequence of SEQ ID NO:163 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:163; and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:164 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:164.

[0113] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first polypeptide chain comprises the amino acid sequence of SEQ ID NO:165 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:165; a second polypeptide chain comprises the amino acid sequence of SEQ ID NO:166 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:166; a third polypeptide chain comprises the amino acid sequence of SEQ ID NO:167 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:167; and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:168 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:168.

[0114] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first polypeptide chain comprises the amino acid sequence of SEQ ID NO:169 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:169; a second polypeptide chain comprises the amino acid sequence of SEQ ID NO:170 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:170; a third polypeptide chain comprises the amino acid sequence of SEQ ID NO:171 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:171; and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:172 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:172.

[0115] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites that specifically bind to one or more HIV target proteins, wherein a first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 173 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 173; a second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 174 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 174; a third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 175 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 175; and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 176 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 176. Exemplary, non-limiting polypeptides that may have use in any of the trispecific binding proteins described herein are provided in Table 4.

[0116] anti-HIV binding site Certain aspects of the present disclosure relate to binding proteins that comprise an antigen-binding site that binds to an HIV target protein or polypeptide.

[0117] In some embodiments, the HIV target protein is glycoprotein 120, glycoprotein 41, or glycoprotein 160. In some embodiments, the binding protein binds to one or more of glycoprotein 120, glycoprotein 41, and glycoprotein 160. Exemplary HIV target proteins include, but are not limited to, the MPER of the HIV-1 gp41 protein, the CD4 binding site of the HIV-1 gp120 protein, a glycan in the V3 loop of the HIV-1 gp120 protein, or the trimer apex of the HIV-1 gp120 protein or gp160. For example, in some embodiments, the binding protein of the present disclosure comprises an antigen binding site that binds to the CD4 binding site of the HIV-1 gp120 protein. Exemplary antigen-binding sites that bind to an HIV target protein contemplated for use herein include, but are not limited to, those described in International Publication WO2017 / 074878, such as those from antibodies CD4BS "a", CD4BS "b", MPER, MPER_100W, V1 / V2 "a", V1 / V2 "b", or V3.

[0118] In some embodiments, a binding protein comprising an antigen-binding site that binds to an HIV target protein is monospecific and / or monovalent, bispecific and / or bivalent, trispecific and / or trivalent, or multispecific and / or multivalent. In some embodiments, a binding protein comprising an antigen-binding site that binds to an HIV target protein is a trispecific binding protein comprising four polypeptides that form three antigen-binding sites.

[0119] In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of NCPIN (SEQ ID NO:1), a CDR-H2 sequence comprising the amino acid sequence of WMKPRHGAVSYARQLQG (SEQ ID NO:2), and a CDR-H3 sequence comprising the amino acid sequence of GKYCTARDYYNWDFEH (SEQ ID NO:3); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of RTSQYGSLA (SEQ ID NO:4), a CDR-L2 sequence comprising the amino acid sequence of SGSTRAA (SEQ ID NO:5), and a CDR-L3 sequence comprising the amino acid sequence of QQYEF (SEQ ID NO:6). In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of NCPIN (SEQ ID NO:1), a CDR-H2 sequence comprising the amino acid sequence of WMKPRHGAVSYARQLQG (SEQ ID NO:2), and a CDR-H3 sequence comprising the amino acid sequence of GKYCTARDYYNWDFEH (SEQ ID NO:3); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of RTSQYGSLA (SEQ ID NO:4), a CDR-L2 sequence comprising the amino acid sequence of SGSTRAA (SEQ ID NO:5), and a CDR-L3 sequence comprising the amino acid sequence of QQYEF (SEQ ID NO:6).

[0120] In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTAHI (SEQ ID NO:7), a CDR-H2 sequence comprising the amino acid sequence of IKPQYGAV (SEQ ID NO:8) or IKPQYGAT (SEQ ID NO:9); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGVGSD (SEQ ID NO:11), a CDR-L2 sequence comprising the amino acid sequence of HTS (SEQ ID NO:12), and a CDR-L3 sequence comprising the amino acid sequence of CQVLQF (SEQ ID NO:13). In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTAHI (SEQ ID NO:7), a CDR-H2 sequence comprising the amino acid sequence of IKPQYGAV (SEQ ID NO:8) or IKPQYGAT (SEQ ID NO:9); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGVGSD (SEQ ID NO:11), a CDR-L2 sequence comprising the amino acid sequence of HTS (SEQ ID NO:12), and a CDR-L3 sequence comprising the amino acid sequence of CQVLQF (SEQ ID NO:13). In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTAHI (SEQ ID NO:7), a CDR-H2 sequence comprising the amino acid sequence of IKPQYGAV (SEQ ID NO:8); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGVGSD (SEQ ID NO:11), a CDR-L2 sequence comprising the amino acid sequence of HTS (SEQ ID NO:12), and a CDR-L3 sequence comprising the amino acid sequence of CQVLQF (SEQ ID NO:13).In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTAHI (SEQ ID NO:7), a CDR-H2 sequence comprising the amino acid sequence of IKPQYGAV (SEQ ID NO:8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGVGSD (SEQ ID NO:11), a CDR-L2 sequence comprising the amino acid sequence of HTS (SEQ ID NO:12), and a CDR-L3 sequence comprising the amino acid sequence of CQVLQF (SEQ ID NO:13). In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTAHI (SEQ ID NO:7), a CDR-H2 sequence comprising the amino acid sequence of IKPQYGAT (SEQ ID NO:9); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGVGSD (SEQ ID NO:11), a CDR-L2 sequence comprising the amino acid sequence of HTS (SEQ ID NO:12), and a CDR-L3 sequence comprising the amino acid sequence of CQVLQF (SEQ ID NO:13). In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTAHI (SEQ ID NO:7), a CDR-H2 sequence comprising the amino acid sequence of IKPQYGAT (SEQ ID NO:9); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGVGSD (SEQ ID NO:11), a CDR-L2 sequence comprising the amino acid sequence of HTS (SEQ ID NO:12), and a CDR-L3 sequence comprising the amino acid sequence of CQVLQF (SEQ ID NO:13).

[0121] In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of DCTLN (SEQ ID NO: 14), a CDR-H2 sequence comprising the amino acid sequence of WLKPRWGAVNYARPLQG (SEQ ID NO: 15), and a CDR-H3 sequence comprising the amino acid sequence of GKNCDYNWDFEH (SEQ ID NO: 16); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of RTSQYGSLA (SEQ ID NO: 17), a CDR-L2 sequence comprising the amino acid sequence of SGSTRAA (SEQ ID NO: 18), and a CDR-L3 sequence comprising the amino acid sequence of QQYEF (SEQ ID NO: 19). In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of DCTLN (SEQ ID NO: 14), a CDR-H2 sequence comprising the amino acid sequence of WLKPRWGAVNYARPLQG (SEQ ID NO: 15), and a CDR-H3 sequence comprising the amino acid sequence of GKNCDYNWDFEH (SEQ ID NO: 16); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of RTSQYGSLA (SEQ ID NO: 17), a CDR-L2 sequence comprising the amino acid sequence of SGSTRAA (SEQ ID NO: 18), and a CDR-L3 sequence comprising the amino acid sequence of QQYEF (SEQ ID NO: 19).

[0122] In some embodiments, a binding protein of the disclosure comprises an antigen-binding site having a VH domain comprising an extended heavy chain FR3 loop of antibody VRC03, e.g., as described in Liu, Q. et al. (2019) Nat. Commun. 10:721.

[0123] In some embodiments, the binding protein of the present disclosure comprises: An antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVRLSQSGGQMKKPGDSMRISCRASGYEFINCPINWIRLAPGKRPEWMGWMKPRHGAVSYARQLQGRVTMTRDMYSETAFLELRSLTSDDTAVYFCTRGKYCTARDYYNWDFEHWGQGTPVTVSS (SEQ ID NO: 43). and / or an antigen-binding site comprising an antibody light chain variable (VL) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SLTQSPGTLSLSPGETAIISCRTSQYGSLAWYQQRPGQAPRLVIYSGSTRAAGIPDRFSGSRWGPDYNLTISNLESGDFGVYYCQQYEFFGQGTKVQVDIK (SEQ ID NO: 45). In some embodiments, a binding protein of the disclosure comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 43, and / or an antigen-binding site comprising an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 45. In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:43, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:45.

[0124] In some embodiments, the binding protein of the present disclosure comprises: An antibody heavy chain variable (VH) molecule comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVRLSQSGGQMKKPGDSMRISCRASGYEFINCPINWIRLAPGKRPEWMGWMKPRHGAVSYARQLQGRVTMTRQLSQDPDDPDWGTAFLELRSLTSDDTAVYFCTRGKYCTARDYYNWDFEHWGQGTPVTVSS (SEQ ID NO: 44). ) domain, and / or an antigen-binding site comprising an antibody light chain variable (VL) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SLTQSPGTLSLSPGETAIISCRTSQYGSLAWYQQRPGQAPRLVIYSGSTRAAGIPDRFSGSRWGPDYNLTISNLESGDFGVYYCQQYEFFGQGTKVQVDIK (SEQ ID NO: 45). In some embodiments, a binding protein of the disclosure comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 44, and / or an antigen-binding site comprising an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 45. In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:44, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:45.

[0125] In some embodiments, the binding protein of the present disclosure comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQYGAVNFGGGFRDRVTLTRDVYREIAYMDIRGLKPDDTAVYYCARDRSYGDSSWALDAWGQGTTVVVSA (SEQ ID NO: 46); and / or an antigen-binding site comprising an antibody light chain variable (VL) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:46, and / or an antigen-binding site comprising an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:49. In some embodiments, a binding protein of the disclosure comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:46, and / or an antigen-binding site comprising an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:49. In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:46, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:49.

[0126] In some embodiments, the binding protein of the present disclosure comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQYGATNFGGGFRDRVTLTRDVYREIAYMDIRGLKPDDTAVYYCARDRSYGDSSWALDAWGQGTTVVVSA (SEQ ID NO: 47); and / or an antigen-binding site comprising an antibody light chain variable (VL) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:47, and / or an antigen-binding site comprising an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:49. In some embodiments, a binding protein of the disclosure comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:47, and / or an antigen-binding site comprising an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:49. In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:47, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:49.

[0127] In some embodiments, the binding protein of the present disclosure comprises: RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQYGAVNFGGGFRDRVTLTRQLSQDPDDPDWGIAYMDIRGLKPDDTAVYYCARDRSYGDSSWALDAWGQGTTVVVSA (SEQ ID NO: 48), In some embodiments, the binding protein of the present disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:48, and / or YIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGVPSRFSGSGFHTSFNLTISDLQADDIATYYCQVLQFFGRGSRLHIK (SEQ ID NO:49). In some embodiments, the binding protein of the present disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising an amino acid sequence of SEQ ID NO:48, and / or an antibody light chain variable (VL) domain comprising an amino acid sequence of SEQ ID NO:49. In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:48, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:49.

[0128] In some embodiments, the binding protein of the present disclosure comprises: QVQLVQSGGQMKKPGESMRISCRASGYEFIDCTLNWIRLAPGKRPEWMGWLKPRWGAVNYARPLQGRVTMTRQLSQDPDDPDWGTAFLELRSLTVDDTAVYFCTRGKNCDYNWDFEHWGRGTPVIVSS (SEQ ID NO:50), In some embodiments, the binding protein of the present disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:50, and / or an antibody light chain variable (VL) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:50. In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:50, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:51.

[0129] In some embodiments of any of the above embodiments, the binding protein is a trispecific binding protein. In some embodiments, the trispecific binding protein comprises an antigen-binding site that binds to an HIV target protein, an antigen-binding site that binds to a CD28 polypeptide, and an antigen-binding site that binds to a CD3 polypeptide. In some embodiments, the binding protein is a trispecific binding protein comprising four polypeptides comprising three antigen-binding sites, wherein the polypeptide of formula I and the polypeptide of formula II form a crossover light chain-heavy chain pair (e.g., as described herein). In some embodiments, the VH and VL domains of any of the anti-CD38 antigen-binding sites described above are VH and VL domains of ... H3 and V L3 and forms a third antigen-binding site that binds to the HIV target protein. H1 and V L1 forms a first antigen-binding site that binds to the CD28 polypeptide, and V H2 and V L2 form a second antigen-binding site that binds to the CD3 polypeptide, and the VH and VL domains of any of the anti-HIV antigen-binding sites described above and / or in Table 2 are V H3 and V L3 which forms a third antigen-binding site that binds to the HIV target protein.

[0130] Exemplary anti-HIV antigen binding site sequences are provided in Table 1. In some embodiments, a binding protein comprising an anti-HIV antigen binding site of the present disclosure comprises one, two, three, four, five, or all six CDR sequences of an anti-HIV antibody set forth in Table 1. In some embodiments, a binding protein comprising an anti-HIV antigen binding site of the present disclosure comprises a VH domain sequence and / or a VL domain sequence of an anti-HIV antibody set forth in Table 1.

[0131] [Table 1] [Table 2]

[0132] Anti-CD28 binding site Certain aspects of the present disclosure relate to binding proteins comprising an antigen-binding site that binds to a CD28 polypeptide. In some embodiments, the CD28 polypeptide is a human CD28 polypeptide also known as Tp44. Human CD28 polypeptides are known in the art, including, but not limited to, the polypeptides represented by NCBI accession numbers XP_011510499.1, XP_011510497.1, XP_011510496.1, NP_001230007.1, NP_001230006.1, or NP_006130.1, or the polypeptides produced from NCBI gene identification number 940. In some embodiments, the binding proteins comprising an antigen-binding site that binds to a CD28 polypeptide are monospecific and / or monovalent, bispecific and / or bivalent, trispecific and / or trivalent, or multispecific and / or multivalent. In some embodiments, the binding protein comprising an antigen-binding site that binds to a CD28 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen-binding sites. In some embodiments, the binding protein comprising an antigen-binding site that binds to a CD28 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen-binding sites, one of the three antigen-binding sites binds to a CD28 polypeptide and one of the antigen-binding sites binds to a CD3 polypeptide. In some embodiments, the binding protein comprising an antigen-binding site that binds to a CD28 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen-binding sites, one of the antigen-binding sites binds to a CD28 polypeptide, one of the antigen-binding sites binds to a CD3 polypeptide, and one of the antigen-binding sites binds to an HIV target protein.

[0133] In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO:33); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of KAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO:36). In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO:33); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of KAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO:36).

[0134] In some embodiments, the binding protein of the present disclosure comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWGKGTTVTVSS (SEQ ID NO: 59); and / or The antigen-binding site comprises an antibody light chain variable (VL) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO: 60). In some embodiments, a binding protein of the disclosure comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 59, and / or an antigen-binding site comprising an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 60. In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:59, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:60.

[0135] In some embodiments of any of the above embodiments, the binding protein is a trispecific binding protein. In some embodiments, the trispecific binding protein comprises an antigen-binding site that binds to an HIV target protein, an antigen-binding site that binds to a CD28 polypeptide, and an antigen-binding site that binds to a CD3 polypeptide. In some embodiments, the binding protein is a trispecific binding protein comprising four polypeptides comprising three antigen-binding sites, wherein the polypeptide of formula I and the polypeptide of formula II form a crossover light chain-heavy chain pair (e.g., as described herein). In some embodiments, the VH and VL domains of any of the above anti-CD28 antigen-binding sites are VH and VL domains. H1 and V L1 and forms a first antigen-binding site that binds to a CD28 polypeptide. In some embodiments, the VH and VL domains of any of the anti-CD28 antigen-binding sites described above represent VH1 and V L1 which forms a first antigen-binding site which binds to the CD28 polypeptide, and V H2 and V L2 forms a second antigen-binding site that binds to the CD3 polypeptide, and V H3 and V L3 forms the third antigen-binding site that binds to the HIV target protein.

[0136] Exemplary anti-CD28 antigen binding site sequences are provided in Table 2. In some embodiments, a binding protein comprising an anti-CD28 antigen binding site of the present disclosure comprises one, two, three, four, five, or all six CDR sequences of an anti-CD28 antibody set forth in Table 2. In some embodiments, a binding protein comprising an anti-CD28 antigen binding site of the present disclosure comprises a VH domain sequence and / or a VL domain sequence of an anti-CD28 antibody set forth in Table 2.

[0137] [Table 3]

[0138] Anti-CD3 binding site Certain aspects of the present disclosure relate to binding proteins comprising an antigen-binding site that binds to a CD3 polypeptide. In some embodiments, the CD3 polypeptide is a human CD3 polypeptide, such as CD3-delta (also known as T3D, IMD19, and CD3-delta), CD3-epsilon (also known as T3E, IMD18, and TCRE), and CD3-gamma (also known as T3G, IMD17, and CD3-gamma). Human CD3 polypeptides are known in the art, including, but not limited to, the polypeptides represented by NCBI accession numbers XP_006510029.1 or NP_031674.1, or the polypeptides produced from NCBI gene identification numbers 915, 916, or 917. In some embodiments, the binding proteins comprising an antigen-binding site that binds to a CD3 polypeptide are monospecific and / or monovalent, bispecific and / or bivalent, trispecific and / or trivalent, or multispecific and / or multivalent. In some embodiments, the binding protein comprising an antigen-binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen-binding sites. In some embodiments, the binding protein comprising an antigen-binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen-binding sites, one of the three antigen-binding sites binds to a CD28 polypeptide and one of the three antigen-binding sites binds to a CD3 polypeptide. In some embodiments, the binding protein comprising an antigen-binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen-binding sites, one of the three antigen-binding sites binds to a CD28 polypeptide, one of the three antigen-binding sites binds to a CD3 polypeptide and one of the three antigen-binding sites binds to an HIV target protein.

[0139] In some embodiments, a binding protein of the disclosure comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 22); and / or a CDR-H4 sequence comprising the amino acid sequence of QSLVHX 1 NX 2 X 3 TY where X 1 is E or Q, and X 2 is A or L, and X 3 QSLVHX is Q, R, or F 1 NX 2 X 3 In some embodiments, the antigen-binding site comprises an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of TY (SEQ ID NO: 293), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 30). L2 The CDR-L1 sequence of the domain comprises an amino acid sequence selected from the group consisting of QSLVHQNAQTY (SEQ ID NO: 24), QSLVHENLQTY (SEQ ID NO: 25), QSLVHENLFTY (SEQ ID NO: 26), and QSLVHENLRTY (SEQ ID NO: 27).

[0140] In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHQNAQTY (SEQ ID NO:24), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30). In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHQNAQTY (SEQ ID NO:24), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30).

[0141] In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLQTY (SEQ ID NO:25), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30). In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLQTY (SEQ ID NO:25), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30).

[0142] In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLFTY (SEQ ID NO:26), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30). In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLFTY (SEQ ID NO:26), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30).

[0143] In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLRTY (SEQ ID NO:27), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30). In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLRTY (SEQ ID NO:27), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30).

[0144] In some embodiments, the binding protein of the present disclosure comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 52); and / or DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 54), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 55), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTF GSGTKVEIK (SEQ ID NO:56), and DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:57). In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and / or an antibody light chain variable (VL) domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57. In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and an antibody light chain variable (VL) domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57.

[0145] In some embodiments, the binding protein of the present disclosure comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 52); and / or DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 54). In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 52, and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 54. In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:54.

[0146] In some embodiments, the binding protein of the present disclosure comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 52); and / or DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:55). In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:52, and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:55. In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:55.

[0147] In some embodiments, the binding protein of the present disclosure comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 52); and / or DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:56). In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:52, and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:56. In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:56.

[0148] In some embodiments, the binding protein of the present disclosure comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 52); and / or DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:57). In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:52, and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:57. In some embodiments, a binding protein of the disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:57.

[0149] Advantageously, anti-CD3 binding sites are described herein with high affinity binding to human CD3 polypeptide and with potential manufacturing disadvantages removed (eg, deamidation sites).

[0150] In some embodiments of any of the above embodiments, the binding protein is a trispecific binding protein. In some embodiments, the trispecific binding protein comprises an antigen-binding site that binds to an HIV target protein, an antigen-binding site that binds to a CD28 polypeptide, and an antigen-binding site that binds to a CD3 polypeptide. In some embodiments, the binding protein is a trispecific binding protein comprising four polypeptides comprising three antigen-binding sites, wherein the polypeptide of formula I and the polypeptide of formula II form a crossover light chain-heavy chain pair (e.g., as described herein). In some embodiments, the VH and VL domains of any of the anti-CD3 antigen-binding sites described above are VH and VL domains of ... H2 and V L2 and forms a second antigen-binding site that binds to the CD3 polypeptide. H1 and V L1 The VH and VL domains of any of the anti-CD3 antigen-binding sites described above and / or in Table 3 form a first antigen-binding site that binds to a CD28 polypeptide. H2 and V L2 which forms a second antigen-binding site which binds to the CD3 polypeptide, and V H3 and V L3 forms the third antigen-binding site that binds to the HIV target protein.

[0151] Exemplary anti-CD3 antigen binding site sequences are provided in Table 3. In some embodiments, a binding protein comprising an anti-CD3 antigen binding site of the disclosure comprises one, two, three, four, five, or all six CDR sequences of an anti-CD3 antibody set forth in Table 3. In some embodiments, a binding protein comprising an anti-CD3 antigen binding site of the disclosure comprises a VH domain sequence and / or a VL domain sequence of an anti-CD3 antibody set forth in Table 3.

[0152] [Table 4] [Table 5]

[0153] Linker In some embodiments, the linker L 1 , L 2 , L 3 , and L 4 L ranges in length from zero amino acids (length=0) to about 100 amino acids, or is less than 100, 50, 40, 30, 20, or 15 amino acids, or less. The linker may be 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid long. 1 , L 2 , L 3 , and L 4 may all have the same amino acid sequence or may all have different amino acid sequences.

[0154] Examples of suitable linkers include, for example, GGGGSGGGGS (SEQ ID NO: 40), GGGGSGGGGSGGGGS (SEQ ID NO: 41), S, RT, TKGPS SEQ ID NO: 39), GQPKAAP (SEQ ID NO: 38), GGSGSSGSGG (SEQ ID NO: 42), and DKTHT (SEQ ID NO: 37), as well as those disclosed in International Publications WO2017 / 074878 and WO2017 / 180913. The above-listed examples are not intended to limit the scope of the disclosure in any way, and linkers containing randomly selected amino acids selected from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, glycine, and proline have been shown to be suitable in binding proteins.

[0155] The identity and sequence of the amino acid residues in the linker can be changed depending on the type of secondary structure element that needs to be achieved in the linker.For example, glycine, serine, and alanine are best for linkers with maximum flexibility.If a more rigid and long linker is required, some combinations of glycine, proline, threonine, and serine are useful.Any amino acid residue can be considered as a linker in combination with other amino acid residues to construct larger peptide linkers that are required depending on the desired properties.

[0156] In some embodiments, L 1 The length of is L 3 In some embodiments, L 2 The length of is L 4 In some embodiments, L 1 The length of is L 3 is at least twice the length of L 2 The length of is L 4 In some embodiments, L 1 is 3 to 12 amino acid residues in length, and L 2 is 3 to 14 amino acid residues in length, and L 3 is 1 to 8 amino acid residues in length, and L 4 In some embodiments, L is 1 to 3 amino acid residues in length. 1 is 5 to 10 amino acid residues in length, and L 2 is 5 to 8 amino acid residues in length, and L 3 is 1 to 5 amino acid residues in length, and L 4 In some embodiments, L is 1 to 2 amino acid residues in length. 1 is 7 amino acid residues long, and L 2 is the length of 5 amino acid residues, and L 3 is the length of one amino acid residue, and L 4 is two amino acid residues in length.

[0157] In some embodiments, L 1, L 2 , L 3 and L 4 are each independently zero amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 40), GGGGSGGGGSGGGGS (SEQ ID NO: 41), S, RT, TKGPS (SEQ ID NO: 39), GQPKAAP (SEQ ID NO: 38), and GGSGSSGSGG (SEQ ID NO: 42). 1 , L 2 , L 3 and L 4 each independently comprises a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 40), GGGSGGGGSGGGGS (SEQ ID NO: 41), S, RT, TKGPS (SEQ ID NO: 39), GQPKAAP (SEQ ID NO: 38), and GGSGSSGSGG (SEQ ID NO: 42). 1 contains the sequence GQPKAAP (SEQ ID NO: 38), 2 contains the sequence TKGPS (SEQ ID NO: 39), 3 contains a sequence S and L 4 contains the sequence RT.

[0158] In some embodiments, L 1 , L 2 , L 3 or L 4 At least one of the L 1 , L 2 , L 3 and L 4 contains the sequence DKTHT (SEQ ID NO:37).

[0159] Fc Region and Constant Domains In some embodiments, the binding protein of the present disclosure is H1 and a second polypeptide chain further comprising an Fc region linked to the hinge region of an immunoglobulin as well as a C H2 and C H3 In some embodiments, the binding proteins of the present disclosure comprise an immunoglobulin heavy chain constant domain.H1 and a third polypeptide chain further comprising an Fc region linked to the hinge region of an immunoglobulin as well as a C H2 and C H3 In some embodiments, the binding proteins of the present disclosure comprise an immunoglobulin heavy chain constant domain. H1 a second polypeptide chain further comprising an Fc region linked to said second polypeptide chain, said Fc region comprising an immunoglobulin hinge region and a C H2 and C H3 a second polypeptide chain comprising an immunoglobulin heavy chain constant domain, and H1 a third polypeptide chain further comprising an Fc region linked to the Fc region, the Fc region comprising an immunoglobulin hinge region and a C H2 and C H3 and a third polypeptide chain comprising an immunoglobulin heavy chain constant domain.

[0160] In some embodiments, the binding proteins of the present disclosure comprise a full-length antibody heavy chain or polypeptide chain comprising an Fc region. In some embodiments, the Fc region is a human Fc region, e.g., an Fc region of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region comprises an antibody hinge, C H1 , C H2 , C H3 , and optionally C H4 In some embodiments, the Fc region comprises a domain. In some embodiments, the Fc region is a human IgG1 Fc region. In some embodiments, the Fc region is a human IgG4 Fc region. In some embodiments, the Fc region comprises one or more of the mutations described below. In some embodiments, the Fc region is an Fc region of one of the heavy chain polypeptides (e.g., polypeptide 2 or 3) of the binding proteins shown in Table 4. In some embodiments, the heavy chain constant region is an Fc region of one of the heavy chain polypeptides (e.g., polypeptide 2 or 3) of the binding proteins shown in Table 4. In some embodiments, the light chain constant region is an Fc region of one of the light chain polypeptides (e.g., polypeptide 1 or 4) of the binding proteins shown in Table 4.

[0161] In some embodiments, the binding protein of the present disclosure comprises one or two Fc variants. The term "Fc variant" as used herein refers to a molecule or sequence that is modified from a native Fc, but still contains a binding site for the salvage receptor, FcRn (neonatal Fc receptor). Exemplary Fc variants and their interactions with the salvage receptor are known in the art. Thus, the term "Fc variant" may include a molecule or sequence that is humanized from a non-human native Fc. Additionally, the native Fc contains regions that can be removed to provide structural features or biological activities that are not required for the antibody-like binding protein of the present invention. Thus, the term "Fc variant" includes molecules or sequences that lack one or more native Fc sites or residues, or have one or more altered Fc sites or residues that affect or are involved in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than the salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0162] In some embodiments, a binding protein of the disclosure (e.g., a trispecific binding protein) comprises a "knob" mutation in the second polypeptide chain and a "hole" mutation in the third polypeptide chain. In some embodiments, a binding protein of the disclosure comprises a "knob" mutation in the third polypeptide chain and a "hole" mutation in the second polypeptide chain. In some embodiments, the "knob" mutation comprises a substitution at a position corresponding to positions 354 and / or 366 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitution is S354C, T366W, T366Y, S354C and T366W, or S354C and T366Y. In some embodiments, the "knob" mutation comprises a substitution at a position corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitution is S354C and T366W. In some embodiments, the "hole" mutation comprises a substitution at a position corresponding to position 407, and optionally positions 349, 366, and / or 368 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitution is Y407V or Y407T and optionally Y349C, T366S, and / or L368A. In some embodiments, the "hole" mutation comprises a substitution at a position corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitution is Y349C, T366S, L368A, and Y407V.

[0163] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising an amino acid substitution at a position corresponding to position 366 and optionally position 354 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being T366W or T366Y and optionally S354C; and a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the second Fc region comprising amino acid substitutions at positions corresponding to position 407 and optionally positions 349, 366, and / or 368 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are Y407V or Y407T and optionally Y349C, T366S, and / or L368A.

[0164] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising amino acid substitutions at positions corresponding to positions 407 and optionally 349, 366, and / or 368 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being Y407V or Y407T and optionally Y349. and the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, the second Fc region comprising an amino acid substitution at a position corresponding to 366 and optionally 354 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitution is T366W or T366Y and optionally S354C.

[0165] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising an amino acid substitution at a position corresponding to position 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitution being T366W; the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the second Fc region comprising an amino acid substitution at a position corresponding to positions 366, 368, and / or 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitution being T366S, L368A, and / or Y407V.

[0166] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising an amino acid substitution at a position corresponding to position 366, 368, and / or 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitution being T366S, L368A, and / or Y407V; the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the second Fc region comprising an amino acid substitution at a position corresponding to position 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitution being T366W.

[0167] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being S354C and T366W; the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the second Fc region comprising amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being Y349C, T366S, L368A, and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being Y349C, T366S, L368A, and Y407V; the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the second Fc region comprising amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being S354C and T366W. In some embodiments, the first and / or second Fc region is a human IgG1 Fc region. In some embodiments, the first and / or second Fc region is a human IgG4 Fc region.

[0168] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising amino acid substitutions at positions corresponding to positions 228, 354, 366, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, S354C, T366W, and R409K; The polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, the second Fc region comprising amino acid substitutions at positions corresponding to 228, 349, 366, 368, 407, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, Y349C, T366S, L368A, Y407V, and R409K. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising amino acid substitutions at positions corresponding to 228, 349, 366, 368, 407, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, Y349C, T366S, L368A, Y4 07V, and R409K; and the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to 228, 354, 366, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, S354C, T366W, and R409K.

[0169] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising amino acid substitutions at positions corresponding to 234, 235, 354, and 366 of human IgG4 according to the EU index, the amino acid substitutions being F234A, L235A, S354C, and T366W; The polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, the second Fc region comprising amino acid substitutions at positions corresponding to positions 234, 235, 349, 366, 368, and 407 of human IgG4 according to the EU index, the amino acid substitutions being F234A, L235A, Y349C, T366S, L368A, and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising amino acid substitutions at positions corresponding to 234, 235, 349, 366, 368, and 407 of human IgG4 according to the EU index, the amino acid substitutions being F234A, L235A, Y349C, T366S, L367S, L368S, L369S, L370S, L371S, L372S, L373S, L374S, L375S, L376S, L377S, L378S, L379S, L379S, L378S, L379S, L379S, L379S, L370S, L371S, L372S, L373S, L374S, L375S, L376S, L377S, L378S, L379 ...9S, L379S, L379S, L379S, L379S, L380S, L381S, L382S, L383S, L384 68A, and Y407V; and the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, the second Fc region comprising amino acid substitutions at positions corresponding to positions 234, 235, 354, and 366 of human IgG4 according to the EU index, the amino acid substitutions being F234A, L235A, S354C, and T366W.

[0170] In some embodiments, the binding proteins of the present disclosure comprise one or more mutations that can reduce effector function, such as Fc receptor-mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and / or antibody-dependent cellular cytotoxicity (ADCC). H1 and a first Fc region linked to the hinge region of an immunoglobulin as well as a C H2 and C H3 the third polypeptide chain comprises an immunoglobulin heavy chain constant domain; H1 and a second Fc region linked to the hinge region of an immunoglobulin as well as a C H2 and C H3 The polypeptide chains each comprise an immunoglobulin heavy chain constant domain; the first and second Fc regions are human IgG1 Fc regions; the first and second Fc regions each comprise an amino acid substitution at a position corresponding to 234 and 235 of human IgG1 according to the EU index, the amino acid substitutions being L234A and L235A. In some embodiments, the Fc regions of the second and third polypeptide chains are human IgG1 Fc regions; the Fc regions each comprise an amino acid substitution at a position corresponding to 234 and 235 of human IgG1 according to the EU index, the amino acid substitutions being L234A and L235A. In some embodiments, the second polypeptide chain comprises a C H1 and a first Fc region linked to the hinge region of an immunoglobulin as well as a C H2 and C H3 the third polypeptide chain comprises an immunoglobulin heavy chain constant domain; H1 and a second Fc region linked to the hinge region of an immunoglobulin as well as a C H2 and C H3the first and second Fc regions are human IgG1 Fc regions; and the first and second Fc regions each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, the amino acid substitutions being L234A, L235A, and P329A. In some embodiments, the Fc regions of the second and third polypeptide chains are human IgG1 Fc regions, and the Fc regions each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, the amino acid substitutions being L234A, L235A, and P329A. In some embodiments, the Fc region of the second and third polypeptide chains is a human IgG4 Fc region, and the Fc region comprises amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, respectively, the amino acid substitutions being F234A and L235A. H1 a second polypeptide chain further comprising a first Fc region linked to said first polypeptide chain, said first Fc region comprising an immunoglobulin hinge region and a C H2 and C H3 a second polypeptide chain comprising an immunoglobulin heavy chain constant domain, and H1 a third polypeptide chain further comprising a second Fc region linked to said third polypeptide chain, said second Fc region being linked to a hinge region and a C region of an immunoglobulin; H2 and C H3 a third polypeptide chain comprising an immunoglobulin heavy chain constant domain; the first and second Fc regions comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, respectively, the amino acid substitutions being F234A and L235A.

[0171] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising amino acid substitutions at positions corresponding to 228, 234, 235, 354, 366, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, F234A, L235A, S354C, T366W, and R409K; The polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, the second Fc region comprising amino acid substitutions at positions corresponding to 228, 234, 235, 349, 366, 368, 407, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, F234A, L235A, Y349C, T366S, L368A, Y407V, and R409K. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising amino acid substitutions at positions corresponding to 228, 234, 235, 349, 366, 368, 407, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, F234A, L235A, Y349C, T366S, L368A, Y4 07V, and R409K; and the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to 228, 234, 235, 354, 366, and 409 of human IgG4 according to the EU index, wherein the amino acid substitutions are S228P, F234A, L235A, S354C, T366W, and R409K.

[0172] In some embodiments, the Fc region is a human IgG4 Fc region comprising one or more mutations that reduce or eliminate FcγI and / or FcγII binding. In some embodiments, the Fc region is a human IgG4 Fc region comprising one or more mutations that reduce or eliminate FcγI and / or FcγII binding but do not affect FcRn binding. In some embodiments, the Fc region is a human IgG4 Fc region comprising an amino acid substitution at a position corresponding to 228 and / or 409 of human IgG4 according to the EU index. In some embodiments, the amino acid substitution is S228P and / or R409K. In some embodiments, the Fc region is a human IgG4 Fc region comprising an amino acid substitution at a position corresponding to 234 and / or 235 of human IgG4 according to the EU index. In some embodiments, the amino acid substitution is F234A and / or L235A. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 228, 234, 235, and / or 409 of human IgG4 according to the EU index. In some embodiments, the amino acid substitutions are S228P, F234A, L235A, and / or R409K. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to the EU index. In some embodiments, the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid mutations at positions corresponding to positions 228, 233-236, and / or 409 of human IgG4 according to the EU index. In some embodiments, the amino acid mutations are S228P; E233P, F234V, L235A, and a deletion at 236; and / or R409K.

[0173] In some embodiments, the Fc region comprises one or more mutations that reduce or eliminate Fc receptor binding and / or effector functions of the Fc region (e.g., Fc receptor-mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and / or antibody-dependent cellular cytotoxicity (ADCC)).

[0174] In some embodiments, the Fc region is a human IgG1 Fc region comprising one or more amino acid substitutions at positions corresponding to positions 234, 235, and / or 329 of human IgG1 according to the EU index. In some embodiments, the amino acid substitutions are L234A, L235A, and / or P329A. In some embodiments, the Fc region is a human IgG1 Fc region comprising amino acid substitutions at positions corresponding to positions 298, 299, and / or 300 of human IgG1 according to the EU index. In some embodiments, the amino acid substitutions are S298N, T299A, and / or Y300S.

[0175] In some embodiments, the binding proteins of the disclosure comprise one or more mutations to improve stability, e.g., stability of the hinge region and / or dimer interface of IgG4 (see, e.g., Spiess, C. et al. (2013) J. Biol. Chem. 288:26583-26593). In some embodiments, the mutations comprise substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P and R409K. In some embodiments, the binding proteins comprise one or more mutations to improve stability, e.g., stability of the hinge region and / or dimer interface of IgG4 (see, e.g., Spiess, C. et al. (2013) J. Biol. Chem. 288:26583-26593). In some embodiments, the mutations comprise substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P and R409K. H1 a second polypeptide chain further comprising a first Fc region linked to said first polypeptide chain, said first Fc region comprising an immunoglobulin hinge region and a C H2 and C H3 a second polypeptide chain comprising an immunoglobulin heavy chain constant domain, and H1 a third polypeptide chain further comprising a second Fc region linked to said third polypeptide chain, said second Fc region being linked to a hinge region and a C region of an immunoglobulin; H2 and CH3 and a third polypeptide chain comprising an immunoglobulin heavy chain constant domain; the first and second Fc regions are human IgG4 Fc regions; the first and second Fc regions comprise amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, respectively, the amino acid substitutions being S228P and R409K. In some embodiments, the binding protein of the present disclosure comprises knob and hole mutations and one or more mutations for improving stability. In some embodiments, the first and / or second Fc regions are human IgG4 Fc regions.

[0176] In some embodiments, the Fc region is a human IgG1 Fc region comprising one or more amino acid substitutions at positions corresponding to positions 234, 235, and / or 329 of human IgG1 according to the EU index. In some embodiments, the amino acid substitutions are L234A, L235A, and / or P329A. In some embodiments, the Fc region is a human IgG1 Fc region comprising amino acid substitutions at positions corresponding to positions 298, 299, and / or 300 of human IgG1 according to the EU index. In some embodiments, the amino acid substitutions are S298N, T299A, and / or Y300S.

[0177] nucleic acid Another aspect of the present disclosure relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding any of the binding proteins described herein. Exemplary and non-limiting nucleic acid sequences are provided in Table 5.

[0178] Other aspects of the present disclosure relate to kits of polynucleotides, for example, encoding one or more polypeptides of binding proteins as described herein. In some embodiments, the kits of polynucleotides of the present disclosure include: (a) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 177, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 178, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 179, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 180; (b) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 181, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 182, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 183, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 184; (c) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 185, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 186, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 187, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 188. (d) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:189, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:190, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:191, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:192; (e) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:193, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:194, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:195, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:196; (f) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:197, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:198, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:199, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:200;(g) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:201, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:202, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:203, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:204; (h) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:205, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:206, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:207, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:208; (i) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:209, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:210, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:211, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:212; (j) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:213, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:214, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:215, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:216; (k) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:217, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:218, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:219, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:220; (l) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:221, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:222, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:223, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:224; (m) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:225, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:226, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:227, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:228;(n) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:229, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:230, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:231, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:232; (o) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:233, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:234, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:235, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:236; (p) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:237, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:238, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:239, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:240; (q) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:241, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:242, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:243, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:244; (r) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:245, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:246, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:247, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:248; (s) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:249, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:250, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:251, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:252; (t) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:253, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:254, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:255, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:256;(u) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:257, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:258, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:259, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:260; (v) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:261, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:262, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:263, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:264; (w) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:265, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:266, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:267, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:268; (x) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:269, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:270, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:271, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:272; (y) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:273, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:274, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:275, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:276; (z) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:277, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:278, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:279, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:280; (aa) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:281, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:282, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:283, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:284;(bb) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:285, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:286, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:287, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:288; or (cc) a kit of polynucleotides comprising one, two, three, or four polynucleotides comprising a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:289, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:290, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:291, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:292;

[0179] Another aspect of the present disclosure relates to a vector system comprising one or more vectors encoding the first, second, third, and fourth polypeptide chains of any of the binding proteins described herein. In some embodiments, the vector system comprises a first vector encoding the first polypeptide chain of the binding protein, a second vector encoding the second polypeptide chain of the binding protein, a third vector encoding the third polypeptide chain of the binding protein, and a fourth vector encoding the fourth polypeptide chain of the binding protein, for example as shown in the polynucleotides of Table 5. In some embodiments, the vector system comprises a first vector encoding the first and second polypeptide chains of the binding protein, and a second vector encoding the third and fourth polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and third polypeptide chains of the binding protein, and a second vector encoding the second and fourth polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and fourth polypeptide chains of the binding protein, and a second vector encoding the second and third polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first, second, third and fourth polypeptide chains of the binding protein. One or more vectors of the vector system may be any vector described herein. In some embodiments, one or more vectors are expression vectors. In some embodiments, the first, second, third and fourth polynucleotides are present on one or more expression vectors, for example, 1, 2, 3 or 4 expression vectors.

[0180] Standard recombinant DNA methodology is used to construct polynucleotides encoding polypeptides that form binding proteins, incorporate these polynucleotides into recombinant expression vectors, and introduce such vectors into host cells. See, for example, Sambrook et al., 2001, MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, 3rd ed.). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications as commonly accomplished in the art or as described herein. Unless specific definitions are provided, the nomenclature utilized in the context of analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein, and the laboratory procedures and techniques thereof, are those well known and commonly used in the art. Similarly, conventional techniques can be used for chemical synthesis, chemical analysis, pharmaceutical manufacturing, formulation, delivery, and treatment of patients.

[0181] In some embodiments, the isolated nucleic acid is operably linked to a heterologous promoter to direct the transcription of the binding protein-encoding nucleic acid sequence. A promoter can refer to a nucleic acid control sequence that directs the transcription of a nucleic acid. A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a binding protein-encoding sequence when the promoter affects the transcription or expression of the coding sequence. Examples of promoters include, but are not limited to, promoters derived from the genomes of viruses (such as polyomavirus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and simian virus 40 (SV40)), heterologous eukaryotic promoters (such as actin promoters, immunoglobulin promoters, and heat shock promoters), CAG-promoter (Niwa et al., Gene 108(2):193-9, 1991), phosphoglycerate kinase (PGK)-promoter, tetracycline-inducible promoter (Masui et al., Nucleic Acid Res. 33:e43, 2005), the lac system, the trp system, the tac system, the trc system, the major operator and promoter regions of phage lambda, the promoter of 3-phosphoglycerate kinase, the promoter of yeast acid phosphatase, and the promoter of yeast alpha mating factor. A polynucleotide encoding a binding protein of the present disclosure may be under the control of a constitutive promoter, an inducible promoter, or any other suitable promoter described herein or readily recognized by one of skill in the art.

[0182] In some embodiments, the isolated nucleic acid is incorporated into a vector. In some embodiments, the vector is an expression vector. The expression vector may include one or more regulatory sequences operably linked to the polynucleotide to be expressed. The term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Examples of suitable enhancers include, but are not limited to, enhancer sequences from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, insulin, etc.), as well as enhancer sequences from eukaryotic cell viruses (e.g., SV40 enhancer on the late side of the replication origin (bp 100-270), cytomegalovirus early promoter enhancer, polyoma enhancer on the late side of the replication origin, adenovirus enhancer, etc.). Examples of suitable vectors can include, for example, plasmid, cosmid, episome, transposon, and virus vectors (e.g., adenovirus, vaccinia virus, Sindbis virus, measles, herpes virus, lentivirus, retrovirus, adeno-associated virus vector, etc.).Expression vectors can be used to transfect host cells, such as bacterial cells, yeast cells, insect cells, and mammalian cells.Biologically functional virus and plasmid DNA vectors capable of expression and replication in hosts are known in the art and can be used to transfect any cell of interest.

[0183] host cell Other aspects of the disclosure relate to a host cell (e.g., an isolated host cell) comprising one or more isolated polynucleotides, vectors, and / or vector systems described herein. In some embodiments, the isolated host cell of the disclosure is cultured in vitro. In some embodiments, the host cell is a bacterial cell (e.g., an E. coli cell). In some embodiments, the host cell is a yeast cell (e.g., a S. cerevisiae cell). In some embodiments, the host cell is an insect cell. Examples of insect host cells can include, for example, Drosophila cells (e.g., S2 cells), Trichoplusia ni cells (e.g., High Five™ cells), and Spodoptera frugiperda cells (e.g., Sf21 or Sf9 cells). In some embodiments, the host cell is a mammalian cell. Exemplary mammalian host cells include, for example, human embryonic kidney cells (e.g., 293 cells or 293 cells subcloned for growth in suspension culture), Expi293™ cells, CHO cells, baby hamster kidney cells (e.g., BHK, ATCC CCL 10), mouse Sertoli cells (e.g., TM4 cells), monkey kidney cells (e.g., CV1 ATCC CCL 70), African green monkey kidney cells (e.g., VERO-76, ATCC CRL-1587), human cervical carcinoma cells (e.g., HELA, ATCC CCL 2), canine kidney cells (e.g., MDCK, ATCC CCL 34), buffalo rat hepatocytes (e.g., BRL 3A, ATCC CRL 1442), human lung cells (e.g., W138, ATCC CCL 75), human liver cells (e.g., Hep G2, HB 8065), mouse mammary tumor cells (e.g., MMT 060562, ATCC CCL51), TRI cells, MRC 5 cells, FS4 cells, human hepatoma lines (eg, Hep G2), and myeloma cells (eg, NS0 and Sp2 / 0 cells).

[0184] Another aspect of the present disclosure relates to a method for producing any of the binding proteins described herein. In some embodiments, the method includes a) culturing a host cell (e.g., any of the host cells described herein) comprising an isolated nucleic acid, vector, and / or vector system (e.g., any of the isolated nucleic acid, vector, and / or vector system described herein) under conditions such that the host cell expresses the binding protein; and b) isolating the binding protein from the host cell. Methods for culturing a host cell under conditions for protein expression are well known to those skilled in the art. Methods for isolating a protein from a cultured host cell are well known to those skilled in the art, and include, for example, affinity chromatography (e.g., two-step affinity chromatography including Protein A affinity chromatography followed by size exclusion chromatography).

[0185] Pharmaceutical Compositions Therapeutic or pharmaceutical compositions comprising the binding proteins are within the scope of this disclosure. Such therapeutic or pharmaceutical compositions can include a therapeutically effective amount of the binding protein, or binding protein-drug conjugate, in combination with a pharma- ceutical or physiologically acceptable formulation agent selected for suitability with the mode of administration.

[0186] Acceptable formulation materials are nontoxic to recipients at the dosages and concentrations employed.

[0187] Pharmaceutical compositions can contain formulation materials to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or permeability of the composition.Suitable formulation materials include amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (e.g., boric acid, bicarbonate, Tris-HCl, citric acid, phosphoric acid, or other organic acids), bulking agents (e.g., mannitol or glycine), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)), complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, or dextrin), proteins (e.g., serum albumin, gelatin, or immunoglobulins), colorants, flavoring agents, and diluents, emulsifiers, hydrophilic polymers (e.g., polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (e.g., sodium), preservatives (e.g., benzalkonium chloride, ammonium chloride, sodium lauryl sulfate ... Examples of suitable pharmaceutical agents include, but are not limited to, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (e.g., glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (e.g., mannitol or sorbitol), suspending agents, surfactants or wetting agents (e.g., pluronics; PEG; sorbitan esters; polysorbates, e.g., polysorbate 20 or polysorbate 80; triton; tromethamine; lecithin; cholesterol or tyloxapol), stability enhancers (e.g., sucrose or sorbitol), tonicity enhancers (e.g., alkali metal halides, e.g., sodium or potassium chloride, or mannitol, sorbitol), delivery vehicles, diluents, excipients, and / or pharmaceutical adjuvants (e.g., see REMINGTON'S TECHNOLOGY ROADMAP FOR SEMICONDUCTORS, 2003, incorporated herein by reference for any purpose). PHARMACEUTICAL SCIENCES (18th Ed., edited by A. R. Gennaro, Mack Publishing Company 1990), and subsequent editions thereof).

[0188] Optimal pharmaceutical compositions can be determined by one of skill in the art depending, for example, on the intended route of administration, delivery format, and desired dosage. Such compositions can affect the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the binding protein.

[0189] The primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, suitable vehicles or carriers for injection can be water, saline solution, or artificial cerebrospinal fluid, optionally supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions include Tris buffer at about pH 7.0-8.5, or acetate buffer at about pH 4.0-5.5, which can further include sorbitol or a suitable substitute. In one embodiment of the present disclosure, the binding protein composition can be prepared for storage by mixing the selected composition having the desired purity with optional formulating agents in the form of a lyophilized cake or aqueous solution. Additionally, the binding protein can be formulated as a lyophilizate using appropriate excipients, such as sucrose.

[0190] The pharmaceutical composition of the present disclosure can be selected for parenteral or subcutaneous delivery.Alternatively, the composition can be selected for inhalation or for delivery through the digestive tract, for example, orally.The manufacture of such pharmacologic acceptable compositions is within the skill of the art.

[0191] Formulation components are present in concentrations that are acceptable for the site of administration, for example, buffers are used to maintain the composition at physiological pH or slightly lower, typically within a pH range of about 5 to about 8.

[0192] When parenteral administration is intended, the therapeutic composition for use can be in the form of a pyrogen-free, parenterally acceptable, aqueous solution containing the desired binding protein in a pharma- ceutically acceptable medium.A particularly suitable medium for parenteral injection is sterile distilled water, in which the binding protein is formulated as a sterile, isotonic solution that is appropriately preserved.Yet another product can involve the formulation of the desired molecule with an agent that provides controlled or sustained release of the product that can be delivered via depot injection, such as injectable microspheres, biodegradable particles, polymeric compounds (e.g., polylactic acid or polyglycolic acid), beads, or liposomes.Hyaluronic acid can also be used, which can have the effect of promoting sustained duration in the circulation.Other suitable means for the introduction of the desired molecule include implantable drug delivery devices.

[0193] In one embodiment, the pharmaceutical composition can be formulated for inhalation.For example, the binding protein can be formulated as a dry powder for inhalation.The binding protein inhalation solution can also be formulated with a propellant for aerosol delivery.In yet another embodiment, the solution can be nebulized.

[0194] It is also contemplated that certain formulations can be administered orally.In one embodiment of the present disclosure, the binding protein administered in this manner can be formulated with or without carriers that are conventionally used in the preparation of solid dosage forms, such as tablets and capsules.For example, capsules can be designed to release the active portion of the formulation at the location in the gastrointestinal tract where bioavailability is maximized and pre-systemic degradation is minimized.Additional agents can be included to promote the absorption of binding protein.Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be used.

[0195] Another pharmaceutical composition can involve an effective amount of binding protein in a mixture with non-toxic excipients suitable for tablet manufacture.By dissolving tablets in sterile water or other suitable medium, the solution can be manufactured into unit dose form.Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binders such as starch, gelatin, or acacia; or lubricants such as magnesium stearate, stearic acid, or talc.

[0196] Additional pharmaceutical compositions of the present disclosure will be apparent to those skilled in the art, including formulations with binding proteins in sustained or controlled delivery formulations. Techniques for formulating various other sustained or controlled delivery means, such as liposome carriers, biodegradable microparticles or porous beads, and depot injections, are also known to those skilled in the art. Additional examples of sustained release preparations include semipermeable polymer matrices in the form of shaped articles, such as films, or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Sustained release compositions can also include liposomes, which can be produced by any of several methods known in the art.

[0197] Pharmaceutical compositions used for in vivo administration must typically be sterile. This can be achieved by filtration through sterile filtration membranes. If the composition is lyophilized, sterilization using this method can be carried out either before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in solution. Additionally, parenteral compositions are generally placed in a container with a sterile access port, for example, an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle.

[0198] Once the pharmaceutical composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form requiring reconstitution prior to administration (e.g., lyophilized).

[0199] The present disclosure also includes kits for producing single-dose administration units.Each kit can contain both a first container with dry protein and a second container with aqueous formulation.Kits containing single and multi-chamber pre-filled syringes (e.g., liquid syringes and lyosyringes) are also included within the scope of the present disclosure.

[0200] The effective amount of the binding protein pharmaceutical composition used therapeutically depends, for example, on the therapeutic context and purpose.Those skilled in the art will understand that the appropriate dosage level for treatment will vary depending in part on the molecule to be delivered, the indication for which the binding protein is used, the route of administration, and the size (weight, body surface, or organ size) and condition (age and general health) of the patient.Therefore, the clinician can titrate the dosage and modify the route of administration to obtain optimal therapeutic effect.

[0201] The frequency of dosing depends on the pharmacokinetic parameters of the binding protein in the formulation being used. Typically, the clinician administers the composition until a dosage is reached that achieves the desired effect. The composition can thus be administered as a single dose, as two or more doses over time (which may or may not contain the same amount of the desired molecule), or as a continuous infusion via an implanted device or catheter. Further refinement of the appropriate dosage is routinely done by those skilled in the art and is within the scope of the tasks routinely performed by those skilled in the art. The appropriate dosage can be ascertained through the use of appropriate dose-response data.

[0202] The route of administration of the pharmaceutical composition is according to known methods, for example, orally; by injection, intravenous, intraperitoneal, intracerebral (intracemall), intraventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; by sustained release system; or by implantation device. If desired, the composition can be administered by bolus injection or continuously by infusion, or by implantation device.

[0203] The compositions can also be administered locally via implantation of a membrane, sponge, or other suitable material into which the desired molecule has been absorbed or encapsulated. When an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed release bolus, or continuous administration.

[0204] The pharmaceutical compositions can be used to prevent and / or treat HIV infection. The pharmaceutical compositions can be used as a stand-alone therapy or in combination with standard antiretroviral therapy.

[0205] The present disclosure also relates to a kit comprising a binding protein and other reagents useful for detecting target antigen levels in a biological sample. Such reagents may include detectable labels, blocking serum, positive and negative control samples, and detection reagents. In some embodiments, the kit comprises a composition comprising any of the binding proteins, polynucleotides, vectors, vector systems, and / or host cells described herein. In some embodiments, the kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container can be formed from a variety of materials, such as glass or plastic. The container holds a composition alone or in combination with another composition effective for treating, preventing, and / or diagnosing a condition (e.g., HIV infection) and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). In some embodiments, the label or package insert indicates that the composition is used to prevent, diagnose, and / or treat a selected condition. Alternatively, or additionally, the article or kit may further comprise a second (or third) container containing a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0206] Methods and Uses of Binding Proteins Certain aspects of the disclosure relate to methods of preventing HIV infection in a patient, treating HIV infection in a patient, preventing AIDS in a patient, and treating AIDS in a patient using any of the binding proteins or pharmaceutical compositions disclosed herein. Any of the binding proteins or pharmaceutical compositions disclosed herein may have use in the methods of the disclosure, such as methods of preventing HIV infection in a patient, treating HIV infection in a patient, preventing AIDS in a patient, and treating AIDS in a patient.

[0207] Figure 1 illustrates an exemplary and non-limiting format for trispecific binding proteins that can be used in the methods and uses described herein. As shown in Figure 2, the proposed mechanism by which the binding proteins shown in Figure 1 may result in the elimination of HIV reservoir cells in patients involves (1) activation of latently infected CD4+ T cells via the anti-CD28 and anti-CD3 arms of the trispecific binding protein; (2) recruitment of CD8+ T cells to activated latently infected CD4+ T cells via the anti-Env and anti-CD3 arms; (3) activation of engaged CD8+ T cells via the anti-CD28 and anti-CD3 arms; and (4) killing of latently infected CD4+ T cells through a perforin / granzyme mechanism. Advantageously, this mechanism is believed to provide a novel strategy to activate and subsequently kill HIV-1 reservoir cells to attack the HIV-1 reservoir in patients.

[0208] In some embodiments, the methods of the disclosure comprise administering to a patient a therapeutically effective amount of at least one of the binding proteins or pharmaceutical compositions described herein.

[0209] In some embodiments, at least one binding protein is administered in combination with antiretroviral therapy (e.g., anti-HIV therapy). In some embodiments, at least one binding protein is administered prior to antiretroviral therapy. In some embodiments, at least one binding protein is administered concurrently with antiretroviral therapy. In some embodiments, at least one binding protein is administered after antiretroviral therapy. In some embodiments, at least one binding protein is administered in combination with any standard antiretroviral therapy known in the art.

[0210] In some embodiments, administration of at least one binding protein or pharmaceutical composition results in elimination of one or more latent and / or chronically HIV infected cells in the patient. In some embodiments, administration of at least one binding protein results in neutralization of one or more HIV virions, resulting in elimination of one or more latent and / or chronically HIV infected cells in the patient. In some embodiments, the patient is a human.

[0211] [Table 6] [Table 7] [Table 8] [Table 9] [Table 10] [Table 11] [Table 12] [Table 13]

Table 14

Table 15

Table 16

Table 17

Table 18

Table 19

Table 20

Table 21

Table 22

Table 23

Table 24

Table 25

Table 26

Table 27

Table 28

[0212]

Table 29

Table 30

Table 31

Table 32

Table 33

Table 34

Table 35

Table 36

Table 37

Table 38

Table 39

Table 40

Table 41

Table 42

Table 43

Table 44

Table 45

Table 46

Table 47

Table 48

Table 49

Table 50

Table 51

Table 52

Table 53

Table 54

Table 55

Table 56

Table 57

Table 58

Table 59

Table 60

Table 61

Table 62

Table 63

Table 64

Table 65

Table 66

Table 67

Table 68

Table 69

Table 70

Table 71

Table 72

Table 73

Table 74

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Table 76

Table 77

Table 78

Table 79

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Table 81

Table 82

Table 83

Table 84

Claims

1. A binding protein comprising four polypeptide chains forming three antigen binding sites, the first polypeptide chain having the formula: V L2 -L 1 -V L1 -L 2 -C L [I] The structure includes: The second polypeptide chain has the formula: V H1 -L 3 -V H2 -L 4 -C H1 - Hinge - C H2 -C H3 [II] The structure includes: The third polypeptide chain has the formula: V H3 -C H1 - Hinge - C H2 -C H3 [III] The structure includes: The fourth polypeptide chain has the formula: V L3 -C L [IV] The structure includes: Where: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V L3 is a third immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; V H3 is a third immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1 a heavy chain constant domain; C H2 is immunoglobulin C H2 a heavy chain constant domain; C H3 is immunoglobulin C H3 a heavy chain constant domain; The hinge is C H1 and C H2 an immunoglobulin hinge region connecting the domains; L 1 , L 2 , L 3 and L 4 is an amino acid linker; a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair; V H1 and V L1 forms a first antigen-binding site; V H2 and V L2 forms a second antigen-binding site which binds to the CD3 polypeptide, V H2 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22), L2 The domain is QSLVHX 1 NX 2 X 3 TY, where X 1 is E or Q, and X 2 is A or L, and X 3 is Q, R, or F; 1 NX 2 X 3 a CDR-L1 sequence comprising the amino acid sequence of TY (SEQ ID NO:293), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30); V H3 and V L3 forms a third antigen-binding site that binds to an HIV target protein.

2. The binding protein of claim 1 , wherein the first antigen-binding site binds to a CD28 polypeptide.

3. V H1 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO: 33), L1 3. The binding protein of claim 2, wherein the domain comprises a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of KAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO:36).

4. V H1 The domain is and / or comprising the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWGKGTTVTVSS (SEQ ID NO:59); L1 The domain is DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO: 60), The binding protein of claim 3.

5. V L2 5. The binding protein of any one of claims 1 to 4, wherein the CDR-L1 sequence of the domain comprises an amino acid sequence selected from the group consisting of QSLVHQNAQTY (SEQ ID NO:24), QSLVHENLQTY (SEQ ID NO:25), QSLVHENLFTY (SEQ ID NO:26), and QSLVHENLRTY (SEQ ID NO:27).

6. V H2 The domain is and / or comprising the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO:52); L2 The domain is DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSW YLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 54), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 55), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHE NLFTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:56), and DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:57), The binding protein of claim 5.

7. 7. The binding protein of any one of claims 1 to 6, wherein the third antigen-binding site binds to an HIV target protein selected from the group consisting of glycoprotein 120, glycoprotein 41 and glycoprotein 160.

8. (a) V H3 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of NCPIN (SEQ ID NO: 1), a CDR-H2 sequence comprising the amino acid sequence of WMKPRHGAVSYARQLQG (SEQ ID NO: 2), and a CDR-H3 sequence comprising the amino acid sequence of GKYCTARDYYNWDFEH (SEQ ID NO: 3), L3 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of RTSQYGSLA (SEQ ID NO:4), a CDR-L2 sequence comprising the amino acid sequence of SGSTRAA (SEQ ID NO:5), and a CDR-L3 sequence comprising the amino acid sequence of QQYEF (SEQ ID NO:6); (b) V H3 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTAHI (SEQ ID NO: 7), a CDR-H2 sequence comprising the amino acid sequence of IKPQYGAV (SEQ ID NO: 8) or IKPQYGAT (SEQ ID NO: 9), and a CDR-H3 sequence comprising the amino acid sequence of DRSYGDSSWALDA (SEQ ID NO: 10), L3 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGVGSD (SEQ ID NO:11), a CDR-L2 sequence comprising the amino acid sequence of HTS (SEQ ID NO:12), and a CDR-L3 sequence comprising the amino acid sequence of CQVLQF (SEQ ID NO:13); or (c) V H3 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of DCTLN (SEQ ID NO: 14), a CDR-H2 sequence comprising the amino acid sequence of WLKPRWGAVNYARPLQG (SEQ ID NO: 15), and a CDR-H3 sequence comprising the amino acid sequence of GKNCDYNWDFEH (SEQ ID NO: 16); L3 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of RTSQYGSLA (SEQ ID NO: 17), a CDR-L2 sequence comprising the amino acid sequence of SGSTRAA (SEQ ID NO: 18), and a CDR-L3 sequence comprising the amino acid sequence of QQYEF (SEQ ID NO: 19); The binding protein of claim 7.

9. (a) V H3 The domain is and / or comprising the amino acid sequence of QVRLSQSGGQMKKPGDSMRISCRASGYEFINCPINWIRLAPGKRPEWMGWMKPRHGAVSYARQLQGRVTMTRDMYSETAFLELRSLTSDDTAVYFCTRGKYCTARDYYNWDFEHWGQGTPVTVSS (SEQ ID NO: 43); L3 The domain comprises the amino acid sequence of SLTQSPGTLSLSPGETAIISCRTSQYGSLAWYQQRPGQAPRLVIYSGSTRAAGIPDRFSGSRWGPDYNLTISNLESGDFGVYYCQQYEFFGQGTKVQVDIK (SEQ ID NO:45); (b) V H3 The domain is QVRLSQSGGQMKKPGDSMRISCRASGYEFINCPINWIRLAPGKRPEWMGWMKPRHGAVSYARQLQGRVTMTRQLSQDPDDPDWGTAFLELRSLTSDDTAVYFCTRGKYCTARDYYNWDFEHWGQGTPVTVSS (SEQ ID NO: 44), and / or L3 The domain comprises the amino acid sequence of SLTQSPGTLSLSPGETAIISCRTSQYGSLAWYQQRPGQAPRLVIYSGSTRAAGIPDRFSGSRWGPDYNLTISNLESGDFGVYYCQQYEFFGQGTKVQVDIK (SEQ ID NO:45); (c) V H3 The domain is and / or comprising the amino acid sequence of V L3 The domain comprises the amino acid sequence of YIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGVPSRFSGSGFHTSFNLTISDLQADDIATYYCQVLQFFGRGSRLHIK (SEQ ID NO:49); (d) V H3 The domain is and / or comprising the amino acid sequence of V L3 The domain comprises the amino acid sequence of YIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGVPSRFSGSGFHTSFNLTISDLQADDIATYYCQVLQFFGRGSRLHIK (SEQ ID NO:49); (e) V H3 The domain is and / or comprising the amino acid sequence of V L3 The domain comprises the amino acid sequence of YIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGVPSRFSGSGFHTSFNLTISDLQADDIATYYCQVLQFFGRGSRLHIK (SEQ ID NO:49); or (f) V H3 The domain is and / or comprising the amino acid sequence of QVQLVQSGGQMKKPGESMRISCRASGYEFIDCTLNWIRLAPGKRPEWMGWLKPRWGAVNYARPLQGRVTMTRQLSQDPDDPDWGTAFLELRSLTVDDTAVYFCTRGKNCDYNWDFEHWGRGTPVIVSS (SEQ ID NO:50); L3 9. The binding protein of claim 8, wherein the domain comprises the amino acid sequence LTQSPGTLSLSPGETAIISCRTSQYGSLAWYQQRPGQAPRLVIYSGSTRAAGIPDRFSGSRWGPDYNLTISNLESGDFGVYYCQQYEFFGQGTKVQVDIK (SEQ ID NO:51).

10. L 1 , L 2 , L 3 Or L 4 10. The binding protein of claim 1, wherein at least one of is independently 0 amino acids in length.

11. (a) L 1 , L 2 , L 3 and L 4 are each independently 0 amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:40), GGGGSGGGGSGGGGGS (SEQ ID NO:41), S,RT,TKGPS (SEQ ID NO:39), GQPKAAP (SEQ ID NO:38), and GGSGSSGSGG (SEQ ID NO:42); or (b) L 1 , L 2 , L 3 and L 4 each independently comprises a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:40), GGGGSGGGGSGGGGGS (SEQ ID NO:41), S,RT,TKGPS (SEQ ID NO:39), GQPKAAP (SEQ ID NO:38), and GGSGSSGSGG (SEQ ID NO:42).

12. L 1 contains the sequence GQPKAAP (SEQ ID NO:38), 2 comprises the sequence TKGPS SEQ ID NO:39), 3 contains a sequence S, and L 4 The binding protein of any one of claims 1 to 9, wherein:

13. L 1 , L 2 , L 3 Or L 4 The binding protein of any one of claims 1 to 9, wherein at least one of said amino acids comprises the sequence DKTHT (SEQ ID NO: 37).

14. L 1 , L 2 , L 3 and L 4 each independently comprises the sequence DKTHT (SEQ ID NO:37).

15. Hinge-C of the second and third polypeptide chains H2 -C H3 The domain is human IgG4 hinge-C H2 -C H3 domain, hinge-C H2 -C H3 15. The binding protein of any one of claims 1 to 14, wherein each of the domains comprises amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, the amino acid substitutions being F234A and L235A.

16. Hinge-C of the second and third polypeptide chains H2 -C H3 The domain is human IgG4 hinge-C H2 -C H3 domain, hinge-C H2 -C H3 15. The binding protein of any one of claims 1 to 14, wherein each of the domains comprises amino acid substitutions at positions corresponding to positions 233 to 236 of human IgG4 according to the EU index, the amino acid substitutions being E233P, F234V, L235A, and a deletion at position 236.

17. Hinge-C of the second and third polypeptide chains H2 -C H3 The domain is human IgG4 hinge-C H2 -C H3 domain, hinge-C H2 -C H3 17. The binding protein of any one of claims 1 to 16, wherein each of the domains comprises amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P and R409K.

18. Hinge-C of the second and third polypeptide chains H2 -C H3 The domain is human IgG1 hinge-C H2 -C H3 domain, hinge-C H2 -C H3 15. The binding protein of any one of claims 1 to 14, wherein the domains comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, respectively, and the amino acid substitutions are L234A, L235A, and P329A.

19. Hinge-C of the second and third polypeptide chains H2 -C H3 The domain is human IgG1 hinge-C H2 -C H3 domain, hinge-C H2 -C H3 15. The binding protein of any one of claims 1 to 14, wherein the domains comprise amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgG1 according to the EU index, respectively, and the amino acid substitutions are S298N, T299A, and Y300S.

20. Hinge-C of the second polypeptide chain H2 -C H3 The domain comprises amino acid substitutions at positions corresponding to 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being Y349C, T366S, L368A, and Y407V; H2 -C H3 The domain is EU 20. The binding protein of any one of claims 1 to 19, comprising amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the CDR index, said amino acid substitutions being S354C and T366W.

21. Hinge-C of the second polypeptide chain H2 -C H3 The domain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being S354C and T366W; H2 -C H3 20. The binding protein of any one of claims 1 to 19, wherein the domain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being Y349C, T366S, L368A, and Y407V.

22. (a) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:73; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:74; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:75; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:76; (b) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:77; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:78; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:79; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:80; (c) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:81; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:82; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:83; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:84; (d) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:85; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:86; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:87; the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:88; (e) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:89; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:90; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:91; the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:92; (f) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:93; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:94; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:95; the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:96; (g) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:97; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:98; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:99; the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:100; (h) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 109; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 110; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 111; the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 112; (i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:113; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:114; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:115; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:116; (j) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:121; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:122; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:123; the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:124; (k) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 131; the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 132; (l) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 133; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 134; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 135; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 136; (m) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 138; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 139; the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 140; (n) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 141; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 142; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 143; the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 144; (o) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:145; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:146; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:147; the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:148; (p) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 150; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 151; the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 152; (q) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 153; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 154; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 155; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 156; (r) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:157; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:158; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:159; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:160; (s) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:161; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:162; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:163; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:164; (t) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 165; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 166; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 167; the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 168; (u) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 169; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 170; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 171; the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 172; or (v) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 173; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 174; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 175; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

176.

23. 23. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a binding protein according to any one of claims 1 to 22.

24. 24. An expression vector comprising the nucleic acid molecule of claim 23.

25. 25. An isolated host cell comprising the nucleic acid molecule of claim 23 or the expression vector of claim 24.

26. 26. The isolated host cell of claim 25, which is a mammalian or insect cell.

27. A pharmaceutical composition comprising the binding protein of any one of claims 1 to 22 and a pharma- ceutically acceptable carrier.

28. Use of at least one binding protein according to any one of claims 1 to 22 in the manufacture of a medicament for preventing and / or treating an HIV infection in a patient.

29. 29. The use of claim 28, wherein the at least one binding protein is administered in combination with a standard antiretroviral therapy.

30. 30. The use according to claim 28 or claim 29, wherein administration of the at least one binding protein results in the elimination of one or more latent and / or chronically HIV infected cells in a patient.

31. The use according to any one of claims 28 to 30, wherein the patient is a human.

32. 28. The pharmaceutical composition according to claim 27 for the prevention and / or treatment of an HIV infection in a patient.

33. 33. The pharmaceutical composition of claim 32, wherein the pharmaceutical composition is administered in combination with a standard antiretroviral therapy.

34. 34. The pharmaceutical composition of claim 32 or claim 33, wherein the pharmaceutical composition causes elimination of one or more latent and / or chronically HIV infected cells in a patient.

35. The pharmaceutical composition according to any one of claims 32 to 34, wherein the patient is a human.

36. A vector system comprising one or more vectors encoding the first, second, third and fourth polypeptide chains of the binding protein of any one of claims 1 to 22.

37. 37. The vector system of claim 36, comprising a first vector encoding a first polypeptide chain of a binding protein, a second vector encoding a second polypeptide chain of the binding protein, a third vector encoding a third polypeptide chain of the binding protein, and a fourth vector encoding a fourth polypeptide chain of the binding protein.

38. A kit comprising a polynucleotide, (a) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:189, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:190, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:191, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:192; (b) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:193, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:194, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:195, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:196; (c) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:197, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:198, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:199, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:200; (d) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:201, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:202, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:203, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:204; (e) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:205, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:206, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:207, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:208; (f) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:209, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:210, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:211, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:212; (g) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:213, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:214, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:215, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:216; (h) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:225, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:226, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:227, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:228; (i) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:229, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:230, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:231, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:232; (j) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:237, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:238, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:239, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:240; (k) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:245, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:246, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:247, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:248; (l) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:249, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:250, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:251, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:252; (m) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:253, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:254, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:255, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:256; (n) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:257, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:258, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:259, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:260; (o) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:261, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:262, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:263, a third polynucleotide comprising the nucleotide sequence, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:264; (p) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:265, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:266, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:267, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:268; (q) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:269, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:270, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:271, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:272; (r) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:273, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:274, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:275, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:276; (s) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:277, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:278, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:279, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:280; (t) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:281, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:282, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:283, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:284; (u) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:285, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:286, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:287, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:288; or (v) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:289, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:290, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:291, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:

292. The kit comprising:

39. 40. The kit of claim 38, wherein the first, second, third, and fourth polynucleotides are present on one or more expression vectors.

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