RABV-g protein binders and compositions and methods of use thereof

Abstract

Provided herein are molecules or antibodies that immunospecifically binds to a surface unit or a transmembrane unit of a rabies virus glycoprotein (RABV-G). The molecules and antibodies typically including an antigen binding region of an antibody having six complementarity determining regions (CDRs). Also provided are antibody-conjugates (e.g., antibody-drug conjugates) and fusion proteins can include an antigen binding domain and a heterologous amino acid sequence. For example, chimeric antigen receptor (CAR) polypeptides including the provided antigen binding domains are also provided. In some forms, the CAR includes an extracellular antigen binding region, a spacer region, a transmembrane region, and intracellular signaling region. In some forms, the CAR includes a co-stimulatory domain. Nucleic acids encoding the molecules, antibodies, and fusions proteins such as CAR, and cells expressing the same are also provided. Pharmaceutical compositions including the provided compositions and methods of use thereof are also provided.

Description

[0001] ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0002] RABV-G PROTEIN BINDERS

[0003] AND COMPOSITIONS AND METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims benefit of and priority to U.S. Provisional Application No. 63 / 724,041, filed November 22, 2024, which is specifically incorporated by reference herein in its entirety.

[0005] REFERENCE TO THE SEQUENCE LISTING

[0006] The Sequence Listing submitted as an XML file named “LJI2024- 112-02_PCT.xml” created on November 24, 2025, and having a size of 32,752 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.834(c)(1).

[0007] FIELD OF THE INVENTION

[0008] The field of the invention is generally directed to RABV-G binding proteins, including but not limited to antibodies, and use thereof in RABV-G related research and therapy.

[0009] BACKGROUND OF THE INVENTION

[0010] Rabies is the most lethal human disease discovered, with a nearly 100% fatality rate for untreated infections. These infections cause over 60,000 deaths annually, with approximately 40% of deaths occurring in children. While lifesaving rabies vaccines and post-exposure treatments are available, both have flaws that prevent their universal use. Rabies vaccines elicit only a short-term protective antibody response, with re-vaccination required as frequently as every six months to maintain protective antibody titers. As a result, most potential human rabies transmissions are prevented through post-exposure treatment instead of pre-exposure vaccination.

[0011] Rabies post-exposure treatment consists of a 4- or 5-dose vaccine series administered in combination with human rabies immunoglobulin (HRIG), polyclonal serum derived from the blood of vaccinated human volunteers. Human rabies immunoglobulin (IIRIG), derived from the serum of vaccinees, is a key component of rabies treatment, but its high cost can make treatment prohibitively expensive, especially in low-income countries where rabies deaths are highest. Because HRIG is human-derived, it poses a risk for the transmission of bloodborne pathogens and can be prohibitively expensive, contributing to fatality rates in the low-income countries where rabies deaths are highest. Both rabies vaccines and HRIG are also specific to rabies virus, showing little or no cross-neutralization for the seventeen rabies- related emerging lyssaviruses, many of which can be transmitted to humans and cause identical clinical symptoms and disease HRIG is also rabies-specific, with little efficacy

[0012] 45803765 1 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US against similar emerging lyssaviruses that can cause clinical symptoms in humans identical to those of rabies. To control rabies transmission and develop therapies for emerging lyssaviruses, there is a need to develop more durable, broadly neutralizing, and affordable rabics / lyssavirus vaccines and antibody therapeutics. However, rational vaccine and therapeutic antibody design require high-resolution structures to guide antibody / glycoprotein interactions.

[0013] Recent research has focused on the development of anti-rabies monoclonal antibodies to replace HRIG and on the production of next-generation rabies vaccines, but these efforts have been hampered by insufficient structural information about how antibodies bind across the surface of rabies virus glycoprotein (RABV-G). Antigenic sites have been previously mapped in different schemes by competition or mutations alone, with too few high-resolution structures to illuminate the antigenic landscape and therapeutic choice opportunities.

[0014] Thus, there remains a need for improvement in this area.

[0015] It is therefore an object of the invention to provide compositions that specifically bind to RABV-G, and methods of use thereof.

[0016] BRIEF SUMMARY OF THE INVENTION

[0017] Provided herein arc molecules or antibodies that immunospccifically binds to a surface unit or a transmembrane unit of a rabies virus glycoprotein (RABV-G). The molecules and antibodies typically including an antigen binding region of an antibody having six complementarity determining regions (CDRs).

[0018] For example, in some forms the CDRs include at least one CDR of the CDRs of anti- RABV-G antibody A2, A4, All, or a variant thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto: with all remaining CDRs independently selected from anti- RABV-G antibodies A2, A4, Al 1, and variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

[0019] In some forms, the CDRs include three heavy chain variable region CDRs independently selected from the heavy chain variable region CDRs of anti-RABV-G antibodies A2, A4, Al l, and variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto in combination with three light chain variable region CDRs independently selected from the light chain variable region CDRs of anti-RABV-G antibodies A2, A4, Al l, and variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

[0020] In some forms, the CDRs include the three heavy chain variable region CDRs of anti- RABV-G antibody A2, A4, Al l, or variants thereof with at least 60, 70, 75, 80, 85, 90, or

[0021] 45803765 2 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0022] 95% sequence identity thereto in combination with the three light chain variable region CDRs for anti- RABV-G antibody A2, A4, Al l or variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

[0023] In some format, the six CDRs arc

[0024] (A) the three heavy chain variable region CDRs and the three light chain variable region CDRs of anti-RABV-G antibody A2;

[0025] (B) the three heavy chain variable region CDRs and the three light chain variable region CDRs of anti-RABV-G antibody A4; or

[0026] (C) the three heavy chain variable region CDRs and the three light chain variable region CDRs of anti-RABV-G antibody Al l.

[0027] The six CDRs can be in the same orientation as in the anti-RABV-G antibody from which they were selected.

[0028] In some forms, the heavy chain variable regions and light chain variable regions of antibody A2, A4, and Al l are the heavy chain variable regions of A2, A4, or Al 1 according to Tables 2 or variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto, and the light chain variable regions of A2, A4, and Al 1 according to Table 4 or variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

[0029] In some forms, the six CDRs are the CDRs according to:

[0030] Table 1: Heavy Chain Variable Region CDR Sequences

[0031] Table 3: Eight Chain Variable Region CDR Sequences

[0032] 45803765 3 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US or variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto. For example, the molecule or antibody can include a heavy chain variable region including three CDRs and a light chain variable region including three CDRs, wherein

[0033] (A) the three heavy chain variable region CDRs include GYTFTNYG (SEQ ID NO: 1), INTYTGEP (SEQ ID NOG), RGDYFGTKYYFDY (SEQ ID NOG), respectively, and the three light chain variable region CDRs include QDVSTA (SEQ ID NO:6), SAS, QQHYNTPT (SEQ ID NO:7) respectively;

[0034] (B) the three heavy chain variable region CDRs include GYSFTDYI (SEQ ID NOTO), INPYYGTT (SEQ ID NO: 11), RADGPDYFDY (SEQ ID NO:12), respectively, and the three light chain variable region CDRs include QDVNTA (SEQ ID NO: 15), SAS, CQQHYNTPPT (SEQ ID NO: 16), respectively;

[0035] (C) the three heavy chain variable region CDRs include GYTFTDYA (SEQ ID NO: 19), ITTYSGDA (SEQ ID NO:20), ARPYYYGNSWFAY (SEQ ID NO:21), respectively, and the three light chain variable region CDRs include QNVGTN (SEQ ID NO:24), SAS, QQYHTYPLT (SEQ ID NO:25), respectively;

[0036] In some forms, the antigen binding region includes the heavy chain variable region of A2, A4, Al 1, or a variant thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

[0037] In some forms, the antigen binding region includes the light chain variable region of A2, A4, Al 1, or a variant thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

[0038] The antigen binding region can include the heavy chain variable region and light chain variable region of A2, A4, Al 1, or variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

[0039] The antigen binding region can include the heavy chain variable region and / or light chain variable region according to Table 2 and / or Table 4, or variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

[0040] The antibody is an intact antibody and functional antibody fragment or fusion protein. Functional fragments or fusion proteins can be, for example, Fab fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain

[0041] 45803765 4 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US antibody fragments optionally single chain variable fragments (scFv), and single region antibodies optionally selected from sdAb, sdFv, and nanobody fragments.

[0042] The antibodies can be intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and hctcroconjugatc antibodies, and multispccific antibodies optionally selected bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, or tandem tri-scFv.

[0043] In some forms, the antibody is an IgM, IgE, IgA, IgD, or IgG optionally an IgGl, IgG2, IgG3, or IgG4.

[0044] The antibody can be detectably labeled or include a conjugated toxin, drug, receptor, enzyme, receptor ligand.

[0045] Also provided are fusion proteins can include a disclosed antigen binding domain and a heterologous amino acid sequence. For example, chimeric antigen receptor (CAR) polypeptides including the provided antigen binding domains are also provided. In some forms, the CAR includes an extracellular antigen binding region, a spacer region, a transmembrane region, and intracellular signaling region. In some forms, the CAR includes a co-stimulatory domain.

[0046] Nucleic acids encoding the molecules, antibodies, and fusions proteins such as CAR, and cells expressing the same are also provided. For example, the nucleic acids can include an expression control sequence operably linked thereto, optionally wherein the expression control sequence includes a promoter. The nucleic acid can be a vector. The cell can be an immune cell.

[0047] Pharmaceutical compositions including the provided molecules, antibodies, fusion proteins, CARs, etc. are also provided. In some forms, the antibodies include a drug conjugated thereto and / or have antibody-dependent cell-mediated cytotoxicity (ADCC) activity, or complement-dependent cytotoxicity activity (GDC), and / or is a bispecific immune cell engager.

[0048] Methods of treatment are also provided and can include administering to a subject in need thereof an effective amount of a pharmaceutical composition. In some embodiments, the subject has a disease or disorder caused by or characterized by increased presence of RABV-G or a fragment thereof. In some forms, the subject has a rabies infection, is at risk for developing symptoms of rabies infection, or complications caused by rabies infection. In

[0049] 45803765 5 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US some forms, the subject has a rabies infection and RABV-G or fragment thereof is an antigen of the rabies infection.

[0050] Methods of detecting RABV-G or fragment thereof are also provided and typically include contacting a biological sample with one or more of the disclosed molecules or antibodies, and detecting binding between the molecule(s) or antibod(ies) and the RABV-G or fragment thereof. In some forms, the methods include determining that the sample includes increased RABV-G or fragment thereof if the level of detected binding is higher in the biological sample than in a control. The binding can be detected by, for example, an immunoassay, immunohistochemistry, Western blotting, surface plasmon resonance, flow cytometry (FACS) analysis, or a biochip. Immunoassays include, but are not limited to, enzyme immunoassay (EIA), radioimmunoassay (RIA), fluoroimmunoassay (FIA), chemiluminescent immunoassay (CLIA) and counting immunoassay (CIA), homogeneous enzyme-multiplied immunoassays (“EMIT”), apoenzyme reactivation immunoassay (“ARIS”), dipstick immunoassays, and immuno-chromatography assays.

[0051] In some forms, the biological sample is cells or a cell lysate or a fraction thereof or a fluid optionally blood, saliva, or urine. The cells or cell lysate or fraction thereof can be derived from a biopsy from a subject. The biopsy sample can contain, or be suspected of containing, rabies infection.

[0052] Methods of diagnosing a subject with a RABV-G-related disease or disorder are also provided and include detecting RABV-G or fragment thereof, i.e., according to the provided detection methods. The methods can further include treating the subject, e.g., with a therapy effective for treating a RABV-G-related diseases and disorders. In some forms, the treatment includes administering the subject an effective amount of a disclosed pharmaceutical composition and / or an antiviral.

[0053] BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 is an illustration showing the docked structures of complexes formed by antibody-glycoprotein interactions .

[0055] Figure 2 is an illustration showing cryo-EM footprints of the nine rabies antibodies.

[0056] Figures 3A-3C are plots showing neutralization and binding / uptake data. Figs. 3A- 3B show neutralization titers, measured by the percentage of infection (Y-axis), across various concentrations (loglO nM) of 9 IgGs (Fig. 3A) and 8 Fabs (Fig. 3B), as indicated. Fig. 3C shows plots comparing the percentage of infection (Y-axis) across various concentrations (logio nM) of IgGs and Fabs derived from the same monoclonal antibody.

[0057] 45803765 6 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0058] Figure 4A is a series of plots showing binding affinity and competition for different monoclonal antibodies as indicated. Figure 4B is a heat map showing the % binding of the antibodies to analyte (in solution) and ligand (on Biosensor).

[0059] Figure 5 arc plots demonstrating the impact of pH on binding affinity, as measured by OD450 (Y-axis), across different concentrations of the indicated monoclonal antibody.

[0060] Figures 6A-6D show antibodies at antigenic sites 11 and IV recognize an overlapping core of amino acids. Mouse monoclonal antibodies A2 (Figure 6A) and Al l (Figure 6B) and human monoclonal antibodies 8C5 (Figure 6C) and 4H3 (Figure 6D) recognize antigenic sites II and IV. Antibody binding footprints (left) are shown depicting all residues on RABV-G within 4A of the bound antibody. Atomic models of antibodies bound to RABV- G are shown in the middle, and the core region of shared amino acids between all four antibodies is shown to the right. Van der Waals interactions between RABV-G and antibodies are depicted with a dotted line.

[0061] Figures 7A and 7B show discovered antigenic sites on the central domain (site V) and fusion domain (site VI). Human monoclonal antibodies 4C12 (Figure 7A) and RVC68 (Figure 7B) recognize previously uncovered binding sites on the central domain and fusion domain, respectively. Antibody binding footprints (top left) are shown depicting all residues on RABV-G within 4A of the bound antibody and atomic models of RAB V-G / antibody complexes are shown on the top right. A magnified view of RAB V-G / antibody binding interactions (bottom) is shown for each antibody, with Van der Waals interactions depicted with a green dotted line and hydrogen bonds with a light blue dotted line.

[0062] Figures 8A and 8B. Site III monoclonal antibodies stabilize the pre-fusion conformation of RABV-G. Low resolution cryo-EM maps of site III monoclonal antibodies A4 (mouse) (Figure 8A) and 10H5 (human) (Figure 8B) show that site III binding monoclonal antibodies other than RVA122 also recognize the pre-fusion conformation of RABV-G. Approximate binding footprints for each monoclonal antibody are shown on the left. Low-resolution cryo-EM maps (right) are shown with new monoclonal antibodies colored teal or yellow, and RABV-G and mAb RVC68 models docked into the map. An additional site H / IV human monoclonal antibody, 7E8 (yellow), and its binding footprint are also shown. 7E8 is not depicted in the site I I / I V Figures 6A-6D due to its lower resolution.

[0063] Figure 9A-9D show rabies antibody cross-reactivity likely results from sequence conservation in the antibody binding site. Amino acid sequence conservation across eight lyssavirus glycoproteins was projected onto RABV-G (Figure 9A), with binding footprints for antibodies at each antigenic site outlined and site-specific sequence conservation shown. 45803765 7 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0064] Site III recognition is to mAb RVA122 as the other site III mAbs are two low in resolution to map specific contacts. For antibodies 4C12 (Figure 9B) and RVC68 (Figure 9C) at antigenic sites V and VI, respectively, sequence conservation was shown among cross-reactive lyssavirus glycoproteins. Sequence conservation across RABV-G antibody binding sites was also depicted on the amino acid sequence (Figure 9D, SEQ ID NO:31), with residues within 4 A of antibodies shown with color-coded circles.

[0065] Figures 10A and 10B show rabies glycoprotein (RABV-G) antigenic sites and domains. (Figure 10A) Rabies antigenic sites identified by the mutagenesis scheme include residues 263-264 (site I, green); residues 198-200 (site Ila, orange); residues 34-42 (site lib, red); residues 330-338 (site III, teal); residues 226-231 (site IV, yellow); and residues 342- 343 (site ‘a’, pink). (Figure 10B) RABV-G domains, with the Pleckstrin Homology Domain (PHD) colored orange, the Central Domain (CD) colored blue, and the Fusion Domain (FD) colored yellow.

[0066] Figure 11 show site III mAbs are pH sensitive, but site II / IV mAbs are not. ELISA assay showing binding of IgGs to RABV-G over a range of pHs. Binding for each antibody was normalized to the maximum OD450 seen for that antibody across all pH values and three experimental replicates.

[0067] Figure 12A-12E shows a map and model statistics for mAb A2. Cryo-EM map at high contour (Figure 12A), and low contour (Figure 12B) with color-coding for local resolution are shown. Atomic model docked into density (Figure 12C), FSC curves (Figure 12D), and Phenix map / model correlation coefficient graphs for unique chains (Figure 12E) are also shown. Site III binding monoclonal antibody RVA122 was also included in complexes to stabilize RABV-G pre-fusion trimers for high-resolution imaging.

[0068] Figures 13A-13E show map and model statistics for mAb Al 1 . Cryo-EM map at high contour (Figure 13A), and low contour (Figure 13B) with color-coding for local resolution are shown. Atomic model docked into density (Figure 13C), FSC curves (Figure 13D), and Phenix map / model correlation coefficient graphs for unique chains (Figure 13E) are also shown. Site III binding monoclonal antibody RVA122 was also included in complexes to stabilize RABV-G pre-fusion trimers for high-resolution imaging.

[0069] Figures 14A-14E show map and model statistics for mAb 8C5. Cryo-EM map at high contour (Figure 14A), and low contour (Figure 14B) with color-coding for local resolution are shown. Atomic model docked into density (Figure 14C), FSC curves (Figure 14D), and Phenix map / model correlation coefficient graphs for unique chains (Figure 14E) are also shown. Site III binding monoclonal antibody RVA122 was also included in complexes to

[0070] 45803765 8 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US stabilize RABV-G pre-fusion trimers for high-resolution imaging.

[0071] Figures 15A-15E show a map and model statistics for mAbs 4C12 and 4H3. Cryo- EM map at high contour (Figure 15A), and low contour (Figure 15B) with color-coding for local resolution arc shown. Atomic model docked into density (Figure 15C), FSC curves (Figure 15D), and Phenix map / model correlation coefficient graphs for unique chains (Figure 15E) are also shown. Site 111 binding monoclonal antibody RVA122 was also included in complexes to stabilize RABV-G pre-fusion trimers for high-resolution imaging.

[0072] Figures 16A-16E show map and model statistics for mAb RVC68. Cryo-EM map at high contour (Figure 16A), and low contour (Figure 16B) with color-coding for local resolution are shown. Atomic model docked into density (Figure 16C), FSC curves (Figure 16D), and Phenix map / model correlation coefficient graphs for unique chains (Figure 16E) are also shown. While this complex formed stable trimers, flexibility in the fusion domain where RVC68 binds necessitated processing the glycoprotein as a monomer with extensive 3D classification sorting to solve a high-resolution structure. Site II binding monoclonal antibody RVA122 was also included in this complex.

[0073] Figures 17A-17D show map statistics for low-resolution maps of mAbs A4, 7E8, and 10H5. Cryo-EM maps at high contour (Figures 17A and 17C), and FSC curves (Figures 17B and 17D) for mAbs A4, 7E8, and 10H5 are shown. Complexes consist of RABV-G trimers with either antibodies A4, 7E8, and RVC68 or antibodies 10H5 and 7E8.

[0074] DETAILED DESCRIPTION OF THE INVENTION

[0075] A. Definitions

[0076] As used herein, the term “binds” in reference to the interaction of a binding protein and an antigen means that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen. For example, a binding protein recognizes and binds to a specific antigen structure rather than to antigens generally. For example, if a binding protein binds to epitope "A", the presence of a molecule containing epitope “A” (or free, unlabeled “A”), in a reaction containing labeled “A” and the binding protein, will reduce the amount of labeled “A” bound to the binding protein.

[0077] As used herein, a molecule is said to be able to “immunospecifically bind” a second molecule if such binding exhibits the specificity and affinity of an antibody to its cognate antigen. Antibodies are said to be capable of “immunospecifically binding” to a target region or conformation (“epitope”) of an antigen if such binding involves the antigen recognition site of the immunoglobulin molecule. An antibody that immunospecifically binds to a particular antigen may bind to other antigens with lower affinity if the other antigen has some 45803765 9 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US sequence or conformational similarity that is recognized by the antigen recognition site as determined by, e.g., immunoassays, BIACORE® assays, or other assays known in the art, but would not bind to a totally unrelated antigen. Preferably, however, antibodies (and their antigen binding fragments) will not cross-rcact with other antigens. Antibodies may also bind to other molecules in a way that is not immunospecific, such as to FcR receptors, by virtue of binding domains in other regions / domains of the molecule that do not involve the antigen recognition site, such as the Fc region.

[0078] The term “substantially,” as used in the context of binding or exhibited effect, is intended to denote that the observed effect is physiologically or therapeutically relevant. Similarly, a molecule is said to have substantially the same immunospecificity and / or characteristic as another molecule, if such immunospecificities and characteristics are greater than 60% identical, greater than 70% identical, greater than 75% identical, greater than 80% identical, greater than 85% identical, greater than 90% identical, greater than 95% identical, or greater than 97% identical).

[0079] The term “antibody” is used in the broadest sense unless clearly indicated otherwise. Therefore, an "antibody" can be naturally occurring or man-made such as monoclonal antibodies produced by conventional hybridoma technology. Antibodies include monoclonal and polyclonal antibodies as well as fragments and polymers containing the antigen binding domain and / or one or more complementarity determining regions of these antibodies. As used herein, the term "antibody" refers to any form of antibody or antigen binding fragment or recombinant protein, and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they specifically bind the target antigen. Any specific antibody can be used in the methods and compositions provided herein. The term “antibody” encompasses an immunoglobulin molecule that possesses a “variable region” antigen recognition site. Thus, the term "antibody" encompasses a molecule having at least one variable region from a light chain immunoglobulin molecule and at least one variable region from a heavy chain molecule that in combination form a specific binding site for the target antigen. The term antibody includes monoclonal antibodies, multi-specific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies (See e.g., Muyldemians et al., 2001, Trends Biochem. Sci. 26:230; Nuttall et al., 2000, Cur. Pharm. Biotech. 1 :253; Reichmann and Muyldermans, 1999, J. Immunol. Meth. 231 :25; International Publication Nos. WO 94 / 04678 and WO 94 / 25591 ; U.S. Patent No. 6,005,079), single-chain Fvs (scFv) (see, e.g., see Pluckthun in The

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[0081] Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer- Verlag, New York, pp. 269-315 (1994)), single chain antibodies, disulfide-linked Fvs (sdFv), intrabodies, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id and anti-anti-Id antibodies to the disclosed antibodies). In particular, such antibodies include immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, lgG2, lgG3, IgG4, IgAl and lgA2) or subclass.

[0082] The term “variable region” is intended to distinguish such domain of the immunoglobulin from domains that are broadly shared by antibodies (such as an antibody Fc domain). The variable region refers to the portions of the light and / or heavy chains of an antibody as defined herein that specifically binds to an antigen and, for example, includes amino acid sequences of CDRs; i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). For example, the variable region can include three or four FRs (e.g., FR1, FR2, FR3 and optionally FR4) together with three CDRs. VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain. The variable region includes a “hypervariable region” whose residues are responsible for antigen binding.

[0083] The hypervariable region includes amino acid residues from a “Complementarity Determining Region” or “CDR” (e.g., typically at approximately residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and at approximately residues 27-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain according to Kabat; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or those residues from a “hypervariable loop” (e.g., residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (Hl), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain according to Chothia; Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917). Conventions that include corrections or alternate numbering systems for variable domains include not only Kabat and Chothia, but also IMGT (Lefranc, et al. (2003), Dev Comp Immunol 27: 55- 77), Chothia (Chothia C, Lesk AM (1987), J Mai Biol 196: 901-917; Chothia, et al. (1989), Nature 342: 877-883) and AHo (Honegger A, Pluckthun A (2001) I Mol Biol 309: 657-670). For convenience, examples of binding proteins of the present disclosure may also be labelled according to Kabat, Chothia, or IMGT. These examples are expressly indicated as such.

[0084] “Framework Region” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined.

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[0086] As used herein, the term “antigen binding fragment” of an antibody refers to one or more portions of an antibody that contain the antibody’s Complementarity Determining Regions (“CDRs”) and optionally the framework residues that include the antibody’ s “variable region” antigen recognition site, and exhibit an ability to immunospccifically bind antigen. Such fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules and multispecific antibodies formed from antibody fragments, and mutants thereof, naturally occurring variants, and fusion proteins including the antibody’s “variable region” antigen recognition site and a heterologous protein (e.g., a toxin, an antigen recognition site for a different antigen, an enzyme, a receptor or receptor ligand, etc.). For example, the term antigen binding fragment may be used to refer to recombinant single chain Fv fragments (scFv) as well as divalent (di-scFv) and trivalent (tri-scFV) forms thereof. Such fragments can be produced via various methods known in the art.

[0087] The term “constant region” as used herein, refers to a portion of heavy chain or light chain of an antibody other than the variable region. In a heavy chain, the constant region generally includes a plurality of constant domains and a hinge region, e.g., an IgG constant region includes the following linked components, a constant heavy CHI, a linker, a CH2 and a CH3. In a heavy chain, a constant region includes a Fc. In a light chain, a constant region generally include one constant domain (a CE1).

[0088] The term “fragment crystallizable” or “Fc” or “Fc region” or “Fc portion” (which can be used interchangeably herein) refers to a region of an antibody including at least one constant domain and which is generally (though not necessarily) glycosylated and which is capable of binding to one or more Fc receptors and / or components of the complement cascade. The heavy chain constant region can be selected from any of the five isotypes: a, 5, s, y, or p. Exemplary heavy chain constant regions are gamma 1 (IgGl), gamma 2 (IgG2) and gamma 3 (IgG3), or hybrids thereof.

[0089] A “constant domain” is a domain in an antibody the sequence of which is highly similar in antibodies / antibodies of the same type, e.g., IgG or IgM or IgE. A constant region of an antibody generally includes a plurality of constant domains, e.g., the constant region of y, a or 5 heavy chain include two constant domains.

[0090] The terms “full-length antibody”, “intact antibody” or “whole antibody” are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antigen binding fragment of an antibody. Specifically, whole antibodies include those with heavy and light chains including an Fc region. The constant domains may be wild-type

[0091] 45803765 12 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US sequence constant domains (e.g., human wild-type sequence constant domains) or amino acid sequence variants thereof.

[0092] A “chimeric antibody” is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules such as antibodies having a variable region derived from a non-human antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods 125:191-202; and U.S. Patent Nos. 6,311,415, 5,807,715, 4,816,567, and 4,816,397. Chimeric antibodies including one or more CDRs from a non-human species and framework regions from a human immunoglobulin molecule can be produced using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; International Publication No. WO 91 / 09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering 7:805; and Roguska et aL, 1994, Proc. Natl. Acad. Sci. USA 91 :969), and chain shuffling (U.S. Patent No. 5,565,332).

[0093] As used herein, the term “humanized antibody” refers to an immunoglobulin including a human framework region and one or more CDR’s from a non-human (usually a mouse or rat) immunoglobulin. The non-human immunoglobulin providing the CDR’s is called the “donor” and the human immunoglobulin providing the framework is called the “acceptor.”

[0094] As used herein, the term “fragment” refers to a peptide or polypeptide including an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.

[0095] As used herein, the term “fusion protein" refers to a polypeptide formed by the joining of two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide or through linking of one polypeptide to another through reactions between amino acid side chains (for 45803765 3 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US example disulfide bonds between cysteine residues on each polypeptide). The fusion protein can be formed by the chemical coupling of the constituent polypeptides or it can be expressed as a single polypeptide from a nucleic acid sequence encoding the single contiguous fusion protein. Fusion proteins can be prepared using conventional techniques in molecular biology to join the two genes in frame into a single nucleic acid sequence, and then expressing the nucleic acid in an appropriate host cell under conditions in which the fusion protein is produced.

[0096] As used herein, the term “variant” refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide, but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and / or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.

[0097] Modifications and changes can be made in the structure of the polypeptides of the in disclosure and still obtain a molecule having similar characteristics as the polypeptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide's biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like properties.

[0098] In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. Those indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1 .3); proline (-1 .6); histidine (-3.2); glutamate (- 3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0099] 45803765 14 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0100] It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and cofactors. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within ± 2 is preferred, those within + 1 are particularly preferred, and those within + 0.5 are even more particularly preferred.

[0101] Substitution of like amino acids can also be made on the basis of hydrophilicity, particularly where the biological functional equivalent polypeptide or peptide thereby created is intended for use in immunological embodiments. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 + 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (-0.5 ± 1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent polypeptide. In such changes, the substitution of amino acids whose hydrophilicity values are within + 2 is preferred, those within + 1 are particularly preferred, and those within + 0.5 are even more particularly preferred.

[0102] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (He: Leu, Vai), (Leu: He, Vai), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Vai: He, Leu). Embodiments of this disclosure thus contemplate functional or biological equivalents of a polypeptide as set forth above. In particular, embodiments of the polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the polypeptide of interest.

[0103] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning 45803765 5 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that arc within the skill of those practicing in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNAS TAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0104] As used herein, the phrase “pharmaceutically acceptable” refers to compositions, polymers and other materials and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0105] As used herein, the phrase “pharmaceutically acceptable carrier” refers to pharmaceutically acceptable materials, compositions or vehicles, such as a liquid or solid filler, diluent, solvent or encapsulating material involved in carrying or transporting any subject composition, from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of a subject composition and not injurious to the patient.

[0106] As used herein, the term “individual,” “subject,” and “patient” are used interchangeably to refer to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient. Preferably, the patient or subject is a human, such as a human at risk for rabies infection or a human infected with rabies or related lyssaviruses.

[0107] As used herein, the term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to 45803765 g ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0108] As used herein, the term “therapeutically effective amount” refers to an amount of the therapeutic agent that, when incorporated into and / or onto particles described herein, produces some desired effect at a reasonable benefit / risk ratio applicable to any medical treatment. The effective amount may vary depending on such factors as the disease or condition being treated, the particular targeted constructs being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art may empirically determine the effective amount of a particular compound without necessitating undue experimentation. In some embodiments, the term “effective amount” refers to an amount of a therapeutic agent or prophylactic agent to reduce or diminish the risk for developing symptoms of rabies infection and / or other RABV-G-related disease and disorder.

[0109] Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a ligand is disclosed and discussed and a number of modifications that can be made to a number of molecules including the ligand are discussed, each and every combination and permutation of ligand and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B- F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Further, each of the materials, compositions, components, etc. contemplated and disclosed as above can also be specifically and independently included or excluded from any group, subgroup, list, set, etc. of such materials.

[0110] 45803765 17 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0111] These concepts apply to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific form or combination of forms of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

[0112] All methods described herein can be performed in any suitable order unless otherwise indicated or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the forms and does not pose a limitation on the scope of the forms unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0113] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0114] Use of the term “about” is intended to describe values cither above or below the stated value in a range of approx. + / - 10%; in other forms the values can range in value either above or below the stated value in a range of approx. + / - 5%; in other forms the values can range in value either above or below the stated value in a range of approx. + / - 2%; in other forms the values can range in value either above or below the stated value in a range of approx. + / - 1%. The preceding ranges are intended to be made clear by context, and no further limitation is implied.

[0115] B. Compositions

[0116] 1. Rabies virus glycoprotein (RABV-G) Binding Proteins

[0117] Disclosed are antibodies that are capable of immunospecifically binding to rabies virus glycoprotein (RABV-G) and preferably neutralizing virus displaying the RABV-G protein, including but not limited to, rabies viruses. Also disclosed are three antibodies that rabies-specific and do not recognize glycoproteins from closely related lyssaviruses, giving them potential to differentiate rabies infections from related lyssaviruses.

[0118] In some fomis, the rabies antibodies are a therapeutic antibody or cocktail to two or more antibodies for rabies post-exposure treatment, as reagents in structural biology, as diagnostic antibodies, or other use provided herein.

[0119] 45803765 18 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0120] As provided are the therapeutic antibodies and cocktails thereof that are variants engineered to increase neutralization potency, strength of binding, and recognition of glycoproteins from related lyssaviruses.

[0121] As will be evident, these molecules arc referred to interchangeably as rabies antibody, RABV-G-binding polypeptides, RABV-G binders, anti- RABV-G polypeptides, anti-RABV- G antibody, anti-RABV-G monoclonal antibody, or simply as a binder, etc. 1’hree specific monoclonal antibodies, referred to as A2, A4, and Al l, were created and exemplified in the Example below. Thus, when used herein, A2, A4, and Al l, refer to the monoclonal antibodies of the Examples, the amino acid sequences they are formed therefrom. Annotated sequences for A2, A4, and Al l are provided in Tables 1-4 and SEQ ID NOS: 1-27 provided below. The provided sequences provide the foundation of and for the additional and alternative binders provided herein.

[0122] As discussed elsewhere herein in more detail, in non-limiting embodiments, such molecules can be, for example, a monoclonal antibody, a human antibody, a chimeric antibody or a humanized antibody, or a fragment thereof, and fusion proteins formed therefrom. The antibodies and antigen binding fragments can be monospecific, bispecific, trispccific or multispccific. Any of the binding polypeptides can be linked to another compound such a drug, label, etc.

[0123] Additionally provided are chimeric antigen receptors (CARs) including such antibodies and antigen binding fragments, and cells having the CARs. i. RABV-G -binding Polypeptides

[0124] Polypeptides that selectively bind rabies virus glycoprotein (RABV-G) are provided. RABV-G is the target of the rabies neutralizing antibody response and includes three domains: a central domain (CD), a fusion domain (FD), and a Pleckstrin homology domain (PHD). The CD contains the a-helix at the core of the glycoprotein and loops which form a trimeric interface with adjacent protomers, the FD contains the fusion loops, and the PIID connects the CD and PHD. On the viral surface, RABV-G adopts multiple conformations, reversibly transitioning between monomeric, trimeric, pre-fusion, and post-fusion conformations in a pH-dependent manner. During the pre- to post-fusion transition, the glycoprotein’s central a-helices elongate and the PHD rearranges to sit directly above the CD, changing the availability of some antigenic sites. However, even at acidic pH, some glycoproteins have been shown to remain in the pre-fusion conformation, and some glycoproteins at neutral or basic pH maintain the post-fusion conformation. An exemplary RABV-G protein has the amino acid sequence represented by SEQ ID NO:31.

[0125] 45803765 19 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0126] KFPYIYTIPDKLGPWP IDIHHLSCPNNLWEDEGCLLISCESYMELKVGYISAIKMNGFTCT GWTEAETYTNFVGYYTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHNYPYDYWHLRTV KTTKESIVTISPVSVADLDYDRSLHSRVFEPGGNCSGVAASYSTYCSTNHDYTIWMPENPRL GMSCENFTNSGRKSKSEETCGFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWVAMQTSNET KWCPPQGLVNLHDFRSDEIEHLIVEELVKKREECLDALESIMTTKSVSRFRRLSHLRKLVGE FGKAYTINRLLMEADAHYKSVRIHWEIIPSKGCERLVGGRCHPHVNGVFFNGIILGIPGDGN VLIPEMQSSLLQQHMELLISVIPLMHPLA (SEQ ID NO:31).

[0127] As demonstrated in the non-limiting Examples, structural mapping significantly expanded the classical antigenic site model of RABV-G. Whereas earlier mutagenesis studies divided RABV-G into four discrete antigenic sites (I-IV), it was discovered that many antibodies previously thought to target separate sites actually converge on shared or overlapping surfaces. Five of the mapped antibodies bind a continuous region encompassing residues historically assigned to sites II and IV, demonstrating that these do not represent two distinct antigenic sites but a single extended epitope. Two additional antibodies engage epitopes centered around residues attributed to site III. Notably, two antibodies recognize previously uncharacterized regions of RABV-G that do not overlap with any classical site, supporting the designation of two new antigenic sites, termed site V and site VI. Together, these discoveries provide a refined and structurally grounded organization of RABV-G antigenicity that extends beyond the original mutagenesis-based scheme. The description of the antigenic sites are discussed below.

[0128] Human antibodies 8C5 (3.16 A), 4H3 (3.24 A), and 7E8 (~4.5 A), and murine antibodies A2 (3.01 A) and Al 1 (2.92 A) recognize the Pleckstrin homology domain (PHD), with footprints spanning residues associated with antigenic sites II and IV (Figures 6A-6D). Although these residue clusters were previously identified as distinct sites via mutagenesis12, these five structures show that antibodies recognize these residues not in two distinct groups, but instead in an overlapping ~25 nm2continuum, with niAb A2 at the apex and niAb 4H3 toward the base (Figures 6A-6D). Atomic models were built of the four site II / IV antibodies that reached high resolution (Figures 6A-6D) to identify the shared amino acid contacts. In these models, it was observed that all four site I I / I V antibodies with high-resolution structures form Van der Waals interactions with a shared set of contacts on RABV-G including residues M44, C228, and V230. Details and additional contacts, including van der Waals interactions of antibodies A2, Al l, 8C5, and 4H3 with Cl 89, N194, and / or L231, are illustrated in Figures 6A-6D. Each of these site I I / I V mAbs binds both pre- and post-fusion RABV-G

[0129] 45803765 20 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0130] (Figure 11); the Pleckstrin homology domain that they target moves as a rigid body in the conformational transition10, n.

[0131] Antigenic site HI

[0132] Human mAb 10H5 (6.69 A) and murine antibody A4 (-4.5 A) both recognize the central domain (CD) of RABV-G with footprints that overlap residues associated with antigenic site 111 (mutagenic scheme) and the RVA122 epitope11, 12(Figures 8A and 8B). All three of these antibodies recognize a conformational epitope only present on the pre-fusion conformation. Although both 10H5 and A4 only bind pre-fusion RABV-G and we observed pre-fusion trimers during imaging, there was greater structural heterogeneity in A4 / RABV-G and 10H5 / RABV-G complexes than in complexes made with RVA122 (Figures 12A-12E; Figures 13A-13E, Figures 14A-14E, Figures 15A-15E, Figures 16A-16E, Figures 17A-17E). As a result of this and lower number of pre-fusion RABV-G trimers, neither 10H5 nor A4 reached a resolution high enough to build an atomic model. The intermediate-resolution footprints, however, indicate that 10H5 and A4 bind solely to the central domain, while in contrast, RVA122 recognizes an epitope bridging the central and Pleckstrin homology domains, likely better stabilizing the pre-fusion trimer.

[0133] Antigenic site V:

[0134] Human mAb 4C12 is also specific for the pre-fusion conformation of RABV-G, but does not compete with RVA122 (Figures 15A-15E). The cryo-EM structure of mAb 4C12 (3.24 A) revealed a new antigenic site below antigenic site III, not associated with any residues previously identified in RABV-G mutagenesis studies. (Figure 7A). This antigenic site was termed ‘site V as a continuation of the classical rabies antigenic site nomenclature (Figure 7B). Here, the 4C12 footprint bridges the lower central domain (CD) to residues W12, P13, P16, and N57 in the Pleckstrin homology domain (PHD). This anchoring of the CD and PHD is similar to the pre-fusion trimer stabilizing effect previously observed with RVA12211. Notably, 4C12 is the only pre-fusion-specific antibody yet characterized that does not recognize antigenic site III.

[0135] Antigenic site VI: mAb RVC68 does not compete with any other mAb in the study. The cryo-EM structure of its complex reveals another new antigenic site, termed ‘site VI’, on the fusion domain (FD) (Figure 7B). RVC68 recognizes RABV-G via four hydrogen bonds and an extensive network of Van der Waals interactions, with almost half of the interactions contributed by six aromatic amino acids in the RVC68 paratope. Residues W32 in CDRE1 , F94 in CDRL3, Y50 and F52 in CDRH2, Y102 and Y103 in CDRH3 mediate contact to

[0136] 45803765 21 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0137] RABV-G residues 110-118 and the horizontal a-helix adjacent to them (Figure 7B). RVC68 binds RABV-G at both neutral and acidic pH (pre- and post-fusion conformations, respectively), with more binding at acidic pH (Figure 11). While the residues in the RVC68 epitope maintain the same shape in both the pre- and post-fusion conformations of RABV- G10, u, the post-fusion conformation may be more sterically accessible to IgG.

[0138] In some embodiments, the disclosed RABV-G-binding polypeptides bind to the RABV-G, or a homologue, paralogue, or ortholog thereof. In some embodiments, the RABV-G -binding polypeptides bind to a variant having at least 60% up to 99% identity to RABV-G polypeptide. For example in some forms, the variant sequence has at least about 60%, 70%, 75%, 80%, 85%, 90%, or 95% identity to RABV-G polypeptide. Therefore, in some forms, the variant consensus amino acid sequence for the RABV-G polypeptide has an amino acid sequence that has one or more amino acids such as one or more substitutions, deletions or additions at any one of the amino acid positions of RABV-G polypeptide.As introduced above, the term antibody herein refers to natural or synthetic polypeptides that bind a target antigen. The term includes polyclonal and monoclonal antibodies, including intact antibodies and functional e.g., antigen-binding) antibody fragments, including Fab fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and sub classes thereof, IgM, IgE, IgA, and IgD. Thus, although typically discussed in the context of IgG, the target antibody of the Ig-Fc-specific immunoglobulin variable domain can be IgM, IgE, IgA, or IgD.

[0139] The disclosed binders typically include six complementarity determining regions (CDRs). The binder can include any six CDRs of the heavy and light chain variable regions of A2, A4, and Al 1, or variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto, in any combination.

[0140] In some forms, the binder includes heavy and light chain variable regions. In some forms, the heavy chain variable region CDRs are independently selected from the heavy chain variable region CDRs of A2, A4, and Al l, and variants thereof with at least 60, 70, 75, 45803765 22 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0141] 80, 85, 90, or 95% sequence identity thereto. In some forms, the light chain variable region CDRs are independently selected from the light chain variable region CDRs of A2, A4, and Al l, and variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

[0142] In some forms, the CDRs are presented in the same orientation (i.e., same N-to-C terminus orientation, e.g., CDR1, CDR2, CDR3) as in the RABV-G binder from which it originated. Thus, in some forms the heavy chain variable region of the binder includes a CDR1 from the heavy chain variable region CDRls of A2, A4, or Al l, or a variant thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto; the heavy chain variable region of the binder includes a CDR2 from the heavy chain variable region CDR2s of A2, A4, or Al 1, or a variant thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto; the heavy chain variable region of the binder includes a CDR3 from the heavy chain variable region CDR3s of A2, A4, or All, or a variant thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto; the light chain variable region of the binder includes a CDR1 from the light chain variable region CDRls of A2, A4, or Al l, or a variant thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto; the light chain variable region of the binder includes a CDR2 from the light chain variable region CDR2s of A2, A4, or Al 1, or a variant thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto; and the light chain variable region of the binder includes a CDR3 from the light chain variable region CDR3s of A2, A4, or Al 1, or a variant thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

[0143] In some forms, the heavy chain variable region of the binder includes the three heavy chain variable region CDRs of a A2, A4, or Al 1, or variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto; and the light chain variable region of the binder includes the three light chain variable region CDRs of the same or different A2, A4, or Al l, or variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto. Thus, in some forms, the heavy chain variable region of the binder includes the three heavy chain variable region CDRs of A2, A4, or Al l, or variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto; and the light chain variable region of the binder includes the three light chain variable region CDRs of A2, A4, or Al 1, or variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

[0144] In some embodiments, the heavy chain variable region and light chain variable regions of antibody A2, A4, or Al 1 are the heavy chain variable regions of A2, A4, or Al 1 according to Table 2, or variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence 45803765 23 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US identity thereto, and the light chain variable regions of A2, A4, or Al 1 according to Table 4, or variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

[0145] In some embodiment, the CDRs have the sequence according to Table 1 and / or Table 3, or variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto. a. Exemplary RABV-G -binding Polypeptide Sequences

[0146] The Examples below illustrate the preparation of a series of antibodies that bind to RABV-G. Amino acid sequences for CDRs and variable region of the heavy and light chains are provided and can be used to prepare the antibodies of the Examples, as well as the alternative binder embodiments.

[0147] (A) Heavy Chain Sequences

[0148] Heavy chain CDR sequences and V-region sequences are provided below in Tables 1-2, respectively. In addition to the sequences provided in Tables 1-2, variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto also provided. Any of these sequences alone or in any combination can be used to construct the RABV-G binders provided herein.

[0149] Table 1: Heavy Chain Variable Region CDR Sequences

[0150] Table 2: Heavy Chain V-Region (polypeptide and exemplary encoding nucleic acid sequence)

[0151] 45803765 24 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0152] (B) Light Chain Sequences

[0153] Light chain CDR sequences and V-region sequences are provided below in Tables 3-

[0154] 4, respectively. In addition to the sequences provided in Tables 3-4, variants thereof with at

[0155] 45803765 25 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto also provided. Any of these sequences alone or in any combination can be used to construct the RABV-G binders provided herein. Table 3: Light Chain Variable Region CDR Sequences

[0156] Table 4: Light Chain V-Region (polypeptide and exemplary encoding nucleic acid sequence)

[0157] 45803765 26 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US b. Chimeric and Humanized Antibodies

[0158] The disclosure particularly concerns chimeric and humanized antibodies. Constant regions need not be present, but if they are, are typically substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, preferably about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDR’ s, are substantially identical to corresponding parts of natural human immunoglobulin sequences.

[0159] A humanized antibody is an antibody having a humanized light chain and a humanized heavy chain immunoglobulin. For example, a humanized antibody would not encompass a typical chimeric antibody, because, e.g., the entire variable region of a chimeric antibody is nonhuman. One says that the donor antibody has been “humanized,” by the process of “humanization,” because the resultant humanized antibody is expected to bind to the same antigen as the donor antibody that provides the CDR’s.

[0160] For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat,

[0161] 45803765 27 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US rabbit or a non-human primate having the desired specificity, affinity, and capacity. In some instances, Framework Region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can include residues which arc not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will include substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will include at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin that immunospecifically binds to an FcyRIIB polypeptide, that has been altered by the introduction of amino acid residue substitutions, deletions or additions (i.e., mutations). See also, e.g., European Patent Nos. EP 239,400, EP 592,106, and EP 519,596; International Publication Nos. WO 91 / 09967 and WO 93 / 17105; U.S. Patent Nos. 5,225.539, 5,530,101, 5,565,332, 5,585,089, 5,766,886, and 6,407,213; and Padlan, 1991, Molecular Immunology 28(4 / 5):489 498; Studnicka et al., 1994, Protein Engineering 7(6):805-814; Roguska et al., 1994, PNAS 91:969 973; Tan et al., 2002, 1. Immunol. 169: 1119-1125; Caldas et al., 2000, Protein Eng. 13:353-360; Morea et al., 2000, Methods 20:267 79; Baca et al., 1997, 1. Biol. Chem. 272: 10678 10684; Roguska et al., 1996, Protein Eng. 9:895 904; Couto et al., 1995, Cancer Res. 55 (23 Supp):5973s 5977s; Couto et al., 1995, Cancer Res. 55: 1717 22; Sandhu, 1994, Gene 150:409 10; Pedersen et al., 1994, 1. Mol. Biol. 235:959-973; Jones et al., 1986, Nature 321 :522-525; Reichmann et al., 1988, Nature 332:323-329; and Presta, 1992, Curr. Op. Struct. Biol. 2:593-596).

[0162] DNA sequences coding for preferred human acceptor framework sequences include but are not limited to FR segments from the human germline VH segment VH1-18 and JH6 and the human germline VL segment VK-A26 and JK4. In a specific embodiment, one or more of the CDRs are inserted within framework regions using routine recombinant DNA techniques. The framework regions can be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., 1998, “Structural Determinants In The Sequences Of Immunoglobulin Variable Domain,” J. Mol. Biol. 278: 457-479 for a listing of human framework regions).

[0163] A humanized or chimeric antibodies can include substantially all of at least one, and typically two, variable domains in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or

[0164] 45803765 28 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US substantially all of the framework regions are those of a human immunoglobulin consensus sequence. Preferably, the antibody also includes at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The constant domains of the antibodies can be selected with respect to the proposed function of the antibody, in particular the effector function which can be required. In some embodiments, the constant domains of the antibodies are (or include) human IgA, IgD, IgE, IgG or IgM domains. In a specific embodiment, human IgG constant domains, especially of the IgGl and IgG3 isotypes are used, when the humanized antibodies are intended for therapeutic uses and antibody effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complementdependent cytotoxicity (GDC) activity are needed. In alternative embodiments, IgG2 and IgG4 isotypes are used when the antibody is intended for therapeutic purposes and antibody effector function is not required. The disclosure encompasses Fc constant domains including one or more amino acid modifications which alter antibody effector functions such as those disclosed in U.S. Patent Application Publication Nos. 2005 / 0037000 and 2005 / 0064514.

[0165] In some embodiments, the antibody contains both the light chain as well as at least the variable domain of a heavy chain. In other embodiments, the antibody can further include one or more of the CHI, hinge, CH2, CH3, and CH4 regions of the heavy chain. The antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgGl, IgG2, IgG3 and IgG4. In some embodiments, the constant domain is a complement fixing constant domain where it is desired that the antibody exhibit cytotoxic activity, and the class is typically IgGl. In other embodiments, where such cytotoxic activity is not desirable, the constant domain can be of the IgG2 class. The antibody can include sequences from more than one class or isotype, and selecting particular constant domains to optimize desired effector functions is within the ordinary skill in the art. In some embodiments, the antibody is not a mouse IgGl or a mouse IgG2a.

[0166] The framework and CDR regions of a humanized antibody need not correspond precisely to the parental sequences, e.g., the donor CDR or the consensus framework can be mutagenized by substitution, insertion or deletion of at least one residue so that the CDR or framework residue at that site does not correspond to either the consensus or the donor antibody. Such mutations, however, are preferably not extensive. Usually, at least 75% of the humanized antibody residues will correspond to those of the parental framework region (FR) and CDR sequences, more often 90%, and most preferably greater than 95%. Humanized antibodies can be produced using variety of techniques known in the art, including, but not limited to, CDR grafting (European Patent No. EP 239,400; International 45803765 29 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0167] Publication No. WO 91 / 09967: and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28(4 / 5) :489-498; Studnicka et al., 1994, Protein Engineering 7(6):805 814; and Roguska ct al., 1994, Proc. Natl. Acad. Sci. 91 :969 973), chain shuffling (U.S. Patent No. 5,565,332), and techniques disclosed in, e.g., U.S. Patent Nos. 6,407,213, 5,766,886, 5,585,089, International Publication No. WO 9317105, Tan et al., 2002, J. Immunol. 169:11 19 25, Caldas et al., 2000, Protein Eng. 13:353 60, Morea et al., 2000, Methods 20:267 79, Baca et al., 1997, 1. Biol. Chem. 272:10678 84, Roguska et al., 1996, Protein Eng. 9:895 904, Couto et al., 1995, Cancer Res. 55 (23 Supp):5973s 5977s, Couto et al., 1995, Cancer Res. 55: 1717 22, Sandhu, 1994, Gene 150:409 10, Pedersen et al., 1994, J. Mol. Biol. 235:959 73, Jones et al., 1986, Nature 321:522-525, Riechmann et al., 1988, Nature 332:323, and Presta, 1992, Curr. Op. Struct. Biol. 2:593-596. Often, framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Patent No. 5,585,089; U.S. Publication Nos. 2004 / 0049014 and 2003 / 0229208; U.S. Patent Nos. 6,350,861; 6,180.370; 5,693,762; 5,693,761; 5,585,089; and 5,530,101 and Riechmann et al., 1988, Nature 332:323). c. Bispecific and Multispecific Antibodies

[0168] The antibodies used in the methods of the present disclosure can be monospecific. Antibodies monospecific for RABV-G can have a targeting moiety conjugated or otherwise linked thereto. In some embodiments, the targeting moiety is an antibody or antigen binding fragment thereof. Thus, in some embodiments, the anti-RABV-G antibody or antigen binding fragment is a bispecific, trispecific or multispecific antibody that includes a second (or third or more) antigen binding fragment that binds to a cell specific antigen. Thus provided are bispecific, trispecific or multispecific antibodies having one or more antigen binding fragments that binds to anti-RABV-G and a second (third or more) antigen binding fragment that binds to a cell specific antigen. For example, such antibodies can bind to both RABV-G and to an antigen that is important for targeting the antibody to a particular cell type or tissue.

[0169] In some embodiments, the antibodies are heterodimeric bi- and tri- (or more) specific Ig antibodies and Fc fusion proteins. Exemplary structures include, but are not limited to,

[0170] 45803765 30 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0171] IgG, IgM, mono-, di-, tri-, or more scFv-Fcs. For example, bispecific, trispecific, and multispecific formats include, but are not limited to, bispecific and trispecific IgG, IgG-scFv, IgG-dAb, scFv-Fc-scFv, knob-in-hole (KIH)-IgG, i< / .-body, KIHOFc-Fab / scFv, tandem scFv, KIH trispccific, bispccific Fc fusion (N- or C -terminal, with or without KIH).

[0172] In embodiments, multispecific antibody molecules can include more than one antigenbinding site, where different sites are specific for different antigens. In embodiments, multispecific antibody molecules can bind more than one (e.g., two or more) epitopes on the same antigen. In embodiments, multispecific antibody molecules include an antigen-binding site specific for a target cell and a different antigen-binding site specific for RABV-G. In some embodiments, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibody molecules can be classified into five different structural groups: (i) bispecific immunoglobulin G (BsIgG); (ii) IgG appended with an additional antigen-binding moiety; (iii) bispecific antibody fragments; (iv) bispecific fusion proteins; and (v) bispecific antibody conjugates.

[0173] BsIgG is a format that is monovalent for each antigen. Exemplary BsIgG formats include but are not limited to crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knobs -in-holcs common LC, knobs-in-holcs assembly, charge pair, Fab-arm exchange, SEEDbody, triomab, LUZ-Y, Fcab, kappa-lambda-body, orthogonal Fab. See Spiess et al. Mol. Immunol. 67(2015):95- 106. Exemplary BsIgGs include catumaxomab (Fresenius Biotech, Trion Pharma, Neopharm), which contains an anti-CD3 arm and an anti- EpCAM arm; and ertumaxomab (Neovii Biotech, Fresenius Biotech), which targets CD3 and HER2.

[0174] In some embodiments, BsIgG includes heavy chains that are engineered for heterodimerization. For example, heavy chains can be engineered for heterodimerization using a "knobs-into-holes" strategy, a SEED platform, a common heavy chain (e.g., in Kk- bodies), and use of heterodimeric Fc regions. See Spiess et al., Mol. Immunol. 67(2015):95- 106. Strategies that have been used to avoid heavy chain pairing of homodimers in BsIgG include knobs-in-holes, duobody, azymetric, charge pair, HA-TF, SEEDbody, and differential protein A affinity. See Id. BsIgG can be produced by separate expression of the component antibodies in different host cells and subsequent purification / assembly into a BsIgG. BsIgG can also be produced by expression of the component antibodies in a single host cell. BsIgG can be purified using affinity chromatography, e.g., using protein A and sequential pH elution.

[0175] 45803765 31 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0176] IgG appended with an additional antigen-binding moiety is another format of bispecific antibody molecules. For example, monospecific IgG can be engineered to have bispecificity by appending an additional antigen-binding unit onto the monospecific IgG, e.g., at the N- or C-tcrminus of either the heavy or light chain. Exemplary additional antigenbinding units include single domain antibodies (e.g., variable heavy chain or variable light chain), engineered protein scaffolds, and paired antibody variable regions (e.g., single chain variable fragments or variable fragments). See Id. Examples of appended IgG formats include dual variable domain (DVD) IgG (DVD-Ig), IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv- (L)IgG, IgG(L,H)-Fv, IgG(H)-V. V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv- IgG, IgG-2scFv, scFv4-Ig, zybody, and DVI-IgG (four-in-one). See Spiess et al. Mol. Immunol. 67(2015):95-106. An example of an IgG-scFv is MM-141 (Merrimack Pharmaceuticals), which binds IGF-1R and HER3. Examples of DVD-Ig include ABT-981 (AbbVie), which binds IL- 1 alpha and IL-lbeta; and ABT- 122 (AbbVie), which binds TNF and IL- 17 A.

[0177] Bispecific antibody fragments (BsAb) are a format of bispecific antibody molecules that lack some or all of the antibody constant domains. For example, some BsAb lack an Fc region. In some embodiments, bispccific antibody fragments include heavy and light chain regions that are connected by a peptide linker that permits efficient expression of the BsAb in a single host cell. Exemplary bispecific antibody fragments include but are not limited to nanobody, nanobody-HAS, BiTE, Diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, triple body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab- scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, Diabody-Fc, tandem scFv-Fc, and intrabody. See Id. For example, the BiTE format includes tandem scFvs, where the component scFvs bind to CD3 on T cells.

[0178] Bispecific fusion proteins include antibody fragments linked to other proteins, e.g., to add additional specificity and / or functionality. An example of a bispecific fusion protein is an immTAC, which includes an anti-CD3 scFv linked to an affinity-matured T-cell receptor that recognizes HLA-presented peptides. In embodiments, the dock-and-lock (DNL) method can be used to generate bispecific antibody molecules with higher valency. Also, fusions to albumin binding proteins or human serum albumin can be extend the serum half-life of antibody fragments. See Id.

[0179] In embodiments, chemical conjugation, e.g., chemical conjugation of antibodies and / or antibody fragments, can be used to create BsAb molecules. See Id. An exemplary bispecific antibody conjugate includes the CovX-body format, in which a low molecular

[0180] 45803765 32 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US weight drug is conjugated site-specifically to a single reactive lysine in each Fab arm or an antibody or fragment thereof. In embodiments, the conjugation improves the serum half-life of the low molecular weight drug. An exemplary CovX-body is CVX-241 (NCT01004822), which includes an antibody conjugated to two short peptides inhibiting cither VEGF or Ang2. See Id.

[0181] In some embodiments the multispecific molecule further includes a heavy chain constant region (e.g., an Fc region) chosen from the heavy chain constant regions of IgGl, IgG2, and IgG4, more particularly, the heavy chain constant region of human IgGl, IgG2 or IgG4. In some embodiments, the heavy chain constant region (e.g., an Fc region) is linked to, e.g., covalently linked to, one or both of the RABV-G-binding antibody molecule and the second antibody molecule.

[0182] In some embodiments, the heavy chain constant region (e.g., an Fc region) is altered, e.g., mutated, to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function. In some embodiments, an interface of a first and second heavy chain constant regions (e.g., Fc region) is altered, e.g., mutated, to increase or decrease dimerization, e.g., relative to a non-cnginccrcd interface. In some embodiments, the dimerization of the heavy chain constant region (e.g., Fc region) is enhanced by providing an Fc interface of a first and a second Fc region with one or more of: a paired cavity-protuberance ("knob-in-a hole"), an electrostatic interaction, or a strand-exchange, such that a greater ratio of heteromultimerhomomultimer forms, e.g., relative to a non-engineered interface. In some embodiments, the heavy chain constant region (e.g., Fc region) includes an amino acid substitution at a position chosen from one or more of 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409, e.g., of the Fc region of human IgGl, numbered based on the Eu numbering system. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0183] In some embodiments, the heavy chain constant region (e.g., Fc region) includes an amino acid substitution chosen from: T366S, L368A, or Y407V (e.g., corresponding to a cavity or hole), or T366W (e.g., corresponding to a protuberance or knob), or a combination thereof, numbered based on the Eu numbering system.

[0184] 45803765 33 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0185] In some embodiments, the heavy chain constant region (e.g., an Fc region) includes one or more mutations that increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function, relative to a naturally-existing heavy chain constant region.

[0186] In some embodiments, the RABV-G-binding molecule includes a first heavy chain constant region (e.g., a first Fc region) and the second antibody molecule includes a second heavy chain constant region (e.g., a second Fc region), wherein the first heavy chain constant region includes one or more mutations that increase heterodimerization of the first heavy chain constant region and the second heavy chain constant region, relative to a naturally- existing heavy chain constant region, and / or wherein the second heavy chain constant region includes one or more mutations that increase heterodimerization of the second heavy chain constant region and the first heavy chain constant region, relative to a naturally-existing heavy chain constant region. In some embodiments, the first and the second heavy chain constant regions (e.g., first and second Fc regions) include one or more of: a paired cavityprotuberance ("knob-in-a hole"), an electrostatic interaction, or a strand-exchange, such that a greater ratio of heteromultimer:homomultimer forms, e.g., relative to naturally-existing heavy chain constant regions.

[0187] In some embodiments, the first and / or second heavy chain constant region (e.g., a first and / or second Fc region, e.g., a first and / or second IgGl Fc region) includes an amino acid substitution at a position chosen from one or more of 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409, numbered based on the Eu numbering system. In some embodiments, the first and / or second heavy chain constant region (e.g., a first and / or second Fc region, e.g., a first and / or second IgGl Fc region) includes an amino acid substitution chosen from: T366S, L368A, Y407V, or Y349C (e.g., corresponding to a cavity or hole), or T366W or S354C (e.g., corresponding to a protuberance or knob), or a combination thereof, numbered based on the Eu numbering system.

[0188] In some embodiments, the multispecific molecule further includes a linker, e.g., a linker between one or more of: the RABV-G-binding molecule and the second antibody molecule, the RABV-G-binding antibody molecule and the heavy chain constant region (e.g., the Fc region), or the second antibody molecule and the heavy chain constant region. In some embodiments, the linker is chosen from: a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, or a non-helical linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker includes Gly and Ser.

[0189] 45803765 34 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US d. Derivatives and Conjugates

[0190] The disclosure particularly contemplates the production and use of derivatives of any of the above-described antibodies and their antigen-binding fragments. The term derivative encompasses an antibody or antigen-binding fragment thereof that immunospccifically binds to an antigen but which includes, one, two, three, four, five or more amino acid substitutions, additions, deletions or modifications relative to a “parental” (or wild-type) molecule (also referred to as variants). Such amino acid substitutions or additions can introduce naturally occurring (i.e., DNA-encoded) or non-naturally occurring amino acid residues.

[0191] The term derivative also encompasses, for example, chimeric or humanized variants of any of the disclosed antibodies, as well as variants having altered CHI, hinge, CH2, CH3 or CH4 regions, so as to form, for example antibodies, etc., having variant Fc regions that exhibit enhanced or impaired effector or binding characteristics.

[0192] The term derivative additionally encompasses non-amino acid modifications, for example, amino acids that may be glycosylated (e.g., have altered mannose, 2-N- acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5- glycolneuraminic acid, etc. content), acetylated, pegylated, phosphorylated, amidated, derivatized by known protccting / blocking groups, proteolytic cleavage, linked to a cellular ligand or other protein, etc. In some embodiments, the altered carbohydrate modifications modulate one or more of the following: solubilization of the antibody, facilitation of subcellular transport and secretion of the antibody, promotion of antibody assembly, conformational integrity, and antibody-mediated effector function. In a specific embodiment the altered carbohydrate modifications enhance antibody mediated effector function relative to the antibody lacking the carbohydrate modification. Carbohydrate modifications that lead to altered antibody mediated effector function are well known in the art (for example, see Shields, R.L. et al. (2002) “Lack Of Fucose On Human IgG N-Linked Oligosaccharide Improves Binding To Human Fcgamma RIII And Antibody-Dependent Cellular Toxicity.,” J. Biol. Chem. 277(30): 26733-26740; Davies J. et al. (2001) “Expression Of GnTIII In A Recombinant Anti-CD20 CHO Production Cell Line: Expression Of Antibodies With Altered Glycoforms Leads To An Increase In ADCC Through Higher Affinity For FC Gamma RIII,” Biotechnology & Bioengineering 74(4): 288-294). Methods of altering carbohydrate contents are known to those skilled in the art, see, e.g., Wallick, S.C. et al. (1988) “Glycosylation Of A VH Residue Of A Monoclonal Antibody Against Alpha (1— -6) Dextran Increases Its Affinity For Antigen,” I. Exp. Med. 168(3): 1099-1 109; Tao, M.H. et al. (1989) “Studies Of Aglycosylated Chimeric Mouse-Human IgG. Role Of Carbohydrate In 45803765 35 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0193] The Structure And Effector Functions Mediated By The Human IgG Constant Region,” J. Immunol. 143(8): 2595-2601; Routledge, E.G. et al. (1995) “The Effect Of Aglycosylation On The Immunogenicity Of A Humanized Therapeutic CD3 Monoclonal Antibody,” Transplantation 60(8):847-53; Elliott, S. ct al. (2003) “Enhancement Of Therapeutic Protein In Vivo Activities Through Glycoengineering,” Nature Biotechnol. 21 :414-21; Shields, R.L. et al. (2002) “Lack Of Fucose On Human IgG N-Linked Oligosaccharide Improves Binding To Human Fcgamma RIII And Antibody-Dependent Cellular Toxicity.,” J. Biol. Chem. 277(30): 26733-26740).

[0194] In some embodiments, a humanized antibody is a derivative. Such a humanized antibody includes amino acid residue substitutions, deletions or additions in one or more nonhuman CDRs. The humanized antibody derivative can have substantially the same binding, better binding, or worse binding when compared to a non-derivative humanized antibody. In specific embodiments, one, two, three, four, or five amino acid residues of the CDRs have been substituted, deleted or added (i.e., mutated).

[0195] A derivative antibody or antibody fragment can be modified by chemical modifications using techniques known to those of skill in the art, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. In one embodiment, an antibody derivative will possess a similar or identical function as the parental antibody. In another embodiment, an antibody derivative will exhibit an altered activity relative to the parental antibody. For example, a derivative antibody (or fragment thereof) can bind to its epitope more tightly or be more resistant to proteolysis than the parental antibody.

[0196] In some forms, derivative antibodies are engineered to increase neutralization potency, strength of binding, and recognition of glycoproteins from related lyssaviruses. In addition to RABV, such viruses include, but are not limited to, Aravan virus (ARAV), Australian bat lyssavirus (ABLV), Bokeloh bat lyssavirus (BBLV), Duvenhage virus (DUVV), European bat lyssavirus 1 (EBLV-1), European bat lyssavirus 2 (EBLV-2), Gannoruwa bat lyssavirus (GBLV), Irkut virus (IRKV), Khujand virus (KHUV), Madagascar bat lyssavirus (MABV), Lagos bat virus (LBV), Mokola virus (MOKV), Shimoni bat virus (SHIBV), West Caucasian bat virus (WCBV), Ikoma lyssavirus (IKOV), and Lleida bat lyssavirus (LLEBV).

[0197] Derivatized antibodies can be used to alter the half-lives (e.g., serum half-lives) of parental antibodies in a mammal, preferably a human. Preferably such alteration will result in a half-life of greater than 15 days, preferably greater than 20 days, greater than 25 days,

[0198] 45803765 36 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months. The increased half- lives of the humanized antibodies of the present disclosure or fragments thereof in a mammal, preferably a human, results in a higher scrum titer of said antibodies or antibody fragments in the mammal, and thus, reduces the frequency of the administration of said antibodies or antibody fragments and / or reduces the concentration of said antibodies or antibody fragments to be administered. Antibodies or fragments thereof having increased in vivo half-lives can be generated by techniques known to those of skill in the art. For example, antibodies or fragments thereof with increased in vivo half-lives can be generated by modifying (e.g., substituting, deleting or adding) amino acid residues identified as involved in the interaction between the Fc domain and the FcRn receptor. The humanized antibodies can be engineered to increase biological half-lives (see, e.g. U.S. Patent No.

[0199] 6,277,375). For example, humanized antibodies can be engineered in the Fc-hinge domain to have increased in vivo or serum half-lives.

[0200] Antibodies or fragments thereof with increased in vivo half-lives can be generated by attaching to said antibodies or antibody fragments polymer molecules such as high molecular weight polyethylene glycol (PEG). PEG can be attached to said antibodies or antibody fragments with or without a multifunctional linker either through site-specific conjugation of the PEG to the N- or C- terminus of said antibodies or antibody fragments or via epsilon- amino groups present on lysine residues. Linear or branched polymer derivatization that results in minimal loss of biological activity will be used. The degree of conjugation will be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibodies. Unreacted PEG can be separated from antibody-PEG conjugates by, e.g., size exclusion or ion-exchange chromatography.

[0201] The antibodies can also be modified by the methods and coupling agents described by Davis et al. (See U.S. Patent No. 4,179,337) in order to provide compositions that can be injected into the mammalian circulatory system with substantially no immunogenic response.

[0202] One embodiment encompasses modification of framework residues of the humanized RABV-G antibodies. Framework residues in the framework regions can be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., U.S. Patent No. 5,585,089; and

[0203] 45803765 37 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0204] Riechmann, L. et al. (1988) “Reshaping Human Antibodies For Therapy,” Nature 332:323- 327).

[0205] Yet another embodiment encompasses anti-RABV-G antibodies (and more preferably, humanized antibodies) and antigen-binding fragments thereof that arc recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a heterologous molecule (i.e., an unrelated molecule). The fusion does not necessarily need to be direct, but can occur through linker sequences.

[0206] In one embodiment such heterologous molecules are polypeptides having at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids. Such heterologous molecules can alternatively be enzymes, hormones, cell surface receptors, drug moieties, such as: toxins (such as abrin, ricin A, pseudomonas exotoxin (i.e., PE-40), diphtheria toxin, ricin, gelonin, or pokeweed antiviral protein), proteins (such as tumor necrosis factor, interferon (e.g., a-interferon, 0-interferon), nerve growth factor, platelet derived growth factor, tissue plasminogen activator, or an apoptotic agent (e.g., tumor necrosis factor-a, tumor necrosis factor-P)), biological response modifiers (such as, for example, a lymphokine (e.g., interleukin- 1 (“IL-1”), interleukin-2 (“IL-2”), intcrlcukin-6 (“IL-6”)), granulocyte macrophage colony stimulating factor (“GM- CSF”), granulocyte colony stimulating factor (“G-CSF”), or macrophage colony stimulating factor, (“M-CSF”)), or growth factors (e.g., growth hormone (“GH”))), cytotoxins (e.g., a cytostatic or cytocidal agent, such as paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof), anti metabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5 -fluorouracil dacarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, BiCNU® (carmustine; BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromo mannitol, streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), or anti-mitotic agents (e.g., vincristine and vinblastine).

[0207] Techniques for conjugating such therapeutic moieties to antibodies are well known: see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs Tn Cancer Therapy”, in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Reisfeld et al.

[0208] 45803765 38 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0209] (eds.), 1985, pp. 243-56, Alan R. Liss, Inc.); Hellstrom et al., “Antibodies For Drug Delivery”, in CONTROLLED DRUG DELIVERY (2nd Ed.), Robinson et al. (eds.), 1987, pp. 623-53, Marcel Dekker, Inc. ); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in MONOCLONAL ANTIBODIES ‘84: BIOLOGICAL AND CLINICAL APPLICATIONS, Pinchera et al. (eds.), 1985, pp. 475-506); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in MONOCLONAL ANTIBODIES FOR CANCER DETECTION AND THERAPY, Baldwin et al. (eds.), 1985, pp. 303-16, Academic Press; and Thorpe et al. (1982) “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates,” Immunol. Rev. 62: 119-158.

[0210] In one embodiment, the RABV-G antibodies or fusion molecules include an Fc portion. The Fc portion of such molecules can be varied by isotype or subclass, can be a chimeric or hybrid, and / or can be modified, for example to improve effector functions, control of half-life, tissue accessibility, augment biophysical characteristics such as stability, and improve efficiency of production (and less costly). Many modifications useful in construction of disclosed fusion proteins and methods for making them are known in the art, sec for example Mueller, J.P. et al. (1997) “Humanized Porcine VCAM-Spccific Monoclonal Antibodies With Chimeric IgG2 / G4 Constant Regions Block Human Leukocyte Binding To Porcine Endothelial Cells,” Mol. Immun. 34(6) :441-452, Swann, P.G. (2008) “Considerations For The Development Of Therapeutic Monoclonal Antibodies,” Curr. Opin. Immun. 20:493- 499 (2008), and Presta, L.G. (2008) “Molecular Engineering And Design Of Therapeutic Antibodies,” Curr. Opin. Immun. 20:460-470. In some embodiments the Fc region is the native IgGl, IgG2, or IgG4 Fc region. In some embodiments the Fc region is a hybrid, for example a chimeric having IgG2 / IgG4 Fc constant regions. Modifications to the Fc region include, but are not limited to, IgG4 modified to prevent binding to Fc gamma receptors and complement, IgGl modified to improve binding to one or more Fc gamma receptors, IgGl modified to minimize effector function (amino acid changes), IgGl with altered / no glycan (typically by changing expression host), and IgGl with altered pH-dependent binding to FcRn, and IgG4 with serine at amino acid resident #228 in the hinge region changed to proline (S228P) to enhance stability. The Fc region can include the entire hinge region, or less than the entire hinge region.

[0211] The therapeutic outcome in patients treated with rituximab (a chimeric mouse / human IgGl monoclonal antibody against CD20) for non-Hodgkin’s lymphoma or Waldenstrom’s macroglobulincmia correlated with the individual’s expression of allelic variants of Fey

[0212] 45803765 39 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US receptors with distinct intrinsic affinities for the Fc domain of human IgGl. In particular, patients with high affinity alleles of the low affinity activating Fc receptor CD16A (FcyRIIIA) showed higher response rates and, in the cases of non-Hodgkin’s lymphoma, improved progression-free survival. In another embodiment, the Fc domain can contain one or more amino acid insertions, deletions or substitutions that reduce binding to the low affinity inhibitory Fc receptor CD32B (FcyRIIB) and retain wild-type levels of binding to or enhance binding to the low affinity activating Fc receptor CD16A (FcyRIIIA).

[0213] Another embodiment includes IgG2-4 hybrids and IgG4 mutants that have reduced binding to FcR which increase their half-life. Representative IG2-4 hybrids and IgG4 mutants are described in Angal, S. et al. (1993) “A Single Amino Acid Substitution Abolishes The Heterogeneity Of Chimeric Mouse / Human (Igg4) Antibody,” Molec. Immunol. 30( 1): 105- 108; Mueller, J.P. et al. (1997) “Humanized Porcine VCAM-Specific Monoclonal Antibodies With Chimeric Igg2 / G4 Constant Regions Block Human Leukocyte Binding To Porcine Endothelial Cells,” Mol. Immun. 34(6):441-452; and U.S. Patent No. 6,982,323. In some embodiments the IgGl and / or IgG2 domain is deleted for example, Angal, s. et al. describe IgGl and IgG2 having serine 241 replaced with a proline.

[0214] Substitutions, additions or deletions in the derivatized antibodies can be in the Fc region of the antibody and can thereby serve to modify the binding affinity of the antibody to one or more FcyR. Methods for modifying antibodies with modified binding to one or more FcyR are known in the art, see, e.g., PCT Publication Nos. WO 04 / 029207, WO 04 / 029092, WO 04 / 028564, WO 99 / 58572, WO 99 / 51642, WO 98 / 23289, WO 89 / 07142, WO 88 / 07089, and U.S. Patent Nos. 5,843,597 and 5,642,821. In one particular embodiment, the modification of the Fc region results in an antibody with an altered antibody-mediated effector function, an altered binding to other Fc receptors (e.g., Fc activation receptors), an altered antibody-dependent cell-mediated cytotoxicity (ADCC) activity, an altered Clq binding activity, an altered complement-dependent cytotoxicity activity (CDC), a phagocytic activity, or any combination thereof.

[0215] In some embodiments, the disclosure encompasses antibodies whose Fc region will have been modified so that the molecule will exhibit altered Fc receptor (FcR) binding activity, for example to exhibit decreased activity toward activating receptors such as FcyRIIA or FcyRIIIA, or increased activity toward inhibitory receptors such as FcyRIIB. Preferably, such antibodies will exhibit decreased antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) activities (relative to a wild-type Fc receptor). 45803765 4Q ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0216] Modifications that affect Fc-mediated effector function are well known in the art (see U.S. Patent No. 6,194,551, and WO 00 / 42072; Stavenhagen, J.B. et al. (2007) “Fc Optimization Of Therapeutic Antibodies Enhances Their Ability To Kill Tumor Cells In Vitro And Controls Tumor Expansion In Vivo Via Low-Affinity Activating Fcgamma Receptors," Cancer Res. 57(18):8882-8890; Shields, R.L. et al. (2001) “High Resolution Mapping of the Binding Site on Human IgGl for FcyRl, FcyRIl, FcyRlIl, and FcRn and Design of IgGl Variants with Improved Binding to the FcyR,” J. Biol. Chem. 276(9):6591 - 6604). Exemplary variants of human IgGl Fc domains with reduced binding to FcyRIIA or FcyRIIIA, but unchanged or enhanced binding to FcyRIIB, include S239A, H268A, S267G, E269A, E293A, E293D, Y296F, R301A, V303A, A327G, K322A, E333A, K334A, K338A, A339A, D376A.

[0217] In some embodiments, the disclosure encompasses antibodies whose Fc region will have been deleted (for example, a Fab or F(ab)2, etc.).

[0218] Any of the molecules of the present disclosure can be fused to marker sequences, such as a peptide, to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, the hemagglutinin “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, LA. et al. (1984) “The Structure Of An Antigenic Determinant In A Protein," Cell, 37:767-778) and the “flag” tag (Knappik, A. et al. (1994) “An Improved Affinity Tag Based On The FLAG Peptide For The Detection And Purification Of Recombinant Antibody Fragments,” Biotechniques 17(4):754- 761).

[0219] The present disclosure also encompasses antibodies or their antigen-binding fragments that are conjugated to a diagnostic or therapeutic agent or any other molecule for which serum half-life is desired to be increased. The antibodies can be used diagnostically (in vivo, in situ or in vitro) to, for example, monitor the development or progression of a disease, disorder or infection as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, and nonradioactive paramagnetic metal ions. The detectable substance can be coupled or conjugated either directly to the antibody or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Patent No. 4,741 ,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present

[0220] 45803765 41 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US disclosure. Such diagnosis and detection can be accomplished by coupling the antibody to detectable substances including, but not limited to, various enzymes, enzymes including, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic group complexes such as, but not limited to, streptavidin / biotin and avidin / biotin; fluorescent materials such as, but not limited to, umbelliferon, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent material such as, but not limited to, luminol; bioluminescent materials such as, but not limited to, luciferase, luciferin, and aequorin; radioactive material such as, but not limited to, bismuth (213Bi), carbon (14C), chromium (51Cr), cobalt (57Co), fluorine (18F), gadolinium (153Gd, 159Gd), gallium (68Ga, 67Ga), germanium (68Ge), holmium (166Ho), indium (115In, 1 13In, 112In, 111 In), iodine (1311, 1251, 1231, 1211), lanthanium (140La), lutetium (177Lu), manganese (54Mn), molybdenum (99Mo), palladium (103Pd), phosphorous (32P), praseodymium (142Pr), promethium (149Pm), rhenium (186Re, 188Re), rhodium (105Rh), ruthenium (97Ru), samarium (153Sm), scandium (47Sc), selenium (75Se), strontium (85Sr), sulfur (35S), technetium (99Tc), thallium (201Ti), tin (113Sn, 117Sn), tritium (3H), xenon (133Xe), ytterbium (169Yb, 175Yb), yttrium (90Y), zinc (65Zn); positron emitting metals using various positron emission tomography, and nonradioactive paramagnetic metal ions.

[0221] The molecules of the present disclosure can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980. Such heteroconjugate antibodies may additionally bind to haptens (such as fluorescein, etc.), or to cellular markers, or to cytokines, or chemokines (e.g., CCL21), etc.

[0222] The molecules of the present disclosed can be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen or of other molecules that are capable of binding to target antigen that has been immobilized to the support via binding to an antibody or antigen-binding fragment of the present disclosure. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. e. Methods of Making Antibodies and Antigen Binding Fragments

[0223] The disclosed antibodies can be produced by any method known in the art useful for the production of polypeptides, e.g., in vitro synthesis, recombinant DNA production, and the like. Preferably, the antibodies are produced by recombinant DNA technology. The antibodies can be produced using recombinant immunoglobulin expression technology. The recombinant production of immunoglobulin molecules, including humanized antibodies are 45803765 42 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US described in U.S. Patent No. 4.816,397 (Boss et al.). U.S. Patent Nos. 6,331,415 and 4,816,567 (both to Cabilly et al.), U.K. patent GB 2,188,638 (Winter et al.), and U.K. patent GB 2,209,757. Techniques for the recombinant expression of immunoglobulins, including humanized immunoglobulins, can also be found, in Gocddcl ct al., Gene Expression Technology Methods in Enzymology Vol. 185 Academic Press (1991), and Borreback, Antibody Engineering, W. H. Freeman (1992). Additional information concerning the generation, design and expression of recombinant antibodies can be found in Mayforth, Designing Antibodies, Academic Press, San Diego (1993).

[0224] An exemplary process for the production of the recombinant chimeric antibodies can include the following: a) constructing, by conventional molecular biology methods, an expression vector that encodes and expresses an antibody heavy chain in which the CDRs and variable region of a murine anti-RABV-G monoclonal antibody are fused to an Fc region derived from a human immunoglobulin, thereby producing a vector for the expression of a chimeric antibody heavy chain; b) constructing, by conventional molecular biology methods, an expression vector that encodes and expresses an antibody light chain of the murine anti- RABV-G monoclonal antibody, thereby producing a vector for the expression of chimeric antibody light chain; c) transferring the expression vectors to a host cell by conventional molecular biology methods to produce a transfected host cell for the expression of chimeric antibodies; and d) culturing the transfected cell by conventional cell culture techniques so as to produce chimeric antibodies.

[0225] An exemplary process for the production of the recombinant humanized antibodies can include the following: a) constructing, by conventional molecular biology methods, an expression vector that encodes and expresses an anti-RABV-G heavy chain in which the CDRs and a minimal portion of the variable region framework that are required to retain donor antibody binding specificity are derived from a non-human immunoglobulin, such as a murine anti-RABV-G monoclonal antibody, and the remainder of the antibody is derived from a human immunoglobulin, thereby producing a vector for the expression of a humanized antibody heavy chain; b) constructing, by conventional molecular biology methods, an expression vector that encodes and expresses an antibody light chain in which the CDRs and a minimal portion of the variable region framework that are required to retain donor antibody binding specificity are derived from a non-human immunoglobulin, such as a murine anti- RABV-G monoclonal antibody, and the remainder of the antibody is derived from a human immunoglobulin, thereby producing a vector for the expression of humanized antibody light chain; c) transferring the expression vectors to a host cell by conventional molecular biology 45803765 43 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US methods to produce a transfected host cell for the expression of humanized antibodies; and d) culturing the transfected cell by conventional cell culture techniques so as to produce humanized antibodies.

[0226] With respect to cither exemplary method, host cells can be co-transfcctcd with such expression vectors, which can contain different selectable markers but, with the exception of the heavy and light chain coding sequences, are preferably identical. Phis procedure provides for equal expression of heavy and light chain polypeptides. Alternatively, a single vector can be used which encodes both heavy and light chain polypeptides. The coding sequences for the heavy and light chains can include cDNA or genomic DNA or both. The host cell used to express the recombinant antibody can be either a bacterial cell such as Escherichia coli, or more preferably a eukaryotic cell (e.g., a Chinese hamster ovary (CHO) cell or a HEK-293 cell). The choice of expression vector is dependent upon the choice of host cell, and can be selected so as to have the desired expression and regulatory characteristics in the selected host cell. Other cell lines that can be used include, but are not limited to, CH0-K1, NSO, and PER.C6 (Crucell, Leiden, Netherlands).

[0227] Any of the above-described antibodies can be used to generate anti-idiotype antibodies using techniques well known to those skilled in the art (sec, e.g., Greenspan, N.S. et al. (1989) “Idiotypes: Structure And Immunogenicity," FASEB J. 7:437-444; and Nisinoff, A. (1991) “Idiotypes: Concepts And Applications " .1. Immunol. 147(8):2429-2438).

[0228] The binding properties of any of the above antibodies can, if desired, be further improved by screening for variants that exhibit such desired characteristics. For example, such antibodies can be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen binding domains, such as Fab and Fv or disulfide-bond stabilized Fv, expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage, including fd and Ml 3. The antigen binding domains are expressed as a recombinantly fused protein to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the immunoglobulins, or fragments thereof, of the present disclosure include those disclosed in Brinkman, U. et al. (1995) “Phage Display Of Disulfide-Stabilized Fv Fragments,” J. Immunol. Methods, 182:41- 45803765 44 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0229] 50, 1995; Ames, R.S. et al. (1995) “Conversion Of Murine Fobs Isolated From A Combinatorial Phage Display Library To Full Length Immunoglobulins,” J. Immunol. Methods, 184:177-186; Kettleborough, C.A. et al. (1994) “Isolation Of Tumor Cell-Specific Single-Chain Fv From Immunized Mice Using Phage-Antibody Libraries And The Reconstruction Of Whole Antibodies From These Antibody Fragments,” Eur. J. Immunol., 24:952-958, 1994; Persic, L. et al. (1997) “An Integrated Vector System For The Eukaryotic Expression Of Antibodies Or Their Fragments After Selection From Phage Display Libraries,” Gene, 187:9-18; Burton, D.R. et al. (1994) “Human Antibodies From Combinatorial Libraries,” Adv. Immunol. 57:191-280; PCT Publications WO 92 / 001047; WO 90 / 02809; WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; WO 95 / 20401; and U.S. Patents Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.

[0230] As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including humanized antibodies, or any other desired fragments, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, c.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab' and F(ab’)2 fragments can also be employed using methods known in the art (such as those disclosed in PCT Publication WO 92 / 22324; Mullinax, R.L. et al. (1992) “Expression Of A Heterodimeric Fab Antibody Protein In One Cloning Step,” BioTechniques, 12(6):864-869; and Sawai et al. (1995) “Direct Production Of The Fab Fragment Derived From The Sperm Immobilizing Antibody Using Polymerase Chain Reaction And cDNA Expression Vectors,” Am. J. Reprod. Immunol. 34:26-34; and Better, M. et al. (1988) “Escherichia coli Secretion Of An Active Chimeric Antibody Fragment,” Science 240:1041-1043). Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Patent Nos. 4,946,778 and 5,258,498; Huston, J.S. et al. (1991) “Protein Engineering Of SingleChain Fv Analogs And Fusion Proteins,” Methods in Enzymology 203:46-88; Shu, L. et al., “Secretion Of A Single-Gene-Encoded Immunoglobulin From Myeloma Cells,” Proc. Natl. Acad. Sci. (USA) 90:7995-7999; and Skerra. A. et al. (1988) “Assembly Of A Functional Immunoglobulin Fv Fragment In Escherichia coli,” Science 240.-1038-1040.

[0231] Phage display technology can be used to increase the affinity of an antibody for RABV-G. This technique would be useful in obtaining high affinity antibodies that could be used in the disclosed methods. This technology, referred to as affinity maturation, employs 45803765 45 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US mutagenesis or CDR walking and re-selection using such receptors or ligands (or their extracellular domains) or an antigenic fragment thereof to identify antibodies that bind with higher affinity to the antigen when compared with the initial or parental antibody (See, e.g., Glaser, S.M. et al. (1992) “ Antibody Engineering By Codon-Based Mutagenesis In A Filamentous Phage Vector System,” J. Immunol. 149:3903-3913). Mutagenizing entire codons rather than single nucleotides results in a semi-randomized repertoire of amino acid mutations. Libraries can be constructed including of a pool of variant clones each of which differs by a single amino acid alteration in a single CDR and which contain variants representing each possible amino acid substitution for each CDR residue. Mutants with increased binding affinity for the antigen can be screened by contacting the immobilized mutants with labeled antigen. Any screening method known in the art can be used to identify mutant antibodies with increased avidity to the antigen (e.g., ELISA) (see, e.g., Wu, H. et al. (1998) “ St ep wise In Vitro Affinity Maturation Of Vitaxin, AnAlphav Beta3-Specific Humanized Mab,” Proc. Natl. Acad. Sci. (USA) 95(l l):6037-6042; Yelton, D.E. et al. (1995) “ Affinity Maturation Of The BR96 Anti-Carcinoma Antibody By Codon-Based Mutagenesis,” I. Immunol. 155: 1994-2004). CDR walking which randomizes the light chain can be used (sec, Schicr et al. (1996) "Isolation Of Picomolar Affinity Anti-C-Erbb-2 Single-Chain Fv By Molecular Evolution Of The Complementarity Determining Regions In The Center Of The Antibody Binding Site,” J. Mol. Biol. 263:551-567).

[0232] The disclosure thus contemplates the use of random mutagenesis to identify improved CDRs. Phage display technology can alternatively be used to increase (or decrease) CDR affinity. This technology, referred to as affinity maturation, employs mutagenesis or “CDR walking’’ and re-selection uses the target antigen or an antigenic fragment thereof to identify antibodies having CDRs that bind with higher (or lower) affinity to the antigen when compared with the initial or parental antibody (see, e.g., Glaser, S.M. et al. (1992) “Antibody Engineering By Codon-Based Mutagenesis In A Filamentous Phage Vector System,” I. Immunol. 149:3903-3913). Mutagenizing entire codons rather than single nucleotides results in a semi-randomized repertoire of amino acid mutations. Libraries can be constructed including of a pool of variant clones each of which differs by a single amino acid alteration in a single CDR and which contain variants representing each possible amino acid substitution for each CDR residue. Mutants with increased (or decreased) binding affinity for the antigen can be screened by contacting the immobilized mutants with labeled antigen. Any screening method known in the art can be used to identify mutant antibodies with increased (or decreased) avidity to the antigen (e.g., ELISA) (see, Wu, H. et al. (1998) “Stepwise In Vitro 45803765 4g ATTORNEY DOCKET NO. LJI 2024-112-01 PRO US

[0233] Affinity Maturation Of Vitaxin, An Alpliav Beta3 -Specific Humanized Mab,” Proc. Natl. Acad. Sci. (USA) 95(11):6037-6042; Yelton, D.E. et al. (1995) “Affinity Maturation Of The BR96 Anti -Carcinoma Antibody By Codon-Based Mutagenesis,” J. Immunol. 155:1994- 2004). CDR walking which randomizes the light chain can be used (sec, Schicr ct al. (1996) “Isolation Of Picomolar Affinity Anti-C-Erbb-2 Single-Chain Fv By Molecular Evolution Of The Complementarity Determining Regions In The Center Of The Antibody Binding Site,” .1. Mol. Biol. 263:551-567).

[0234] Methods for accomplishing such affinity maturation are described for example in: Krause, J.C. et al. (2011) “An Insertion Mutation Thai Distorts Antibody Binding Site Architecture Enhances Function Of A Human Antibody,” MBio. 2(1) pii: e00345-10. doi: 10.1128 / mBio.00345-10; Kuan, C.T. et al. (2010) “Affinity-Matured Anti -Glycoprotein NMB Recombinant Immunotoxins Targeting Malignant Gliomas And Melanomas,” Int. J. Cancer 10.1002 / ijc.25645 ; Hackel, B.J. et al. (2010) “Stability' And CDR Composition Biases Enrich Binder Functionality Landscapes,” J. Mol. Biol. 401(l):84-96; Montgomery, D.L. et al. (2009) “Affinity Maturation And Characterization Of A Human Monoclonal Antibody Against HIV-1 gp41,” MAbs l(5):462-474; Gustchina, E. et al. (2009) “Affinity Maturation By Targeted Diversification Of The CDR-H2 Loop Of A Monoclonal Fab Derived From A Synthetic Naive Human Antibody Library And Directed Against The Internal Trimeric Coiled-Coil OfGp41 Yields A Set Of Fobs With Improved HIV-1 Neutralization Potency And Breadth,” Virology 393(1): 112-119; Finlay, W.J. et al. (2009) “Affinity Maturation Of A Humanized Rat Antibody For Anti-RAGE Therapy: Comprehensive Mutagenesis Reveals A High Level Of Mutational Plasticity Both Inside And Outside The Complementarity- Determining Regions,” J. Mol. Biol. 388(3):541 -558; Bostrom, J. et al. (2009) “Improving Antibody Binding Affinity And Specificity For Therapeutic Development,” Methods Mol. Biol. 525:353-376; Steidl, S. et al. (2008) “In Vitro Affinity Maturation Of Human GM-CSF Antibodies By Targeted CDR-Diversification,” Mol. Immunol. 46(1): 135-144; and Barderas, R. et al. (2008) “Affinity maturation of antibodies assisted by in silico modeling,’’ Proc. Natl. Acad. Sci. (USA) 105(26):9029-9034.

[0235] 2. Anti-RABV-G Chimeric Antigen Receptors (CAR)

[0236] Chimeric Antigen Receptor (CAR) proteins including the disclosed RABV-G-binding proteins as an antigen binding domain, and cells expressing the same are also provided. Typically, CARs also include a transmembrane domain and one or more intracellular / cytoplasmic domains.

[0237] 45803765 47 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0238] CARs are engineered receptors that possess both antigen-binding and T cell-activating functions. Immunotherapy using T cells genetically engineered to express a CAR is rapidly emerging as a promising new treatment for hematological and non- hematological malignancies. Based on the location of the CAR in the membrane of the cell, the CAR can be divided into three main distinct domains, including an extracellular antigen-binding domain, followed by a space region, a transmembrane domain, and the intracellular signaling domain. The antigen-binding domain, most commonly derived from variable regions of immunoglobulins, typically contains VH and VL chains that are joined up by a linker to form the so called “scFv.” The segment interposing between the antigen-binding domain (e.g., scFv) and the transmembrane domain is a “spacer domain.” The spacer domain can include the constant IgGl hinge CH2-CH3 Fc domain. In some cases, the spacer domain and the transmembrane domain are derived from CD8. The intracellular signaling domains mediating T cell activation can include a CD3^ co receptor signaling domain derived from C region of the TCR a and 0 chains and one or more costimulatory domains.

[0239] In the disclosed CARs, the antigen binding domain is typically a disclosed anti- RABV-G binding protein. In some forms, the antigen-binding domain is derived from an antibody, e.g., a disclosed RABV-G antibody. As introduced above, the term antibody herein refers to natural or synthetic polypeptides that bind a target antigen, and such antibodies can form part or all of the antigen binding domain of the CAR. The term includes polyclonal and monoclonal antibodies, including intact antibodies and functional (e.g., antigen-binding) antibody fragments, including Fab fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di scFv, tandem tri scFv. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and sub classes thereof, IgM, IgE, IgA, and IgD. The antigen-binding domain of a CAR can contain complementary determining regions (CDR) of an antibody, variable regions of an antibody, and / or antigen binding fragments thereof. For example, the antigen-binding domain for an RABV-G CAR can be derived from a disclosed RABV-G antibody as described above. In some forms, the antigen-binding domain can include an F(ab')2, Fab', Fab, Fv or scFv.

[0240] 45803765 48 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0241] In some forms, the CAR includes one or more spacer domain(s) (also referred to as hinge domain) that is located between the extracellular antigen-binding domain and the transmembrane domain. A spacer domain is an amino acid segment that is generally found between two domains of a protein and may allow for flexibility of the protein and movement of one or both of the domains relative to one another. Any amino acid sequence that provides such flexibility and movement of the extracellular antigen-binding domain relative to the transmembrane domain can be used. The spacer domain can be a spacer or hinge domain of a naturally occurring protein. In some forms, the hinge domain is derived from CD8a, such as, a portion of the hinge domain of CD8a, e.g., a fragment containing at least 5 (e.g., 5, 10, 15, 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8a. Hinge domains of antibodies, such as an IgG, IgA, IgM, IgE, or IgD antibodies can also be used. In some forms, the hinge domain is the hinge domain that joins the constant CHI and CH2 domains of an antibody. Non naturally occurring peptides may also be used as spacer domains. Tor example, the spacer domain can be a peptide linker, such as a (GxS)n linker, wherein x and n, independently can be an integer of 3 or more, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more.

[0242] In some forms, the CAR includes a transmembrane domain that can be directly or indirectly fused to the antigen-binding domain. The transmembrane domain may be derived either from a natural or a synthetic source. In some forms, the transmembrane domain of the CAR includes a transmembrane domain of an alpha, beta or zeta chain of a T cell receptor, CD8, CD4, CD28, CD137, CD80, CD86, CD152 (CTLA-4) or PD1, or a portion thereof. Transmembrane domains can also contain at least a portion of a synthetic, non-naturally occurring protein segment. In some forms, the transmembrane domain is a synthetic, non- naturally occurring alpha helix or beta sheet. In some forms, the protein segment is at least about 15 amino acids, e.g., at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids. Examples of synthetic transmembrane domains are known in the art, for example in U.S. Patent No. 7,052,906 and PCT Publication No. WO 2000 / 032776.

[0243] The intracellular signaling domain is responsible for activation of at least one of the normal effector functions of the immune effector cell expressing the CAR. The term effector function refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. In some forms, an intracellular signaling domain includes the zeta chain of the T cell receptor or any of its homologs (e.g., eta, delta, gamma or epsilon), MB1 chain, B29, Fc RIII, Fc RI and combinations of signaling molecules such as CD3ij and CD28, 4 IBB, 0X40 and combination thereof, as well as other similar molecules and fragments. Intracellular signaling 45803765 49 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US portions of other members of the families of activating proteins can be used, such as FcyRIII and FceRI.

[0244] Many immune effector cells require co stimulation, in addition to stimulation of an antigen-specific signal, to promote cell proliferation, differentiation and survival, as well as to activate effector functions of the cell. Therefore, in some fomis, the CAR includes at least one co stimulatory signaling domain. The term co stimulatory signaling domain, refers to at least a portion of a protein that mediates signal transduction within a cell to induce an immune response such as an effector function. The co stimulatory signaling domain can be a cytoplasmic signaling domain from a co stimulatory protein, which transduces a signal and modulates responses mediated by immune cells, such as T cells, NK cells, macrophages, neutrophils, or eosinophils. In some forms, the co-stimulatory signaling domain is derived from a co stimulatory molecule selected from CD27, CD28, CD 137, 0X40, CD30, CD40, CD3, LFA 1, ICOS, CD2, CD7, LIGHT, NKG2C, B7 H3, ligands of CD83 and combinations thereof.

[0245] CARs can be used in order to generate immuno-responsive cells, such as T cells, specific for selected targets, such as malignant cells, with a wide variety of receptor chimera constructs having been described (sec U.S. Patent Nos. 5,843,728; 5,851,828; 5,912,170; 6,004,811; 6,284,240; 6,392,013; 6,410,014; 6,753,162; 8,211,422; and PCT Publication WO 9215322, each of which is specifically incorporated by reference herein in its entirety). Alternative CAR constructs can be characterized as belonging to successive generations. First-generation CARs typically include a single-chain variable fragment of an antibody specific for an antigen, for example including a VL linked to a VH of a specific antibody, linked by a flexible linker, for example by a CD8a hinge domain and a CD8a transmembrane domain, to the transmembrane and intracellular signaling domains of either CD3 or FcRy (scFv CD3^ or scFv FcRy; see U.S. Patent No. 7,741,465; U.S. Patent No. 5,912,172; U.S. Patent No. 5,906,936, each of which is specifically incorporated by reference herein in its entirety). Second-generation CARs incorporate the intracellular domains of one or more costimulatory molecules, such as CD28, 0X40 (CD134), or 4 IBB (CD 137) within the endodomain (for example scFv CD28 / OX40 / 4 IBB CD3^; see U.S. Patent Nos.8, 911,993; 8,916,381; 8,975,071; 9,101,584; 9,102,760; 9,102,761, each of which is specifically incorporated by reference herein in its entirety). Third-generation CARs include a combination of costimulatory endodomains, such a CD3^ chain, CD97, GDI la CD 18, CD2, ICOS, CD27, CD154, CDS, 0X40, 4 IBB, or CD28 signaling domains (for example scFv see U.S. Patent No.8,906,682; U.S. Patent ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0246] No.8,399,645; U.S. Pat. No. 5,686,281; PCT Publication No. WO2014134165; PCT Publication No. W02012079000, each of which is specifically incorporated by reference herein in its entirety). Alternatively, co stimulation can be orchestrated by expressing CARs in antigen-specific T cells, chosen so as to be activated and expanded following engagement of their native aPTCR, for example by antigen on professional antigen-presenting cells, with attendant co stimulation. Any of the first, second, or third generation CARs described above can be used in accordance with the disclosed compositions and methods.

[0247] In some forms, the CAR cells target and / or is used for reducing rabies symptoms, complete elimination or neutralization of the virus, or other infections / disease related to RABV-G proteins (e.g., rabies virus and / or other lyssaviruses).

[0248] 3. Nucleic Acids

[0249] Isolated nucleic acids and vectors encoding or expressing RABV-G binding molecules and RABV-G-CAR fusion proteins are also provided. As used herein, “isolated nucleic acid’’ refers to a nucleic acid that is separated from other nucleic acid molecules that are present in a mammalian genome, including nucleic acids that normally flank one or both sides of the nucleic acid in a mammalian genome.

[0250] An isolated nucleic acid can be, for example, a DNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule independent of other sequences (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease treatment), as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include an engineered nucleic acid such as a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid. A nucleic acid existing among hundreds to millions of other nucleic acids within, for example, a cDNA library or a genomic library, or a gel slice containing a genomic DNA restriction digest, is not to be considered an isolated nucleic acid.

[0251] Nucleic acid encoding the disclosed polypeptide sequences are expressly provided. Nucleic acids can be single strand or double stranded, and can be in sense or antisense orientation, or can be complementary to a reference sequence.

[0252] Nucleic acids can be DNA, RNA, or nucleic acid analogs. Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone. Such modification can 45803765 5 J ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US improve, for example, stability, hybridization, or solubility of the nucleic acid. Modifications at the base moiety can include deoxyuridine for deoxythymidine, and 5-methyl-2’- deoxycytidine or 5-bromo-2’-deoxycytidine for deoxycytidine. Modifications of the sugar moiety can include modification of the 2’ hydroxyl of the ribose sugar to form 2’ -O-mcthyl or 2’-O-allyl sugars. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. See, for example, Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7: 187-195; and Hyrup el al. (1996) Bioorgan. Med. Chem. 4:5-23. In addition, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.

[0253] Nucleic acids, such as those described above, can be inserted into vectors for expression in cells. As used herein, a “vector” is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. Vectors can be expression vectors. An “expression vector” is a vector that includes one or more expression control sequences, and an “expression control sequence” is a DNA sequence that controls and regulates the transcription and / or translation of another DNA sequence.

[0254] Nucleic acids in vectors can be operably linked to one or more expression control sequences. As used herein, “operably linked” means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest. Examples of expression control sequences include promoters, enhancers, and transcription terminating regions. A promoter is an expression control sequence composed of a region of a DNA molecule, typically within 100 nucleotides upstream of the point at which transcription starts (generally near the initiation site for RNA polymerase II). To bring a coding sequence under the control of a promoter, it is necessary to position the translation initiation site of the translational reading frame of the polypeptide between one and about fifty nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function when located at various distances from the transcription site. An enhancer also can be located downstream from the transcription initiation site. A coding sequence is “operably linked” and “under the control” of expression control sequences in a cell when RNA polymerase is able to transcribe

[0255] 45803765 52 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US the coding sequence into mRNA, which then can be translated into the protein encoded by the coding sequence.

[0256] Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruscs, tobacco mosaic virus, herpes viruses, cytomegalo virus, retroviruses, vaccinia viruses, adenoviruses, and adeno-associated viruses. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen Life Technologies (Carlsbad, CA).

[0257] An expression vector can include a tag sequence. Tag sequences, are typically expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino terminus. Examples of useful tags include, but are not limited to, green fluorescent protein (GFP), glutathione S- transferase (GST), polyhistidine, c-myc, hemagglutinin, Flag™ tag (Kodak, New Haven, CT), maltose E binding protein and protein A. In one embodiment, the variant PD-L2 fusion protein is present in a vector containing nucleic acids that encode one or more domains of an Ig heavy chain constant region, preferably having an amino acid sequence corresponding to the hinge, CH2 and CH3 regions of a human immunoglobulin Cyl chain.

[0258] Vectors containing nucleic acids to be expressed can be transferred into host cells. The term “host cell” is intended to include prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced. As used herein, “transformed” and “transfected” encompass the introduction of a nucleic acid molecule (e.g., a vector) into a cell by one of a number of techniques. Although not limited to a particular technique, a number of these techniques are well established within the art. Prokaryotic cells can be transformed with nucleic acids by, for example, electroporation or calcium chloride mediated transformation. Nucleic acids can be transfected into mammalian cells by techniques including, for example, calcium phosphate co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, or microinjection. Host cells (e.g., a prokaryotic cell or a eukaryotic cell such as a CHO cell) can be used to, for example, produce the PD-1 antagonist polypeptides described herein.

[0259] 4. Host Cells

[0260] In some embodiments, polypeptides, nucleic acids, or vectors provided herein are present within a host cells. The term “host cell” is intended to include prokaryotic and eukaryotic cells into which a nucleic acid such as a recombinant expression vector can be introduced. As used herein, “transformed” and “transfected” encompass the introduction of a 45803765 53 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US nucleic acid molecule (e.g., an mRNA, or a vector, etc.) into a cell by one of a number of techniques. Although not limited to a particular technique, a number of these techniques are well established within the art. Prokaryotic cells can be transformed with nucleic acids by, for example, electroporation or calcium chloride mediated transformation. Nucleic acids can be transfected into mammalian cells by techniques including, for example, calcium phosphate co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, or microinjection. Host cells (e.g., a prokaryotic cell or a eukaryotic cell) can be used to produce polypeptides described herein.

[0261] In some forms, the cell is from an established cell line, or a primary cell. The term “primary cell,” refers to cells and cell cultures derived from a subject and allowed to grow in vitro for a limited number of passages, i.e. splitting, of the culture.

[0262] In some embodiments, particularly CAR embodiments, the cells are obtained from a human subject. Therefore, human cells expressing and / or including the disclosed polypeptides are provided. In preferred embodiments, the human cells include or express a RABV-G-CAR. For example, in some forms, the cells are autologous cells, i.e., cells obtained from a subject prior to introduction of the RABV-G-CAR, and / or nucleic acids, or vectors encoding the same, and re-introduction to the same subject following modification. In other forms, the cells are heterologous cells, i.e., cells obtained from a different subject than the intended recipient. In some forms, the cells are frozen prior to or after introduction of the RABV-G-CAR. Methods and compositions for freezing and thawing viable eukaryotic cells are known in the art. In some forms, the cells are autologous immune cells, such as T cells or progenitor cells / stem cells.

[0263] In some forms, cells are obtained from a healthy subject. In other forms, cells are obtained from a subject identified as having or at risk of having a disease or disorder or other disease or condition such as those mentioned elsewhere herein.

[0264] In some embodiments, the introduction of the polypeptides to the cells occurs through genetic modification of the cells. In some embodiments, genetic modification of the cell includes introduction of nucleic acids, or vectors encoding the polypeptides to the cell for expression of the polypeptides within the cell. Therefore, genetically modified (transgenic) cells including the disclosed proteins, e.g., RABV-G-CAR fusion proteins, are described.

[0265] In some forms, the cells are human immune cells, such as T cells, Natural Killers (NK) cells, macrophages, dendritic cells etc. Therefore, human immune cells that include or express the disclosed polypeptides including the RABV-G-CAR polypeptides are described. In some forms, prior to expansion and genetic modification, immune cells such as T cells are 45803765 54 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US obtained from a diseased or healthy subject. The cells can be obtained from a number of samples, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some forms, the immune cells arc obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as F1COLL™ separation. In one preferred form, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The cells collected by apheresis can be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some forms, the cells are washed with phosphate buffered saline (PBS). In some forms, the wash solution lacks calcium and can lack magnesium or can lack many if not all divalent cations. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as, for example, Ca2+ free, Mg2+ free PBS, PLASMALYTE A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample are removed and the cells directly resuspended in culture media.

[0266] In some forms, T cells arc isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. In specific forms, a specific subpopulation of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, is further isolated by positive or negative selection techniques. For example, in some forms, T cells are isolated by incubation with anti CD3 / anti CD28 (i.e., 3x28) conjugated beads, such as DYNABEADS® M 450 CD3 / CD28 T, for a time period sufficient for positive selection of the desired T cells.

[0267] 5. Pharmaceutical Compositions

[0268] The compositions can be formulated with appropriate pharmaceutically acceptable carriers into pharmaceutical compositions for administration to an individual in need thereof. The formulations can be administered enterally (e.g., oral) or parenterally (e.g., by injection or infusion).

[0269] The compositions can be formulated for parenteral administration. “Parenteral administration”, as used herein, means administration by any method other than through the digestive tract or non-invasive topical or regional routes. For example, parenteral administration may include administration to a patient intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly,

[0270] 45803765 55 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US intraprostatically, intrapleurally, intratracheally, intravitreally, intratumorally, intramuscularly, subcutaneously, subconjunctivally, intravesicularly, intrapericardially, intraumbilically, or transmucosal (nasal, vaginal, pulmonary, or rectal), e.g., by injection, and by infusion.

[0271] In some embodiments, the compositions are administered systemically by, for example, injection or infusion. In some embodiments, the compositions are administered locally by injection or infusion.

[0272] Parenteral formulations can be prepared as aqueous compositions using techniques known in the art. Typically, such compositions can be prepared as injectable formulations, for example, solutions or suspensions: solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection; emulsions, such as water-in-oil (w / o) emulsions, oil-in-water (o / w) emulsions, and microemulsions thereof, liposomes, or emulsomes.

[0273] The earner can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils, such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.), and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required nanocarrier size in the case of dispersion and / or by the use of surfactants. In many cases, isotonic agents, for example, sugars or sodium chloride, are included.

[0274] Solutions and dispersions of the active compounds as the free acid or base or pharmacologically acceptable salts thereof can be prepared in water or another solvent or dispersing medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersants, emulsifiers, pH modifying agents, viscosity modifying agents, and combination thereof.

[0275] Suitable surfactants may be anionic, cationic, amphoteric or nonionic surface active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene 45803765 5 g ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG- 150 laurate, PEG-400 monolaurate, polyoxyethylene monolauratc, polysorbates, polyoxyethylene octylphcnylcthcr, PEG- 1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodccyl-bcta-alaninc, sodium N-lauryl-0- iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

[0276] The formulation can contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The formulation may also contain an antioxidant to prevent degradation of the active agent(s).

[0277] The formulation is typically buffered to a pH of 3-8 for parenteral administration upon reconstitution. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.

[0278] Water soluble polymers are often used in formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol.

[0279] Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, exemplary methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0280] Enteral formulations are prepared using pharmaceutically acceptable carriers. As generally used herein “carrier” includes, but is not limited to, diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof. Polymers used in the dosage form include hydrophobic or hydrophilic polymers and pH dependent or independent polymers. Hydrophobic and hydrophilic polymers include, but are not limited to, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, carboxy methylcellulose, polyethylene glycol, ethylcellulose, microcrystalline 45803765 57 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US cellulose, polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl acetate, and ion exchange resins.

[0281] Carrier also includes all components of the coating composition, which may include plasticizers, pigments, colorants, stabilizing agents, and glidants. Formulations can be prepared using one or more pharmaceutically acceptable excipients, including diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof.

[0282] Controlled release dosage formulations can be prepared as described in standard references such as “Pharmaceutical dosage form tablets’’, eds. Liberman et. al. (New York, Marcel Dekker, Inc., 1989), “Remington - The science and practice of pharmacy”, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, and “Pharmaceutical dosage forms and drug delivery systems”, 6th Edition, Ansel et al., (Media, PA: Williams and Wilkins, 1995). These references provide information on excipients, materials, equipment and process for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules. These references provide information on carriers, materials, equipment and process for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules.

[0283] Stabilizers are used to inhibit or retard drug decomposition reactions which include, by way of example, oxidative reactions. Suitable stabilizers include, but are not limited to, antioxidants, butylated hydroxytoluene (BHT); ascorbic acid, its salts and esters; Vitamin E, tocopherol and its salts; sulfites such as sodium metabisulphite; cysteine and its derivatives; citric acid; propyl gallate, and butylated hydroxyanisole (BHA).

[0284] In some embodiments, the compositions are formulated for mucosal administration, such as through nasal, pulmonary, or buccal delivery.

[0285] Mucosal formulations may include one or more agents for enhancing delivery through the nasal mucosa. Agents for enhancing mucosal delivery are known in the art, see for example U.S. Patent Application No. 20090252672 to Eddington, and U.S. Patent Application No. 20090047234 to Touitou. Acceptable agents include, but are not limited to, chelators of calcium (EDTA), inhibitors of nasal enzymes (boro-leucin, aprotinin), inhibitors of muco-ciliar clearance (preservatives), solubilizers of nasal membrane (cyclodextrin, fatty acids, surfactants) and formation of micelles (surfactants such as bile acids, Laureth 9 and taurodehydrofusidate (STDHF)). Compositions may include one or more absorption enhancers, including surfactants, fatty acids, and chitosan derivatives, which can enhance delivery by modulation of the tight junctions (TJ) (B. J. Aungst, et al., J. Pharm. Sci.

[0286] 45803765 58 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0287] 89(4):429-442 (2000)). In general, the optimal absorption enhancer should possess the following qualities: its effect should be reversible, it should provide a rapid permeation enhancing effect on the cellular membrane of the mucosa, and it should be non-cytotoxic at the effective concentration level and without deleterious and / or irreversible effects on the cellular membrane or cytoskeleton of the TJ.

[0288] Any of the disclosed compositions including, but not limited to the provided RAB V-G binder polypeptides and CAR fusion proteins formed therefrom and / or nucleic acids encoding the same, can be delivered to target cells using a delivery vehicle. The delivery vehicles can be, for example, polymeric particles, inorganic particles, silica particles, liposomes, micelles, multilamellar vesicles, etc.

[0289] Delivery vehicles may be microparticles or nanoparticles. Nanoparticles are often utilized for intertissue application, penetration of cells, and certain routes of administration. The nanopaiticles may have any desired size for the intended use. The nanoparticles may have any diameter from 10 nm up to about 1,000 nm. The nanoparticle can have a diameter from 10 nm to 900 nm, from 10 nm to 800 nm, from 10 nm to 700 nm, from 10 nm to 600 nm, from 10 nm to 500 nm, from 20 nm from 500 nm, from 30 nm to 500 nm, from 40 nm to 500 nm, from 50 nm to 500 nm, from 50 nm to 400 nm, from 50 nm to 350 nm, from 50 nm to 300 nm, or from 50 nm to 200 nm. In some embodiments the nanoparticles can have a diameter less than 400 nm, less than 300 nm, or less than 200 nm. The range can be between 50 nm and 300 nm.

[0290] Thus, in some embodiments, the delivery vehicles are nanoscale compositions, for example, 10 nm up to, but not including, about 1 micron. However, it will be appreciated that in some embodiments, and for some uses, the particles can be smaller, or larger (e.g., microparticles, etc.). Although many of the compositions disclosed herein are referred to as nanoparticle or nanocarrier compositions, it will be appreciated that in some embodiments and for some uses the carrier can be somewhat larger than nanopaiticles. Such compositions can be referred to as microparticulate compositions. For example, a nanocarriers according to the present disclosure may be a microparticle. Microparticles can a diameter between, for example, 0.1 and 100 pm in size.

[0291] As provided are pharmaceutical packs and kits including one or more containers filled with antibody or fusion protein or nucleic acid. Additionally, one or more other prophylactic or therapeutic agents useful for the treatment of a disease can also be included in the pharmaceutical pack or kit. One embodiment provides a pharmaceutical pack or kit including one or more containers filled with one or more of the ingredients of the

[0292] 45803765 59 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US pharmaceutical compositions. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[0293] Also provided are kits that can be used in the below methods. In one embodiment, a kit includes one or more antibodies or fusion proteins or nucleic acids. In another embodiment, a kit further includes one or more other prophylactic or therapeutic agents useful for reducing rabies symptoms, complete elimination or neutralization of the virus, or other infections / disease related to RABV-G proteins. In other embodiments, the prophylactic or therapeutic agent is a biological or hormonal therapeutic.

[0294] C. Methods of Use

[0295] 1. Methods of Detecting RABV-G

[0296] The disclosed RABV-G binders can be used to detect RABV-G in various research, diagnostic, and prognostic applications. Typically the binders are used to detect RABV-G protein or fragment thereof, e.g., an fragment thereof in a biological sample.

[0297] Likewise, the disclosed binders can used to isolate or purify RABV-G, e.g., by immunoprecipitation, etc.

[0298] Exemplary biological sources for detection of RABV-G protein and fragments thereof are samples that include, but are not limited to saliva, urine, serum, and blood which may or may not include blood cells such as white blood cells such as immune cells, or plasma cells.

[0299] A sample may be obtained and processed using well-known and routine clinical methods. In some aspects, the biological sample includes a plurality of cells. The cells can be intact and / or permeabilized. In certain aspects, the biological sample includes fresh or frozen tissue. In specific aspects, the biological sample includes formalin fixed, paraffin embedded tissue.

[0300] In some embodiments, the cells are permeabilized. Thus, in some embodiments, a cell lysate or homogenate is subjected to RABV-G protein or fragment detection.

[0301] RABV-G protein or fragment thereof is typically detected using one or more of the antibodies or other antigen binding molecules provided herein. The RABV-G protein or fragment thereof can be detected by any suitable method utilizing the provided antibodies. In preferred embodiments, the RABV-G protein or fragment thereof is detected and / or measured by an immunoassay. Immunoassays utilize biospecific capture reagents, such as antibodies, to capture or locate the RABV-G protein or fragment.

[0302] 45803765 60 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0303] The steps of various useful immunodetection methods have been described in the scientific literature. In general, the immunobinding methods include obtaining a sample, and contacting the sample with an antibody specific for the protein to be detected, as the case may be, under conditions effective to allow the formation of immunocomplcxcs. In general, the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any of those radioactive, fluorescent, biological and enzymatic tags. Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody and / or a biotin / avidin ligand binding arrangement, as is known in the art.

[0304] The antibody employed in the detection may itself be linked to a detectable label (also referred to as a detectable substance or reporter), wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined. Alternatively, the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is itself often an antibody, which may thus be termed a “secondary” antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non- specific ally bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.

[0305] Traditional immunoassays including, for example, sandwich immunoassays including ELISA or fluorescence-based immunoassays, as well as other enzyme immunoassays can be used for detecting the RABV-G protein or fragment thereof. In other embodiments, the detection of the RABV-G protein or fragment thereof is earned out on slides of test material (e.g., immunohistochemistry), immunoblotting (e.g., Western blotting), surface plasmon resonance (e.g. Biacore), or by flow cytometry (FACS) analysis).

[0306] Other specific examples include, but are not limited to, enzyme immunoassay (EIA), radioimmunoassay (RIA), fluoroimmunoassay (FIA), chemiluminescent immunoassay (GLIA) and counting immunoassay (CIA), homogeneous enzyme-multiplied immunoassays (“EMIT”), apoenzyme reactivation immunoassay (“ARIS”), dipstick immunoassays, and immuno-chromatography assays. Most assays now use nonradioactive labels. Enzyme

[0307] 45803765 61 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US immunoassays (enzyme-linked immunosorbent assays, or ELISA; immunometric assays) can use enzymes as labels, such as, for example, horseradish peroxidase or alkaline phosphatase. Chemiluminescent immunoassays (CIA) can use luminol. Fluorimetric immunoassays (FIA) use fluorescent compounds (c.g., fluorescein) as labels.

[0308] The assays can be homogenous or heterogeneous assays, competitive and noncompetitive assays. There are four main kinds of ELISA: sandwich, competitive, direct, and indirect assays. These methods differ in how the antibody or antigen is attached to the solid plate, and how the signal is detected. For example, in a sandwich ELISA, for example, an antibody is immobilized on a plate. The sample containing the target antigen is added, which binds to the antibody and so is immobilized on the plate. Next, a second type of antibody is added, which also binds to the target antigen on the plate, forming a ‘sandwich’ with the target antigen in the middle. The second antibody is linked to an enzyme, called a reporter enzyme, which allows the binding reaction to be measured by creating a color signal. To create this signal, first any unbound antibody is washed away, and a colorimetric substrate is added. The enzyme catalyzes a reaction of the substrate, creating a color change. A stronger color signal indicates more target antigen is present. An example of this is a home pregnancy test. In some embodiments, the first or second antibody is one of the disclosed antibodies, and the other antibody is one that detects RABV-G protein or fragment thereof, but may or may not detect its phosphorylated state, and thus may target a different antigen of the protein (e.g., a non-phosphorylated antigen).

[0309] In some embodiments, the assay is in the form of a sandwich assay, which is a noncompetitive immunoassay, wherein the molecule to be detected and / or quantified is bound to a first antibody and to a second antibody. The first antibody may be bound to a solid phase, e.g., a bead, a surface of a well or other container, a chip or a strip, and the second antibody is an antibody which is labeled, e.g. with a dye, with a radioisotope, or a reactive or catalytically active moiety. The amount of labeled antibody bound to the analyte is then measured by an appropriate method. The general composition and procedures involved with “sandwich assays” are well-established and known to the skilled person.

[0310] Immunohistochemistry (IHC) is a process of localizing antigens (e.g., proteins) in tissue utilizing antigen-specific antibodies. The antigen-binding antibody can be conjugated or fused to a tag that allows its detection, e.g., via visualization. In some embodiments, the tag is an enzyme that can catalyze a color-producing reaction, such as alkaline phosphatase or horseradish peroxidase. The enzyme can be fused to the antibody or non-covalently bound, e.g., using a biotin-avidin system. Alternatively, the antibody can be tagged with a

[0311] 45803765 62 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US fluorophore, such as fluorescein, rhodamine, DyLight Fluor or Alexa Fluor. The antigenbinding antibody can be directly tagged or it can itself be recognized by a detection antibody that carries the tag.

[0312] Quantitative immunochemical techniques can also be used. For example, the Quantitative Tissue Biomarker Platfomi from HistoRx and / or measuring immunofluorescence level(s) can be used to quantify levels of RABV-G protein or a fragment thereof.

[0313] Western blotting can be used and can be quantitative or qualitative. A typical Western blotting procedure includes the steps of immunoprecipitating a target protein from a lysate of cells expressing the protein, performing an SDS-PAGE with said protein, transferring the protein to a nitrocellulose membrane, incubating the nitrocellulose membrane with said antibody, detecting said antibody with a secondary antibody conjugated to a fluorescent or chromogenic compound (e.g. peroxidases such as horseradish peroxidase (HRP), alkaline phosphatase (AP), IRDye near-infrared (NIR) fluorescent dyes), and quantifying the respective signal of said compound (e.g. fluorescence, luminescence, chromogenic enzyme substrate).

[0314] The ratio of two signals generated by Western blotting employing the same antibody but two different samples can be calculated, thereby detemiining how much more / less (foldchange) of the RABV-G protein or fragment thereof is present in one sample compared to another.

[0315] In some embodiments, the methods provided herein involve determining the presence, absence, and / or concentration of RABV-G protein or a fragment thereof in a cell, and / or the number of cell positive for RABV-G protein or fragment thereof in sample by fluorescence activated cell sorting (FACS) using a flow cytometry device (e.g., Beckman Coulter Z2 Coulter Counter, Beckman Coulter Inc.). In some embodiments, a FACs-based method include the step of preparing the output composition for detection by flow cytometry before the RABV-G protein or fragment thereof can be detected. For example, the output composition can be incubated with a fluorescently labeled RABV-G binder, and then the sample can be analyzed using a flow cytometer. In some embodiments, the cells are permeabilized to facilitate antibody access to intracellular RABV-G protein or fragment thereof. In flow cytometry, cells bound by fluorescently labeled affinity reagents are carried in a fluidic stream, are separated based on size and / or fluorescent signal and are subsequently analyzed and counted using a FACS software program (e.g., Flowlo software). The number or approximate number of cells can be determined by detection of the fluorescent signal,

[0316] 45803765 63 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US which optionally can be determined or processed by the FACS software program to provide the total or approximate number of particles in the output composition.

[0317] In some embodiments, a sample is analyzed by means of a biochip. Biochips generally include solid substrates and have a generally planar surface, to which a capture reagent (also called an adsorbent or affinity reagent) is attached. Frequently, the surface of a biochip includes a plurality of addressable locations, each of which has the capture reagent bound there.

[0318] Protein biochips are biochips adapted for the capture of polypeptides. Many protein biochips are described in the art. These include; for example, protein biochips produced by Ciphergen Biosystems, Inc. (Fremont, Calif.), Packard BioScience Company (Meriden Conn.), Zyomyx (Hayward, Calif.), Phylos (Lexington, Mass.) and Biacore (Uppsala, Sweden).

[0319] Photonic biosensors can be used in label-free assays. For example, photonic biosensors combine photonic sensing with bio recognition technology to create label-free testing on-chip. Instead of moving electrons around on silicon chips, light is moved around on silicon chips via waveguides. This technology has allowed the development of miniature lab-on-a-chip label-free immunoassay (LFIA) devices. These devices arc functionalized with capture antibodies and have a resonance condition of light. This resonance wavelength will be shifted by a reaction between the capture antibody and the target antigen due to the change in refractive index. Measuring the shift in resonance wavelength provides a readout of a binding event. Label-free assays therefore enable the detection of antigen- antibody binding without the use of an additional label, resulting in increased assay sensitivity and decreased working time.

[0320] 2. Methods of Diagnosis

[0321] The RABV-G and fragments thereof can be used in diagnostic tests to assess RABV- G -related infections, disease and disorder status in a subject, e.g., to distinguish between normal cells and diseased cells, and disease status. For example, disease status includes, without limitation, the presence or absence of disease (e.g., infection v. non-infection), the risk of developing disease, the stage of the disease, the progress of disease (e.g., progress of disease or remission of disease over time) and the effectiveness or response to treatment of disease. Based on this status, further procedures may be indicated, including additional diagnostic tests or therapeutic procedures or regimens.

[0322] The RABV-G can be present and / or expressed in certain cells of a subject including but not limited to those mentioned elsewhere herein, and therefore, detection thereof is useful 45803765 4 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US in aiding in the determination of infection and / or other RABV-G -related diseases and disorders. An exemplary method involves, first, measuring RABV-G protein or a fragment thereof in a subject sample using the methods described herein, and, second, comparing the measurement with a diagnostic amount or cut-off that distinguishes a positive infection and / or other RABV-G-related disease and disorder (e.g., rabies) status from a negative infection and / or other RABV-G -related disease and disorder status. The diagnostic amount represents a measured amount of RABV-G protein or an fragment thereof above which a subject is classified as having a particular status. For example, because the RABV-G protein or a fragment thereof or viral load is higher compared to non-infected subjects, then a measured amount above the diagnostic cutoff provides a diagnosis or status of the disease and / or other RABV-G -related diseases and disorders. As is well understood in the art, by adjusting the particular diagnostic cut-off used in an assay, one can increase sensitivity or specificity of the diagnostic assay depending on the preference of the diagnostician. The particular diagnostic cut-off can be determined, for example, by measuring the amount of the RABV-G protein or fragment thereof in a statistically significant number of samples from subjects and drawing the cut-off to suit the diagnostician's desired levels of specificity and sensitivity.

[0323] 3. Determining Risk of Developing Disease

[0324] Methods for determining the risk of developing disease in a subject are also provided. RABV-G protein or fragment thereof amounts or patterns can be characteristic of various risk states, e.g., high, medium, or low. The risk of developing a disease is determined by measuring the RABV-G protein or fragment thereof and then either submitting them to a classification algorithm or comparing them with a reference amount and / or pattern of RABV- G protein or an fragment thereof that is associated with the particular risk level.

[0325] 4. Determining Stage of Disease

[0326] Another embodiment provides methods for determining the stage of disease in a subject. Each stage of the disease can have a characteristic amount of RABV-G protein or fragment thereof. The stage of a disease is determined by measuring the RABV-G protein or a fragment thereof and then either submitting them to a classification algorithm or comparing them with a reference amount and / or pattern of the RABV-G protein or fragment thereof that is associated with the particular stage.

[0327] 5. Determining Course (Progression / Remission) of Disease

[0328] Still another embodiment provides methods for determining the course of disease in a subject. Disease course refers to changes in disease status over time, including disease

[0329] 45803765 65 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US progression (worsening) and disease regression (improvement). Over time, the amounts or relative amounts (e.g., the pattern) of the RABV-G protein or fragment thereof changes. This method involves measuring RABV-G protein or a fragment thereof in a subject at least two different time points, c.g., a first time and a second time, and comparing the change in amounts, if any. The course of disease is determined based on these comparisons. Similarly, this method is useful for determining the response to treatment. If a treatment is effective, then the RABV-G protein or a fragment thereof will trend toward normal, while if treatment is ineffective, the RABV-G protein or a fragment thereof will trend toward disease indications.

[0330] 6. Subject Management

[0331] In certain embodiments of the method including the detection and / or analysis of RABV-G protein or a fragment thereof further includes managing subject treatment based on the status. Such management includes the actions of the physician or clinician subsequent to determining infections and / or other RABV-G-related disease and disorder status. For example, if a physician makes a diagnosis of RABV-G-related disease and disorder, then a certain regime of treatment, such as prescription or administration of chemotherapy, radiation, immunotherapy, including, but not limited to administration of the compositions discussed in more detail below, might follow. Alternatively, a diagnosis of non- RABV-G- related disease might be followed with further testing to determine a specific disease that the patient might be suffering from. Also, if the diagnostic test gives an inconclusive result on RABV-G -related disease and disorder, further tests may be required.

[0332] One embodiment provides a method for selecting a subject for treatment of RABV-G- related disease and disorder by detecting the presence or quantity of RABV-G protein or an fragment thereof in a sample from a subject suspected of RABV-G-related disease and disorder, comparing the levels of RABV-G protein or a fragment thereof in the sample to a predetermined standard, wherein the patient is selected RABV-G-related disease and disorder if RABV-G protein or an fragment thereof, or a certain level thereof, is detected in the sample. Such treatments can be those known to be effective and / or preferred for treating subjects with aberrant RABV-G protein or an fragment thereof expression. Exemplary treatments are discussed below.

[0333] In some embodiments, the methods additionally or alternatively include identifying the subject as not having a RABV-G-related disease and disorder, when the test is negative. Thus, although the subject may have another diseases or disorder, the subject can be identified as negative for aberrant RABV-G -related disease and other diseases and disorder. 45803765 gg ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0334] Such embodiments may lead to selection of alternative treatments and may avoid treatments known to be effective or preferred for treating subjects with aberrant RABV-G -positive conditions, and / or may include treatments that are known not to be effective and / or preferred for treating subjects with aberrant RABV-G-positivc conditions.

[0335] Additional embodiments relate to the communication of assay results or diagnoses or both to technicians, physicians or patients, for example. In certain embodiments, computers will be used to communicate assay results or diagnoses or both to interested parties, e.g.: physicians and their patients. In some embodiments, the assays will be performed or the assay results analyzed in a country or jurisdiction which differs from the country or jurisdiction to which the results or diagnoses are communicated.

[0336] In a preferred embodiment a diagnosis based on the presence or absence in a test subject of RABV-G protein or a fragment thereof is communicated to the subject as soon as possible after the diagnosis is obtained. The diagnosis may be communicated to the subject by the subject's treating physician. Alternatively, the diagnosis may be sent to a test subject by email or communicated to the subject by phone. A computer may be used to communicate the diagnosis by email or phone. In certain embodiments, the message containing results of a diagnostic test may be generated and delivered automatically to the subject using a combination of computer hardware and software which will be familiar to artisans skilled in telecommunications. In certain embodiments all or some of the method steps, including the assaying of samples, diagnosing of diseases, and communicating of assay results or diagnoses, may be carried out in diverse (e.g., foreign) jurisdictions.

[0337] 7. Screening Assays

[0338] The RABV-G binders can be used to screen for compounds that modulate the expression of the RABV-G protein or fragments thereof in vitro or in vivo, which compounds in turn may be useful in treating or preventing RABV-G-related diseases and disorders in patients. Compounds suitable for therapeutic testing may be screened initially by identifying compounds which reduce the presence of RABV-G or a fragment in RABV-G-related disease and disordered cells.

[0339] Test compounds capable of modulating the presence and / or expression of RABV-G or a fragment thereof in RABV-G-related disease and disorder may be administered to patients who are suffering from or are at risk for developing symptoms of rabies infection and / or other RABV-G-related disease and disorder. For example, the administration of a test compound that decreases the presence or activity of RABV-G or a fragment thereof may decrease the risk of RABV-G-related disease and disorder in a patient if the increased

[0340] 45803765 67 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US presence or activity of the RABV-G or a fragment thereof is responsible or indicative, at least in part, for the onset of RABV-G-related disease and disorder.

[0341] At the clinical level, screening a test compound includes obtaining samples from test subjects before and after the subjects have been exposed to a test compound. The levels in the samples of RABV-G or a fragment thereof can be measured and analyzed to determine whether the levels of the RABV-G or a fragment thereof changes after exposure to a test compound. The samples can be analyzed by any appropriate means known to one of skill in the art including e.g., by the means described herein. In a further embodiment, the changes in the level of expression of RABV-G or a fragment thereof can be measured using in vitro methods and materials. For example, human tissue cultured cells which express, or are capable of expressing, RABV-G or a fragment thereof may be contacted with test compounds. Subjects who have been treated with test compounds can be routinely examined for any physiological effects which may result from the treatment. In particular, the test compounds are evaluated for their ability to decrease disease likelihood in a subject. Alternatively, if the test compounds are administered to subjects who have previously been diagnosed with RABV-G-related disease and disorder, test compounds will be screened for their ability to slow or stop the progression of the disease.

[0342] 8. Assessing the Effectiveness of Treatment or Risk for Developing Symptoms of Rabies Infection and / or RABV-G-related Diseases and Disorders

[0343] Methods for determining the course of Rabies and / or other RABV-G-related diseases and disorders in a subject are also provided. Disease course refers to changes in disease status over time, including disease progression (worsening) and disease regression (improvement). Over time, the amounts or relative amounts (e.g., the pattern) of RABV-G protein or an fragment thereof changes. Accordingly, this method involves measuring RABV-G protein or an fragment thereof in a subject at least two different time points, e.g., a first time and a second time, and comparing the change in amounts, if any. The course of disease is determined based on these comparisons. Similarly, this method is useful for determining the response to treatment. If a treatment is effective, then the RABV-G protein or fragment thereof will trend toward normal, while if treatment is ineffective, the RABV-G protein or fragment thereof will trend toward disease indications.

[0344] In yet another example, the binders can be used in studies to determine if the subject is at risk for developing symptoms of rabies infection and / or other RABV-G-related disease and disorder.

[0345] 45803765 68 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0346] D. Diseases to be Detected and / or Treated

[0347] The rabies virus glycoprotein (RABV-G) forms part of infectious rabies virus, which is implicated in rabies infection and pathogenesis, but it’s also linked to certain stages and mechanisms within this disease.

[0348] RABV-G is important for the virus's entry into host cells, particularly neurons. RABV-G facilitates attachment to cellular receptors, such as the nicotinic acetylcholine receptor, leading to viral uptake and spread in the nervous system. RABV-G is implicated in the development of rabies encephalitis, the severe inflammation of the brain associated with rabies infection, leading to symptoms such as aggression, hydrophobia, and paralysis. As RABV-G enables the virus to travel along neuronal pathways, it contributes to neurological symptoms, including confusion, agitation, muscle spasms, and paralysis. It is associated with the neurotoxic effects that occur as the virus spreads through the central nervous system. RABV-G is involved in the vims's immune evasion strategies, allowing it to persist within the host by avoiding immune detection, particularly through its interactions within neural tissues. When treatment is delayed, RABV-G’s role in rapid viral propagation in neural tissues can lead to progressive and fatal complications, often without the host's immune system effectively combating it.

[0349] Any of the disclosed compositions and methods can be used to detect, diagnose, prognose, and / or treat any stage, mechanisms of rabies infection, symptoms of rabies infection or complications caused by rabies vims, RABV-G protein or RABV-G related disease or disorder.

[0350] E. Methods of Treatment

[0351] Methods of treatment are also provided and can be used alone or in combination with other methods disclosed herein such as the disclosed methods of detection, diagnosis, prognosis, and treatment monitoring. The methods typically include administering a subject in need thereof an effective amount of a disclosed composition, e.g., a RABV-G binder or CAR T cell formed therefrom, to treat the subject. This is particularly tme where disease or condition is characterized by increased expression or presence of RABV-G. Additionally, or alternatively, particularly where increased live RABV-G vims has been detected, antivirals can be employed.

[0352] Cell-mediated immune responses can be directed against RABV-G antigens to treat viral infection. Similarly, RABV-G antigens can be targeted by antibody-conjugates, such as antibody drag conjugates.

[0353] 45803765 69 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0354] In cases where viral assembly occurs, the use of antiretroviral drugs to inhibit productive viral replication can also be considered for use alone or in combination with an immunotherapeutic approach.

[0355] An exemplary method involves treating a subject (e.g., a human) having a disease, disorder, or condition by administering to the subject an effective amount of a pharmaceutical composition including genetically-modified cells including a RAB V-G binder, optionally in a bispecific format and / or conjugated to an active agent such as drug (e.g., chemotherapeutic drug).

[0356] In some embodiments, the methods administer cells (e.g., T cells) engineered to express recombinant RABV-G-CAR to a subject (e.g., a human) having a disease, disorder, or condition in an amount effective to treat the disease, disorder, or condition. For example, in some embodiments, the methods treat a disease or disorder associated with an elevated expression or specific expression of RAB V-G or an antigenic fragment thereof by administering to the subject an effective amount of a pharmaceutical composition including cells modified to express recombinant RABV-G-CAR. For example, in some forms, the methods treat a subject having a disease, disorder, or condition by administering to the subject an effective amount of a pharmaceutical composition having a genetically modified cell, where the cell is modified by introducing to the cell:

[0357] (i) a nucleic acid (e.g., vector or mRNA) encoding a RABV-G-CAR; and

[0358] (ii) causing the RABV-G-CAR to be expressed by the cell. The cell can have been isolated from the subject having the disease, disorder, or condition, or from a healthy donor, prior to genetic modification.

[0359] In some embodiments, the methods administer cells a RABV-G binder optionally to a subject (e.g., a human) having a disease, disorder, or condition in an amount effective to treat the disease, disorder, or condition. In some embodiments, the molecule or antibody includes a drug conjugated thereto and / or has antibody-dependent cell-mediated cytotoxicity (ADCC) activity, or complement-dependent cytotoxicity activity (CDC), and / or is a bispecific engager optionally a T cell engager.

[0360] 1. Effective Amounts

[0361] In some forms the methods administer the composition in an effective amount. The effective amount or therapeutically effective amount of a pharmaceutical compositions including modified cells, such as therapeutic T cells, or RABV-G-binder such as an antibody, antibody-drag conjugate or bispecific antibody, etc. can be a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of a disease or disorder, such as a rabies viral

[0362] 45803765 70 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US infection or related complications caused by the virus, RABV-G protein or RABV-G related disease or disorder which could also include, but not limited to, neurological disorder, immune disease, or other condition mentioned herein, or to otherwise provide a desired pharmacologic and / or physiologic effect, for example, reducing, inhibiting, or reversing one or more of the underlying pathophysiological mechanisms underlying a disease or disorder.

[0363] In some forms, when administration of the composition elicits an anti-viral response, the amount administered can be expressed as the amount effective to achieve a desired antiviral effect in the recipient. For example, in some forms, the amount of the composition, is effective to inhibit the viability or proliferation of virus in the recipient. In some forms, the amount of the pharmaceutical composition is effective to reduce the viral burden in the recipient, or reduce the total number of virus, and combinations thereof. In other forms, the amount of the composition is effective to reduce one or more symptoms or signs of viral infection in a patient.

[0364] The effective amount of the composition can vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the disorder being treated, and its mode of administration. Thus, it is not possible to specify an exact amount for every pharmaceutical composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. For example, effective dosages and schedules for administering the pharmaceutical compositions can be determined empirically, and making such determinations is within the skill in the art. In some forms, the dosage ranges for the administration of the compositions are those large enough to effect reduction in viral load, symptoms of the infection, or to reduce viral burden for example.

[0365] The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, and sex of the patient, route of administration, whether other drugs are included in the regimen, and the type, stage, and location of the disease to be treated. The dosage can be adjusted by the individual physician in the event of any counter-indications. It will also be appreciated that the effective dosage of the composition can increase or decrease over the course of a particular treatment. Changes in dosage can result and become apparent from the results of diagnostic assays.

[0366] Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. Optimal dosing schedules can be calculated from 45803765 7 J ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US measurements of drug accumulation in the body of the subject or patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages can vary depending on the relative potency of individual pharmaceutical compositions, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models.

[0367] In a non- limiting example, a composition containing CAR cells described herein can be administered at a dosage of 104to 109cells / kg body weight, preferably 105to 107cells / kg body weight, including all integer values within those ranges. In some forms, patients can be treated by infusing a disclosed pharmaceutical composition containing CAR expressing cells (e.g., T cells) in the range of about 104to 1012or more cells per square meter of body surface (cells / m).

[0368] In particular embodiments, for antibodies and other proteins, the dosage administered to a patient is typically 0.0001 mg / kg to 100 mg / kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.0001 mg / kg and 20 mg / kg, 0.0001 mg / kg and 10 mg / kg, 0.0001 mg / kg and 5 mg / kg, 0.0001 and 2 mg / kg, 0.0001 and 1 mg / kg, 0.0001 mg / kg and 0.75 mg / kg, 0.0001 mg / kg and 0.5 mg / kg, 0.0001 mg / kg to 0.25 mg / kg, 0.0001 to 0.15 mg / kg, 0.0001 to 0.10 mg / kg, 0.001 to 0.5 mg / kg, 0.01 to 0.25 mg / kg or 0.01 to 0.10 mg / kg of the patient' s body weight. Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies or fragments thereof, or fusion proteins may be reduced by enhancing uptake and tissue penetration of the antibodies or fusion proteins by modifications such as, for example, lipidation.

[0369] The administration can be repeated as often and as many times as the patient can tolerate until the desired response is achieved. Compositions can also be administered once or multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988).

[0370] The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly. In some forms, the unit dosage is in a unit dosage form for intravenous injection. In some forms, the unit dosage is in a unit dosage form for oral administration. In some forms, the unit dosage is in a unit dosage form for inhalation.

[0371] 45803765 72 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0372] Treatment can be continued for a duration adequate to achieve specific therapeutic objectives, such as reducing rabies symptoms compared to baseline levels at the start of treatment, or achieving the complete elimination of the vims in the patient. Treatment can be continued for a desired period of time, and the progression of treatment can be monitored using any means known for monitoring the progression of infection treatment in a patient. In some fomrs, administration is carried out every day of treatment, or every week, or every fraction of a week. In some forms, treatment regimens are carried out over the course of up to two, three, four or five days, weeks, or months, or for up to 6 months, or for more than 6 months, for example, up to one year, two years, three years, or up to five years.

[0373] The efficacy of administration of a particular dose of the pharmaceutical compositions according to the methods described herein can be determined by evaluating the aspects of the medical history, signs, symptoms, and objective laboratory tests that are known to be useful in evaluating the status of a subject in need for the treatment of rabies symptoms or other diseases and / or conditions. These signs, symptoms, and objective laboratory tests will vary, depending upon the particular disease or condition being treated or prevented, as will be known to any clinician who treats such patients or a researcher conducting experimentation in this field. For example, if, based on a comparison with an appropriate control group and / or knowledge of the normal progression of the disease in the general population or the particular individual: (1) a subject’s physical condition is shown to be improved, (2) the progression of the disease or condition is shown to be stabilized, or slowed, or reversed, or (3) the need for other medications for treating the disease or condition is lessened or obviated, then a particular treatment regimen will be considered efficacious. In some forms, efficacy is assessed as a measure of the reduction in reducing rabies symptoms and / or viral load or viral titer at a specific time point (e.g., 1-5 days, weeks, or months) following treatment.

[0374] 2. Modes of Administration

[0375] In some embodiments the methods administer the composition in combination with a pharmaceutically acceptable carrier. The compositions described herein can be conveniently formulated into pharmaceutical compositions composed of one or more of the compounds in association with a pharmaceutically acceptable earner. See, e.g., Remington's Pharmaceutical Sciences, latest edition, by E.W. Martin Mack Pub. Co., Easton, PA, which discloses typical carriers and conventional methods of preparing pharmaceutical compositions that can be used in conjunction with the preparation of formulations of the therapeutics described herein and which is incorporated by reference herein. These most typically would be standard carriers for administration of compositions to humans. In one aspect, for humans and non-humans,

[0376] 45803765 73 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US these include solutions such as sterile water, saline, and buffered solutions at physiological pH. Other therapeutics can be administered according to standard procedures used by those skilled in the art.

[0377] The pharmaceutical compositions can include, but arc not limited to, carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the therapeutic(s) of choice.

[0378] Pharmaceutical compositions can be administered to the subject in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Thus, for example, a pharmaceutical composition including modified cells, such as therapeutic T cells, can be administered as an intravenous infusion, or directly injected into a specific site. Moreover, a pharmaceutical composition can be administered to a subject as an ophthalmic solution and / or ointment to the surface of the eye, vaginally, rectally, intranasally, orally, by inhalation, or parenterally, for example, by intradermal, subcutaneous, intramuscular, intraperitoneal, intrarectal, intraarterial, intralymphatic, intravenous, intrathecal and intratracheal routes.

[0379] Parenteral administration, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, cither as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein. Suitable parenteral administration routes include intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue injection (e.g., intraocular injection, intra-retinal injection, or sub-retinal injection); subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps); direct application by a catheter or other placement device (e.g., an implant including a porous, non-porous, or gelatinous material).

[0380] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions which can also contain buffers, diluents and other suitable additives. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous 45803765 74 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0381] Administration of the pharmaceutical compositions can be localized (i.c., to a particular region, physiological system, tissue, organ, or cell type) or systemic.

[0382] It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0383] 3. Combination Therapy

[0384] In some embodiments the compositions are administered in combination with other therapeutic agents or treatment modalities. Any of the disclosed pharmaceutical compositions can be used alone, or in combination with other therapeutic agents or treatment modalities, for example, chemotherapy or stem-cell transplantation. As used herein, “combination” or “combined” refer to either concomitant, simultaneous, or sequential administration of the therapeutics.

[0385] In some forms, the pharmaceutical compositions and other therapeutic agents are administered separately through the same route of administration. In other forms, the pharmaceutical compositions and other therapeutic agents are administered separately through different routes of administration. The combinations can be administered either concomitantly (e.g., as an admixture), separately but simultaneously (e.g., via separate intravenous lines into the same subject; one agent is given orally while the other agent is given by infusion or injection, etc.,), or sequentially (e.g., one agent is given first followed by the second).

[0386] Examples of preferred additional therapeutic agents include other conventional therapies known in the art for treating the desired disease, disorder or condition. The compositions and methods described herein may be used as a first therapy, second therapy, third therapy, or combination therapy with other types of therapies known in the art.

[0387] The disclosed pharmaceutical compositions and / or other therapeutic agents, procedures or modalities can be administered during periods of active disease, or during a period of remission or less active disease. The pharmaceutical compositions can be administered before the additional treatment, concurrently with the treatment, post-treatment, or during remission of the disease or disorder. When administered in combination, the

[0388] 45803765 75 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US disclosed pharmaceutical compositions and the additional therapeutic agents (e.g., second or third agent), or all, can be administered in an amount or dose that is higher, lower or the same than the amount or dosage of each agent used individually, e.g., as a monotherapy. In certain forms, the administered amount or dosage of the disclosed pharmaceutical composition, the additional therapeutic agent (e.g., second or third agent), or all, is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent used individually, e.g., as a monotherapy (e.g., required to achieve the same therapeutic effect).

[0389] The compositions and methods can be further understood by the following numbered paragraphs:

[0390] Paragraph 1. A molecule or antibody containing an antigen binding region of an antibody comprising six complementarity determining regions (CDRs), that immunospecifically binds to a surface unit or a transmembrane unit of a rabies virus glycoprotein (RABV-G).

[0391] Paragraph 2. A molecule or antibody containing an antigen binding region of an antibody that immunospecifically binds to RABV-G, the antigen binding region comprising six complementarity determining regions (CDRs), wherein the CDRs include at least one CDR of the CDRs of anti-RABV-G antibody A2, A4, Al l, or a variant thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto; with all remaining CDRs independently selected from anti- RABV-G antibodies A2, A4, Al l, and variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

[0392] Paragraph 3. The molecule or antibody of paragraph 1 or 2, wherein the CDRs include three heavy chain variable region CDRs independently selected from the heavy chain variable region CDRs of anti-RABV-G antibodies A2, A4, Al l, and variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto in combination with three light chain variable region CDRs independently selected from the light chain variable region CDRs of anti-RABV-G antibodies A2, A4, Al l, and variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

[0393] Paragraph 4. The molecule or antibody of any one of paragraphs 1-3, wherein the CDRs include the three heavy chain variable region CDRs of anti-RABV-G antibody A2, A4, Al l, or variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto in combination with the three light chain variable region CDRs for anti- RABV-G antibody A2, A4, Al 1 or variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

[0394] 45803765 76 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0395] Paragraph 5. The molecule or antibody of any one of paragraphs 1-4, wherein the six CDRs are

[0396] (A) the three heavy chain variable region CDRs and the three light chain variable region CDRs of anti-RABV-G antibody A2;

[0397] (B) the three heavy chain variable region CDRs and the three light chain variable region CDRs of anti-RABV-G antibody A4; or

[0398] (C) the three heavy chain variable region CDRs and the three light chain variable region CDRs of anti-RABV-G antibody All.

[0399] Paragraph 6. The molecule or antibody of any one of paragraphs 1-5, wherein the six CDRs are oriented and in the same orientation as in the anti-RABV-G antibody from which they were selected.

[0400] Paragraph 7. The molecule or antibody of any one of paragraphs 1-6, wherein the heavy chain variable regions and light chain variable regions of antibody A2, A4, and Al 1 are the heavy chain variable regions of A2, A4, or Al 1 according to Tables 2 or variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto., and the light chain variable regions of A2, A4, and Al 1 according to Table 4 or variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

[0401] Paragraph 8. The molecule or antibody of any one of paragraphs 1-7, wherein the six CDRs are the CDRs according to:

[0402] Table 1: Heavy Chain Variable Region CDR Sequences

[0403] Table 3: Light Chain Variable Region CDR Sequences

[0404] 45803765 77 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US or variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

[0405] Paragraph 9. A molecule or antibody comprising a heavy chain variable region comprising three CDRs and a light chain variable region containing three CDRs, wherein

[0406] (A) the three heavy chain variable region CDRs include GYTFTNYG (SEQ ID NO:1), INTYTGEP (SEQ ID NO:2), RGDYFGTKYYFDY (SEQ ID NO:3), respectively, and the three light chain variable region CDRs include QDVSTA (SEQ ID NO:6), SAS, QQHYNTPT (SEQ ID NO:7) respectively;

[0407] (B) the three heavy chain variable region CDRs include GYSFTDYI (SEQ ID NO: 10), INPYYGTT (SEQ ID NO:11), RADGPDYFDY (SEQ ID NO:12), respectively, and the three light chain variable region CDRs comprise QDVNTA (SEQ ID NO: 15), SAS, CQQHYNTPPT (SEQ ID NO: 16). respectively;

[0408] (C) the three heavy chain variable region CDRs include GYTFTDYA (SEQ ID NO: 19), ITTYSGDA (SEQ ID NO:20), ARPYYYGNSWFAY (SEQ ID NO:21), respectively, and the three light chain variable region CDRs comprise QNVGTN (SEQ ID NO:24), SAS, QQYHTYPLT (SEQ ID NO:25), respectively:

[0409] Paragraph 10. The molecule or antibody of any one of paragraphs 1-9, wherein the antigen binding region comprises the heavy chain variable region of A2, A4, Al 1 , or a variant thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

[0410] Paragraph 11. The molecule or antibody of any one of paragraphs 1-10, wherein the antigen binding region comprises the light chain variable region of A2, A4, Al 1, or a variant thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

[0411] Paragraph 12. The molecule or antibody of any one of paragraphs 1-11, wherein the antigen binding region comprises the heavy chain variable region and light chain variable region of A2, A4, Al 1, or variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

[0412] Paragraph 13. The molecule or antibody of any one of paragraphs 1-12, wherein the antigen binding region comprises the heavy chain variable region and / or light chain variable region according to Table 2 and / or Table 4, or variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

[0413] Paragraph 14. The molecule or antibody of any one of paragraphs 1-13, wherein the molecule or antibody is an antibody.

[0414] 45803765 78 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0415] Paragraph 15. The molecule or antibody of paragraph 14, wherein the antibody is an intact antibody and functional antibody fragment or fusion protein.

[0416] Paragraph 16. The molecule or antibody of paragraph 15, wherein the functional fragment or fusion protein is selected from Fab fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments optionally single chain variable fragments (scFv), and single region antibodies optionally selected from sdAb, sdFv, and nanobody fragments.

[0417] Paragraph 17. The molecule or antibody of any one of paragraphs 14-16, wherein the antibody is selected from intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, and multispecific antibodies optionally selected bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, and tandem tri-scFv.

[0418] Paragraph 18. The molecule or antibody of any one of paragraphs 14-17 wherein the antibody is an IgM, IgE, IgA, IgD, or IgG optionally an IgGl, IgG2, IgG3, or IgG4. Paragraph 19. The molecule or antibody of any one of paragraphs 1-18, wherein the molecule or antibody is delectably labeled or comprises a conjugated toxin, drug, receptor, enzyme, receptor ligand.

[0419] Paragraph 20. A fusion protein containing the molecule or antibody of any one of claims 1- 19 and a heterologous amino acid sequence.

[0420] Paragraph 21. A chimeric antigen receptor (CAR) polypeptide containing the molecule or antibody of any one of paragraphs 1-20.

[0421] Paragraph 22. The chimeric antigen receptor of paragraph 21 containing an extracellular antigen binding region, a spacer region, a transmembrane region, and intracellular signaling region.

[0422] Paragraph 23. The chimeric antigen receptor of paragraphs 22 containing a co-stimulatory domain.

[0423] Paragraph 24. A nucleic acid or acids encoding the molecule or antibody of any one of claims 1-19, fusion protein of paragraphs 20, or CAR of any one of paragraphs 21-23. Paragraph 25. The nucleic acid or acids of paragraphs 24 including an expression control sequence operably linked thereto, optionally wherein the expression control sequence comprises a promoter.

[0424] Paragraph 26. A vector containing the nucleic acid or acids of claims 24 or 25.

[0425] 45803765 79 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0426] Paragraph 27. A host cell containing the molecule or antibody of any one of paragraphs 1-19, fusion protein of paragraphs 20, CAR of any one of paragraphs 21-23, nucleic acid or acids(s) of paragraphs 24-25, or vector of paragraph 26.

[0427] Paragraph 28. A CAR immune cell comprising the CAR of any one of paragraphs 21-23, optionally wherein the immune cell is a T cell.

[0428] Paragraph 29. A pharmaceutical composition comprising the CAR immune cells of paragraph 28, or the molecule or antibody of any one of paragraphs 1-1 optionally wherein the molecule or antibody comprises a drug conjugated thereto and / or has antibody-dependent cell-mediated cytotoxicity (ADCC) activity, or complement-dependent cytotoxicity activity (CDC), and / or is a bispecific immune cell engager.

[0429] Paragraph 30. A method of treating a subject in need thereof comprising administering the subject an effective amount of pharmaceutical composition of paragraph 29.

[0430] Paragraph 31. The method of paragraph 30, wherein the subject has a disease or disorder caused by or characterized by increased presence of RABV-G or a fragment thereof.

[0431] Paragraph 32. The method of paragraph 30 or 31, wherein the subject has a rabies infection, is at risk for developing symptoms of rabies infection, or complications caused by rabies infection.

[0432] Paragraph 33. The method of paragraphs 32, wherein the subject has rabies infection and RABV-G or fragment thereof is an antigen of the rabies infection.

[0433] Paragraph 34. A method of detecting RABV-G or fragment thereof comprising contacting a biological sample with one or more molecules or antibodies of any one of paragraphs 1-19, and detecting binding between the molecule(s) or antibod(ies) and the RABV-G or fragment thereof.

[0434] Paragraph 35. The method of paragraph 34 further comprising determining that the sample includes increased RABV-G or fragment thereof if the level of detected binding is higher in the biological sample than in a control.

[0435] Paragraph 36. The method of paragraph 34 or 35, wherein binding is detected by an immunoassay, immunohistochemistry, Western blotting, surface plasmon resonance, flow cytometry (FACS) analysis, or a biochip.

[0436] Paragraph 37. The method of paragraph 36, wherein the immunoassay is selected from an enzyme immunoassay (EIA), radioimmunoassay (RIA), fluoroinmiunoassay (FIA), chemiluminescent immunoassay (CLIA) and counting immunoassay (CIA), homogeneous enzyme-multiplied immunoassays (“EMIT”), apoenzyme reactivation immunoassay (“ARIS”), dipstick immunoassays, or immuno-chromatography assays.

[0437] 45803765 80 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0438] Paragraph 38. The method of any one of paragraphs 34-37, wherein the biological sample is cells or a cell lysate or a fraction thereof or a fluid optionally blood, saliva, or urine.

[0439] Paragraph 39. The method of paragraph 38, wherein the cells or cell lysate or fraction thereof arc derived from a biopsy from a subject.

[0440] Paragraph 40. The method of paragraph 39, wherein the biopsy sample contains, or is suspected to contain, rabies infection.

[0441] Paragraph 41 . A method of diagnosing a subject with a RAB V-G-related disease or disorder comprising detecting RABV-G or fragment thereof according to the method of any one of paragraphs 34-40.

[0442] Paragraph 42. The method of paragraph 41, wherein the RAB V-G-related disease or disorder is a rabies infection, is a risk for developing symptoms of rabies infection, or complications caused by rabies infection.

[0443] Paragraph 43. The method of any one of paragraphs 34-42 further comprising treating the subject.

[0444] Paragraph 44. The method of paragraph 43, wherein the treatment comprises a therapy effective for treating a RABV-G-related diseases and disorders.

[0445] Paragraph 45. The method of paragraph 43 or 44, wherein the treatment comprises administering the subject an effective amount of the pharmaceutical composition of paragraph 29 and / or an antiviral.

[0446] Examples

[0447] Example 1: Development of rabies virus glycoprotein (RABV-G) binding proteins Materials and Methods

[0448] Cells and plasmids

[0449] Soluble RABV-G ectodomains and pseudo viruses used in neutralization assays were produced in 293T cells (ATCC) (Homo sapiens; American Type Culture Collection, no. CRL-3216, RRID:CVCL_0063) grown in Dulbecco’s modified Eagle’s medium (DMEM; ThermoFisher Scientific) with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (ThermoFisher Scientific). Pseudovirus titration and neutralization assays were performed in Vero cells (Cercopithecus aethiops; American Type Culture Collection, no. CCL-81) grown in DMEM with 10% FBS. Whole IgGs were expressed in ExpiCho cells (Cricetulus griseus; ThermoFisher Scientific) grown in ExpiCHO Expression Medium (ThermoFisher Scientific). Fab fragments with C-terminal strep tags were prepared in S2 insect cells (ATCC) grown in Schneider’s Drosophila media (Drosophila melanogaster; Invitrogen, no. R69007; ThermoFisher 21720-024) for adherent cultures and grown in Insect-XPRESS medium

[0450] 45803765 81 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0451] (Lonza #12-730Q) with 1% penicillin-streptomycin (ThermoFisher Scientific) at 27°C in a rotary shaker for suspension cultures.

[0452] Full length glycoproteins and strains

[0453] Full length, wild-type glycoproteins for rabies and seven additional lyssaviruscs were synthesized with human codon adjustment under the CMV promoter in the pCDNA 3.1 (-) backbone. These glycoproteins included PV-strain RABV-G (PDB: 7u9g), Irkut lyssavirus glycoprotein (Genbank: AFP74571), Ikoma lyssavirus glycoprotein (Genbank: YP_006742183), Eastern bat lyssavirus 1 glycoprotein (Genbank: AAK97857 with L244Q, P520A, and P521T), Duvenhage lyssavirus glycoprotein (Genbank: ACF32424 with S244L, H322R, R498M; numbering includes signal peptide), Australian Bat Lyssavirus (Genbank: NP_478342.1 with E432K), Mokola lyssavirus glycoprotein (Genbank: AAA67271), and West Caucasian bat lyssavirus glycoprotein (Genbank: YP_009094271).

[0454] Antibody production and purification

[0455] Whole IgGs were cloned into phCMV expression vectors containing the human IgGl heavy chain, kappa light chain, or lambda light chain. IgGs were expressed in ExpiCho cells via transfection with 15ug plasmid DNA (8ug light chain and 7ug heavy chain) in 25mL culture volume according to the manufacturer’s protocol. Antibodies were purified via Protein A affinity chromatography (Cytiva) and buffer exchanged into PBS prior to experiments.

[0456] Fab fragments were cloned into pMT Puro (heavy chain) and pMT (light chain) insect cell expression vectors after the BiP signal sequence. The heavy chain Fab sequence was followed by a double StrepII tag for purification. Stable cell lines were produced by transfecting S2 cells in 6-well plates with heavy chain and light chain plasmids using Effectene transfection reagent (Qiagen: 301425). Following transfection, cells were selected using Puromycin at 6 pg / mL and expanded into suspension cultures in Insect-XPRESS media. When cultures reached a density of l*107cells / cm2or 2xl06cells / cm2, protein expression was induced by the addition of CuSO4 to a final concentration of 500pM. At 3-4 days after induction, supernatants were collected, adjusted to pH 8.0 with NaOH, and run over a StrepTactin Superflow affinity column to purify Fab fragments. Fragments were eluted from the column in 2.5 mM d-desthiobiotin, and buffer exchanged into PBS using a lOkDa MWCO concentrator.

[0457] Mouse immunization and antibody isolation

[0458] To produce murine antibodies, female BALB / c mice (Jackson labs) were immunized with DNA plasmids encoding full-length PV-strain RABV-G in the pcDNA 3. l(-) vector

[0459] 45803765 82 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US under the CMV promoter. Endotoxin free DNA was produced using a Qiagen Endofree Gigaprep kit. Mice were injected intramuscularly in both quadriceps with about 50 pl. of 50 pg or about 25pg of 0.5pg / mL plasmid DNA in PBS. Following injection of DNA, an AgilcPulsc Waveform Elcctroporator (BTX) was used to electroporate DNA for improved uptake. Mice were immunized with DNA at day 0 and at day 14, and two mice were sacrificed on day 19 (5 days post-boost) to collect splenocytes for plasma cell isolation.

[0460] Spleens were briefly stored in DMEM with 10% FBS on ice prior to the start of the antibody discovery workflow. Spleens were homogenized via passage three times, pooled from both mice, and the resulting single cells were centrifuged. Plasma cells were then purified from bulk splenocytes using the EasySep Release Mouse CD138+ kit according to the manufacturer’s instructions. Following release of plasma cells from kit beads, cells were counted and injected onto the Beacon Optifluidics System (Berekley Lights) for rabies specific plasma cell isolation.

[0461] Biotinylated PV-strain RABV-G ectodomains were incubated with streptavidin coated polystyrene beads (Sphereotech) and used to detect rabies-specific antibody secreting cells in nanoliter pens on OptoSelect 4K chips (Berekeley Lights, Inc.). Secreted antibodies were detected over a 30 minute time course assay with an anti-mouse Alexa (Alexa 488, 594, or 647) antibody added to antigen-coated beads resuspended in cell culture media. Thirteen RABV-G positive plasma cells were exported from the Beacon, three of which ultimately yielded unique, paired antibody heavy and light chain sequences encoding RABV-G positive antibodies. Following individual cell export, cells were lysed in lysis buffer and antibody heavy chain and light chain mRNA was reverse transcribed into cDNA. Heavy chain variable sequences were then amplified from cDNA using primers and light chains using primers for kappa and lambda light chains to produce DNA with overhangs for insertion into human IgG heavy and light chain expression vectors via Gibson Assembly.

[0462] Plasmids from cells that yielded paired heavy and light chain sequences were transfected into ExpiCho cells in 24- well block format according to the manufacturer’s protocol for small-scale antibody expression. Supernatants containing secreted IgGs were collected 4-5 days post-transfection and were tested for RABV-G specificity via ELISA assay.

[0463] RABV-G production and purification

[0464] Soluble PV-strain RABV-G ectodomains consisting of residues 1-439 were transiently expressed in adherent 293T cells, as previously described. Sequences were codon optimized for expression in human cell lines, and the first 19 amino acids of the sequence

[0465] 45803765 83 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US correspond to the glycoprotein’s native signal peptide. The glycoprotein ectodomain was either tagged (for complexes) or followed by a C-terminal double strep-tag II and Avi-tag, with linkers to add flexibility between tags (G-StrepTagll-GGGSGGGSGGGS (SEQ ID NO:28)-StrepTagII -GSGS (SEQ ID NO:29)-AviTag; the resulting sequence has the amino acid sequence: GWSHPQFEKGGGSGGGSGGGSGGGSWSHPQFEKGGGSGSGSGLNDIFE AQKIEWHE (SEQ ID NO:30) (for all other experiments with soluble ectodomains). An exemplary amino acid sequence for the StrepTagll is WSHPQFEK (SEQ ID NO:32). An exemplary amino acid sequence for the AviTag is GLND IFEAQKIEWHE (SEQ ID NO:33) There were cloned into a pcDNA 3. l(-) vector under the control of the CMV promoter. To produce glycoproteins, 293T cells were seeded into T75 flasks at a concentration of 4xlOA4 cells / cmA2 and grown overnight in a standard, humidified CO2 incubator with 5% CO2 at 37C. The following day, cells were transfected with 9.8ug plasmid per flask using Polyethylenimine (PEI) at a ratio of 3:1 DNA:PEI. Cell supernatant was collected and exchanged at two days post transfection, and collected again at four days post transfection.

[0466] Pooled cell supernatants were centrifuged to remove debris, adjusted to pH 8.0 with NaOH, and incubated with StrepTactin Superflow beads overnight on a shaker at 4C. The following day, beads were collected in a gravity flow column, washed with PBS, and eluted in PBS with 25mM d-desthiobiotin. For bio-layer interferometry experiments, glycoproteins were then concentrated to 40uM in a lOOkDa MWCO concentrator and biotinylated at the Avi Tag with BirA ligase (Avidity), according to the manufacturer’s instructions.

[0467] For cryo-EM structure determination, strep-tagged Fabs were bound to StrepTactin beads and washed to remove unbound antibodies. These beads were then incubated overnight with tissue culture supernatant containing untagged soluble RABV-G ectodomains. The following day, beads were collected in a gravity flow column, washed with PBS, and eluted with 25mM d-desthiobiotin. To trimerize glycoproteins or create high molecular weight complexes for cryo-EM, glycoprotein / antibody complexes were then incubated with additional Fab fragments in multiple (3-fold or higher) molar excess for one hour at room temperature. Following incubation, complexes were buffer exchanged into PBS and concentrated via three passes through a 500uL, 100 kDa MWCO concentrator.

[0468] Cryo-EM specimen preparation and imaging

[0469] Complexes concentrated to ~150qg / mL in PBS were mixed 1:3 with 0.36mM Lauryl maltose neopentyl glycol (LMNG) to a final concentration of 0.09mM immediately prior to freezing. 4pL of complexes were loaded onto C-flat 2 / 1 grids and frozen with a FEI Vitrobot

[0470] 45803765 84 ATTORNEY DOCKET NO. LJI 2024-112-01 PRO US at 100% humidity, a blot force of 0, and 10s blot time. Grids were imaged on a Titan Krios 300kV cryo-electron microscope with a Gatan K3 direct electron detector and energy filter, at a total dose of ~50e7A2, 45,000x or 75,900x magnification and a pixel size of 1.1 or 0.66 (Tabic 5). Table 5: CryoEM map and model statistics

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[0472] Data processing

[0473] Cryo-EM maps were reconstructed in CryoSPARC15. To avoid model bias, particles for each dataset were picked first with a blob picker, then with 2D classes generated within the dataset, and finally via Topaz 0.2.5a16. Ab-initio 3D classes were also generated entirely within the dataset to avoid model bias. Maps were refined iteratively in CryoSPARC, using a combination of 2D and 3D classification (3D Hetero-refinement) to remove junk particles and debris, and 3D classification to account for particles with partial antibody occupancy on the RABV-G turner. For maps except for the one with antibody A2, which had between 1-2 Fabs bound per trimer, trimers with partial antibody occupancy were excluded from the reconstruction. 3D homogenous refinement with C3 symmetry was used to reconstruct maps containing antibodies 4C12 / 4H3, 8C5, A4 / 7E8, and 10H5 / 7E8, with local refinement with C3 symmetry used to obtain the final map. For maps containing antibodies A2, Al 1, and RVC68, particles were symmetry expanded using C3 symmetry, then refined in 3D. The map for antibody A2 was refined using a combination of homogeneous refinement (C3), followed by C3 symmetry expansion, non-uniform refinement (Cl), and local refinement (Cl). The map for antibody Al 1 was refined first with homogenous refinement (C3), followed by C3 symmetry expansion, and local refinement (Cl). The map for antibody RVC68 was refined as a monomer to account for flexibility in the fusion domain where RVC68 binds. It was refined first with a combination of homogenous, non-uniform, and local refinement (C3), then symmetry expanded (C3), and masked around the monomer / Fab complex, and refined with local refinement (Cl) to obtain the final map. After reconstruction, maps were sharpened using DeepEMHancer, and models were built using COOT17and refined in Phenix18. Maps and models were validated in Phenix and in the PDB18(Table 5).

[0474] Neutralization Assays

[0475] Rabies pseudoviruses used in neutralization assays were prepared in 293T cells and titered on Vero cells. 293T cells were seeded in 6- well plates at a concentration of 9*105cells / well in 2 mL DMEM with 10% FBS (Day 1). Prior to seeding, wells were incubated with O.lmg / mL poly-L-lysine (# P4832) for 1 hour at room temperature and washed with PBS. Cells were incubated overnight at 37°C and transfected with a plasmid encoding full- length PV-strain RABV-G the following day (Day 2). Each well was transfected with 1.5pg plasmid DNA and TransIT-LTl (Minis) at a 1:3 DNA:transfection reagent ratio, according to the manufacturer’s protocol. The following day (Day 3), transfected cells were infected with VSV-AG GFP parent virus (Kerafast) at an MOI of 1 -2. Vims was incubated on cells in Opti- MEM (Gibco) with 2% FBS and lx Penicillin / Streptomycin, in a total volume of 500pL / well 45803765 gg ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US for 1 hour, with plates rocked every 15 minutes. Following incubation, supernatant was removed and cells were washed once in Opti-MEM / 2% FBS. 2mL of Opti-MEM / 2% FBS were added to cells, and cells were incubated overnight at 37°C. At approximately 18 hours post-infection (Day 4), supernatant was collected from cells and frozen at -80°C.

[0476] Pseudoviruses were titered on Vero cells seeded in DMEM / 10% FBS in 96-well plates at a concentration of 2*10’’ cells / well. Plates were incubated at 37°C for at least 4 hours prior to infection to allow for cells to attach to plates. 1:10 serial dilutions of virus stocks were made by diluting thawed supernatant in Optimem / 2% FBS. Media was removed from cell plates and 50jiL of serial dilutions were added to each well. Each serial dilution was plated in triplicate and plates were incubated overnight at 37°C. The following day, supernatant was removed from plates and cells were fixed in 4% paraformaldehyde / PBS and 20pg / mL Hoescht for 30 minutes at room temperature. Plates were washed twice in PBS to remove the fixative, and infected cells were counted in a ThermoFisher CX5 high-content cell imager to calculate viral titers.

[0477] For neutralization assays, Vero cells were seeded into 96-well plates, as described above. IgGs or Fab fragments were diluted to a starting concentration of 1 - 125pg / mL for IgGs and 0.24-30pg / mL for Fabs and serially diluted 1 part to 4 parts in Optimcm / 2% FBS in 60pL in 96-well dilution plates. Following serial dilution, 15,000 ffu of pseudovirus in 60pL Optimem / 2% FBS was added to each dilution and plates were incubated at 37°C for 1 hour. After incubation, media was removed from Vero cells and replaced with 50pL of pseudovirus / antibody mixture. Plates were incubated for 16 hours at 37°C, then cells were fixed and counted as described above to determine antibody neutralization titer. Three experimental replicates were performed, each consisting of two technical replicates

[0478] Bio-layer interferometry

[0479] Bio-layer interferometry experiments were performed on an Octet Red 384 instrument (Sartorius) using streptavidin biosensors (Sartorius). Biosensors were hydrated in kinetics buffer (PBS with 0.1% BSA and 0.02% CHAPS) for a minimum of 10 minutes prior to experiments. Kinetics buffer was also used for all subsequent dilutions of proteins and washes in this protocol. For each experiment, the instrument was run in 8-biosensor mode, with 7 biosensors for data collection and one biosensor for reference subtraction. A baseline reading was collected for 30s, and then soluble RABV-G ectodomains biotinylated at the C- terminal Avi-tag were loaded onto biosensors to ~lnm of binding over 5 minutes. Biosensors were washed for 1 minute, then incubated with Fab fragments at various concentrations (40, 20, 10, 5, 2.5, 1.25, and 0.625nM) in kinetics buffer for 5 minutes to measure association.

[0480] 45803765 87 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US

[0481] Biosensors were then washed in kinetics buffer for 5 minutes to measure disassociation.

[0482] For competition assays, biosensors were loaded with RABV-G, as described above, and then saturated with 40nM of a Fab fragment. Biosensors were then washed in kinetics buffer and binding 40nM of additional Fab fragments from antibodies in our panel were measured. Competition was determined by comparing Fab binding to RABV-G / Fab complexes with Fab binding to RABV-G alone.

[0483] Data was analyzed in the Octet Data Analysis 11.1 software using single reference subtraction, with biosensors with RABV-G and without Fabs serving as references for nonspecific binding. Data was fit with a 1 : 1 binding kinetics model with global fitting, and experiments were performed in duplicate.

[0484] Fusion Assays

[0485] For fusion assays, two populations of 293T cells each expressing part of a split mNeonGreen protein (mNGi-io or mNGn) were co-seeded in 24-well plates in a 1: 1 ratio and a concentration of 2*104cells / cm2each (4*104cells / cm2total). The following day, cells were transfected with full-length RABV-G using TransIT-LTl transfection reagent at a 1 :3 DNA:transfection reagent ratio, according to the manufacturer’s protocol. Three days posttransfection, cells were incubated with IgGs or Fabs (1.2-150 pg / mL IgG and 0.39-50 pg / mL Fab) in PBS for 1 hour at room temperature. Following incubation, cells were washed with PBS and incubated in citric acid buffer (0.1M citric acid / Na citrate, pH 5.5) for 10 minutes at room temperature. Following incubation, citric acid buffer was removed from cells, and cells were washed in PBS and then incubated in DMEM with 10% FBS for 1 hour at 37 °C to facilitate cell fusion. Cells were fixed in 4% paraformaldehyde / PBS for 30 minutes, washed with PBS, and imaged on a Keyence epifluorescence microscope to visualize syncytia formation.

[0486] Binding and Uptake Assay

[0487] 293T cells were seeded on 8-well glass bottom plates (IB ID I) at a density of l*104cells / well and grown overnight at 37°C. The following day, rabies pseudoviruses 2*106ffu / well were incubated with IgG at a concentration of 4 times the IC90 for 1 hour on ice, then incubated with pre-cooled cells on coverslips for 1 hour on ice. Following incubation, cells were returned to 37°C for 15 minutes, then fixed in 4% paraformaldehyde / PBS for 15 minutes at room temperature and quenched in 20mM glycine / PBS for 5 minutes. Cells were permeabilized with 0.1% Triton X-100 in PBS for 5 minutes and blocked in 1% normal goat serum / PBS for 1 hour. Cells were then stained with stained with both 1 pg / mE RVC68 and Ipg / mL RVA122 Fab fragments in PBS with 1% normal goat serum for 1 hour at room

[0488] 45803765 88 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US temperature, washed twice with PBS, and stained with 1:2,000 Streptactin DY549, 1:2,000 Phalloidin Alexa 488, and 20pg / mL Hoescht in PBS with 1% normal goat serum for 1 hour. Coverslips were then washed twice in PBS and mounted on slides with Prolong Antifade Gold mounting reagent (Invitrogcn). Cells were imaged on a Zeiss 880 confocal microscope at 63x magnification.

[0489] Immunofluorescence Assay

[0490] 293T cells expressing half of a split mNeonGreen protein were seeded in 96-well plates at a concentration of 4*104cells / cm2(2*104cells / cm2each population) and incubated overnight at 37°C. The following day, cells were transfected with full-length PV-strain RABV-G or glycoprotein from Irkut lyssavirus, Ikoma lyssavirus, Eastern bat lyssavirus 1, Duvenhage lyssavirus, Mokola lyssavirus, or West Caucasian bat lyssavirus with TransIT- LT1 transfection reagent, as described above. Two days post-transfection, cells were either were fixed in 4% paraformaldehyde for 30 minutes for cell staining or incubated with 0.1M citric acid / sodium citrate buffer at pH 5.5 to induce fusion. For cell staining, cells were washed with PBS, and stained with either 5 pg / mL of one of the nine monoclonal antibodies from our panel, 1 :500 HRIG, or 1 :500 of a rabbit anti-RABV-G polyclonal antibody (a kind gift from Dr. Matthias Schnell, Thomas Jefferson University) in 1% BSA / PBS for 1 hour at room temperature. Cells were washed twice with PBS, then stained with 1 : 1,000 goat antihuman DY594 (Invitrogen) or goat anti-rabbit Alexa 568 and 20pg / mL Hoescht for 1 hour at room temperature.

[0491] Results

[0492] For multiple viruses, administration of highly purified and well-characterized potent monoclonal antibodies (mAbs) provides a superior treatment option to polyclonal human sera (Mire et al. 2017; Mulangu et al. 2019; Cross et al. 2019). Individual mAbs with breadth and potency of neutralization than polyclonal serum have been identified for rabies and multiple related lyssaviruses (De Benedicts et al. 2016)(Zorzan et al. 2023). However, these studies lack key structural information about what epitopes antibodies target, what residues they interact with, and how antibody interactions neutralize virus. Identifying the specific amino acid contacts that control viral neutralization will greatly facilitate the development of rabies therapeutic antibody cocktails by identifying antibodies that target spatially distinct regions of the glycoprotein and that avoid regions of high residue variability or frequent escape mutations.

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[0494] High-resolution structural information will also allow directed mutagenesis of antibodies to increase binding strength, neutralization potency, and ability to recognize and neutralize related lyssaviruses.

[0495] Previously determined structures of mAbs in complex with RABV-G include two antibodies that recognize antigenic site III (as defined in the mutation scheme; mAbs RVA12211and 17C714) and three that recognize antigenic site 11 (RVC2013, 523-1110, and 1112-114). The present study aimed at characterizing additional mAbs to determine the span of neutralizing rabies antibody recognition and identify new rabies antigenic sites. RABV-G antigenic sites are classified either by antibody sensitivity to RABV-G point mutations or by antibody competition. The mutation-based naming scheme identified five major and one minor antigenic site: site I (residues 263-264), site Ila (198-200), site lib (34-42), site III (330-338), site IV (226-231), and site ‘a’ (342-343) (Figure IB) (Evans et al. 2012). The antibody competition-based naming scheme, which used a large antibody panel to define competition groups, separated antibodies as groups I, III, III.2, A, B and C (De Benedictis et al. 2016). Note that groups named I and III in the competition-based scheme are distinct from antigenic sites I and III in the mutation-based scheme. Both the mutation-based and competition-based naming schemes were developed prior to the availability of 3D structures for RABV-G. Crystal structures of alternate conformation pre- and post-fusion RABV-G became available in 2020 (Yang et al. 2020), a PHD / antibody complex in 2020 (Hellert et al. 2020), and the first structures of pre-fusion, trimeric RABV-G in 2022 (Callaway et al. 2022; Ng et al. 2022), in complex with stabilizing antibodies. Thus far, just five individual mAbs, two against the same antigenic site, have been illuminated, and the different pre-structure naming schemes have not yet been reconciled. Thus, many antibody epitopes were largely inferred, rather than directly visualized. mAb Discovery and Characterization

[0496] Human monoclonal antibodies (mAbs) from publicly available sequence information (mAbs 4C12, 4H3, 7E8, 8C5, 10H5, and RVC68, and complemented them with three additional novel mAbs elicited for this study by immunizing mice (mAbs A2, A4, and Al l) were synthesized. Bio-layer interferometry was used to organize antibodies into competition groups, observing four groups across the panel: one competition group with mAbs 10H5 and A4 (Figure 11); a second with mAbs 4H3, 7E8, 8C5, A2, and Al l; and two more with mAbs RVC68 and 4C12 each forming their own distinct competition groups. Interestingly, 4C12 exhibited directional competition: when placed on the sensor first, it blocks binding of 10H5 and A4, but placing 10H5 and A4 on the biosensor first does not block binding of 4C12. Each 45803765 9Q ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US of these mAbs were mapped in complex with RABV-G by cryo-EM.

[0497] In these studies, a wild-type RABV-G which rapidly transitions into conformationally heterogeneous monomers if not stabilized by an antibody was used. To retain the trimeric, pre-fusion conformation of RABV-G, one of several pre-fusion stabilizing mAbs for structure determination was employed. Binding of mAb RVA122, binding site III (as defined by the mutagenic scheme) from prior work11, allowed high-resolution structure determination of six additional mAbs against other sites. EM studies revealed that 10H5 and A4 also bound site III. Binding of these mAbs to stabilize trimers allowed intermediate (6- 10 A) resolution structures. Even the intermediate-resolution structures, however, allow mapping of the general antibody binding footprints. Overall, nine complexes were determined by cryoEM, each containing a site III mAb plus one other mAb.

[0498] The instant application reveals high-resolution cryo-EM structures for six additional mAbs, and low-resolution structures for three more. In this work, the number of new antibodies, combined with and in comparison to the handful of prior structures, now allow assignment of actual, 3D antigenic footprints to the previously proposed antigenic sites. These nine structures span five of the previously proposed antigenic sites, reconcile antigenic site nomenclature across the mutagenesis-based and antibody competition-based studies, and include two new antigenic sites not previously identified. Through complementary functional studies, it was identified the likely mechanisms of neutralization against each antigenic site and determine which rabies antibodies recognize glycoproteins from other lyssaviruses. Combining these distinct structures and earlier work yields a more complete map of the rabies antigenic landscape, through which potent and broad rabies and lyssavirus neutralization may be understood, better candidates can be identified for therapeutic antibody cocktails, and select better metrics to evaluate next-generation vaccines for rabies and related lyssaviruses.

[0499] In this application, six high-resolution and three additional low-resolution structures of RABV-G / antibody complexes were solved, yielding an immunogenic landscape of rabies virus recognition and key information about the antibody binding sites and contacts that control rabies neutralization. The cryo-EM structures of nine different mAbs in complex with RABV-G (six high-resolution and three low-resolution), that together span the breadth of the rabies immunological landscape. This work allowed the characterization of multiple unvisualized epitopes and reconcile disparate RABV-G antibody classification schemes from the literature. Five of the nine antibodies bind to the pleckstrin homology domain (PHD) in a continuum that spans the previously proposed antigenic sites II and IV (previously defined by 45803765 <) | ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US amino acid mutations); two bind to the upper central domain (CD), spanning antigenic sites III and ’a’ (also previously defined by amino acid mutations); one binds to a previously unrecognized antigenic site on the lower central domain; and one binds to a second previously unrecognized antigenic site on the fusion domain (ED). After mapping the epitopes, the mechanisms were explored through which this array of antibodies neutralizes virus and recognizes glycoproteins from related lyssaviruses. Two antibodies neutralize by bridging the PHD and CD on the RABV-G monomer, stabilizing the pre-fusion conformation. All nine antibodies block fusion as IgGs, five also block fusion strongly as Fab fragments, four block viral uptake, and another four capitalize on IgG bivalency for higher potency neutralization. Six antibodies are also broadly reactive, recognizing glycoproteins from phylogroup I and II lyssaviruses. These structures and experimental results provide a roadmap for selection of potent, pan-lyssavirus antibody cocktails to replace HRIG and to treat infections of rabies-related lyssaviruses for which no vaccines yet exist.

[0500] Rabies antibody binding sites and discovered antigenic sites

[0501] In an effort to more fully characterize the antibody landscape of RABVG mice were immunized with DNA encoding recombinant, wild-type RABV G, identifying three previously unrecognized mAbs, termed A2, A4 and Al l. Six other mAbs identified in vaccinated humans were gathered, but not yet structurally characterized, which include mAbs 4C12, 4H3, 7E8, 8C5, 10H5, and RVC68 . Antibodies were expressed as both IgGs and as strep-tagged Fabs for structural and functional studies, and determined structures of RABV- G / Fab complexes via cryo-electron microscopy (cryo-EM).

[0502] Complexes were prepared for cryo-EM by immuno-precipitating soluble PV strain RABV-G ectodomains with strep-tagged Fab fragments, as previously described (Callaway et al. 2022). For Fab fragments that failed to trimerize RABV-G protomers, additional Fab fragments (RVA122 (Callaway et al. 2022), 4C12, or 10H5) were then added from a nonoverlapping binding site to induce trimerization, and ran complexes over a 100 kDa MWCO concentrator to remove monomeric glycoproteins and unbound Fabs. Complexes were frozen in PBS with 0.09mM LMNG on 2 / 1 C-flat grids, and data were collected on a Titan Krios with a Gatan K3 direct electron detector and energy filter, yielding structures ranging from 2.9 to -7 A in resolution.

[0503] Five of the nine antibodies bound to the PHD (antibodies A2, Al l, 4H3, 7E8 and 8C5), three to the CD (antibodies A4, 10H5, and 4C12), and one to the FD (antibody RVC68). However, instead of binding to discrete antigenic sites identified in prior work via glycoprotein mutagenesis, it was observed that the antibodies recognize a broader continuum 45803765 92 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US of antigenic sites within protein domains (Figures 1-2). PHD-binding antibodies A2, Al l, and 8C5, for example, overlap both antigenic sites Ila / b and IV, and while PHD antibody 7E8 largely targets the traditional antigenic site IV alone. Another PHD-binder, 4H3, has additional contacts extending into the fusion domain. Similarly, CD-binding antibodies 10H5 and A4 overlap traditional antigenic sites III and ‘a,’ while 10H5 also has contacts in the PHD. The remaining two antibodies, 4C12 and RVC68, recognize new antigenic sites not previously identified via mutagenesis; antibody 4C12 binds to a site on the lower CD, below traditional antigenic sites III and ‘a’, and antibody RVC68 binds to the FD.

[0504] The antibodies that bind primarily to the PHD, A2, Al l, 4H3, 7E8, and 8C5, do not bridge domains. They do not stabilize the pre-fusion conformation, and further, they bind to glycoproteins in a largely pH-independent manner (Figure 5). These results indicate that antigenic sites on the PHD are readily available in both the pre- and post-fusion conformations of the glycoprotein.

[0505] In contrast, two of the three antibodies that primarily recognize the CD, 4C12 and 10H5, do bridge domains. Both antibodies link the CD and PHD together to stabilize the prefusion conformation and facilitate glycoprotein trimerization. Unsurprisingly, these antibodies bind in a pH-dcpcndcnt manner, with progressively less attachment at decreasing pH (Figure 5). However, both 4C12 and 10H5 antibodies do maintain some binding at acidic pHs. Because the binding site they recognize on the CD is unavailable in the post-fusion conformation, it is likely they bind the small proportion of pre-fusion RABV-G that exists at acidic pH. Over time, as unbound glycoproteins breathe and shift conformation, more antibodies may bind, leading to similar levels of bound antibodies in ELISA assays, but requiring progressively higher antibody concentrations with more acidic pHs. In contrast, the remaining CD-binding antibody, A4, does not stabilize glycoprotein trimers. During cryo-EM data processing with complexes at neutral pH, RABV-G trimers in complex with A4 were difficult to find, indicating that any pre-fusion stabilizing interactions with the PHD may be weak.

[0506] The cryo-EM structure of RABV-G in complex with antibody RVC68 reveals a new antigenic site, located on the FD. RVC68, the first antibody revealed in complex with the FD, is notable because it targets highly conserved residues across the lyssavirus family. As a result, it has broad binding and in-vitro neutralizing activity against several rabies-related lyssaviruses. Although the RVC68 epitope exists in both the pre- and post- fusion glycoprotein conformations, there is more antibody binding at acidic pHs (Fig. 5), indicating likely greater availability of the epitope in the post-fusion conformation. In the prefusion

[0507] 45803765 93 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US complex, RVC68 binds to RABV-G at an angle that would cause the antibody Fc to sterically clash with cellular membranes; binding of the IgG may be better accommodated in the postfusion conformation.

[0508] The previous mapping by mutagenesis, before the availability of RABV-G structures, identified residues on RABV-G important for antibody binding and clustered them into four antigenic sites. Residues 263-264 were named “antigenic site 1”, residues 34-42 and 198-200 “antigenic site II”, residues 330-338 “antigenic site III”, and residues 226-231 “antigenic site IV” (Figures 10A-10B)12. Structures of RABV-G in complex with RVC20 (Competition group I) and RVA122 (Competition group III) (have linked group I from the competitionbased naming scheme to the Pleckstrin-homology domain (sites II and IV from the mutagenesis-based naming scheme) and competition group III to site III from the mutagenesis-based scheme. The nine cryo-EM complexes described here substantially expand on that work. Five antibodies from these nine complexes target antigenic sites II and IV, revealing an overlapping epitope rather than two distinct antigenic sites. Two other mAbs recognize epitopes containing residues associated with antigenic site III. The remaining two antibodies recognize new epitopes not overlapping with any classical antigenic site, which we have named ‘antigenic site V’ and ‘antigenic site VI’, expanding on the classic mutagenesisbased nomenclature. The details of these antigenic sites are described in the detailed description above.

[0509] Mechanisms of Neutralization

[0510] As IgGs, all antibodies neutralize rabies pseudoviruses (Vesicular Stomatitis Virus pseudotyped with full-length PV-strain RABV-G) with IC50s around lOpM, with the exception of RVC68. As Fabs, however, some antibodies maintain high neutralization titers, while others drop in potency, indicating that bivalent antibody binding can be important for rabies neutralization. The three murine antibodies, A2, A4, and A 11 neutralize substantially worse as Fabs than as IgGs. Notably, Fab potency was not linked to epitope: other antibodies targeting the same antigenic sites neutralized strongly as Fabs. Instead, individual antibody binding kinetics likely affect neutralization outcomes. A4 and Al l Fabs have a higher off- rate in bio-layer interferometry experiments than other antibodies targeting the same sites. Further, while A2 Fabs had relatively low off-rates in these experiments, only partial occupancy was observed of A2 Fabs on RABV-G trimers in cryo-EM, indicating disassociation from RABV-G. Because binding angles of A2, A4, and Al 1 would not allow both arms of the IgG to simultaneously bind the same trimer, the increased potency of IgGs compared to Fabs likely comes from IgG cross-linking neighboring trimers or viruses,

[0511] 45803765 94 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US although greater steric hindrance by the IgG bulk may also play a role in neutralization.

[0512] To identify mechanisms of neutralization for all antibodies and to determine if antigenic site or bivalency affected mechanisms of neutralization, additional experiments were performed to measure fusion inhibition and cellular binding / uptakc. In the fusion assays, two cell populations were co-seeded each expressing half of a split GFP protein, then transfected the cells with RABV-G and incubated those cells with IgGs or Tabs at neutral pH. After incubation, cells were washed to remove unbound Fabs, and exposed them to pH 5.5 to trigger RABV-G-mediated fusion of the cell membranes and association of the split GFP proteins. It was found that all IgGs blocked fusion and inhibited with similar potencies, consistent with the potent neutralization it was observed with all of the IgGs except RVC68. As Fabs, A2, A4, Al 1, and RVC68 inhibited fusion substantially worse than their IgG counterparts. For the murine antibodies A2, A4, and Al l, loss of fusion inhibition as Fabs is consistent with the decreased neutralization potency as Fabs (Figures 3A-3C). For RVC68, IgGs and Fabs neutralize with equal potency, but only Fabs have poor fusion inhibition, indicating that RVC68 may primarily neutralize through another mechanism.

[0513] To determine if antibody binding blocks uptake, assays were performed in which rabies pscudoviruscs were incubated with IgGs, incubated complexes with cells to allow internalization, and stained cells with a polyclonal rabbit anti-RABV-G antibody to visualize uptake. It was found that A4, 7E8, 10H5, and RVC68 IgGs effectively block attachment of rabies pseudoviruses to cells, whereas A2, Al l, 4C12, 4H3, and 8C5 IgGs do not. In comparing binding sites between these antibodies, those that failed to block uptake consist of four of the five PHD-binders (7E8 excepted), and the single lower CD binder 4C12. Both upper CD binders, A4 and 10H5, and the FD binder, RVC68 blocked uptake, indicating that these antibodies either directly overlap receptor-binding sites or indirectly block receptors through steric hindrance, by crosslinking RABV-G trimers, or by crosslinking pseudoviruses. The sole PHD-binder to block uptake, 7E8, binds at a slightly different angle than its counterparts, which might allow receptor blocking via crosslinking.

[0514] Broad Reactivity

[0515] A major goal of rational rabies therapeutic antibody and vaccine design is to develop therapeutics effective not only against rabies, but also the many rabies-related lyssaviruses that cause the same clinical disease as rabies virus. To determine whether specific antigenic sites or amino acid contacts facilitate broad recognition of rabies-related lyssaviruses, a panel of mAbs were screened for binding to glycoproteins from seven additional lyssaviruses. Glycoproteins (G) were chosen from lyssaviruses across all four lyssavirus phylogroups to 45803765 95 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US measure broad antibody recognition. These included four members of phylogroup I, the largest group which also includes rabies virus [Duvenhage lyssavirus (DUVV), Eastern Bat Lyssavirus-1 (EBLV-1), Australian Bat Lyssavirus (ABLV) and Irkut Lyssavirus (Irkut)]; one member of phylogroup II [Mokola lyssavirus (Mokola)]; one member of phylogroup III [West Caucasian Bat Lyssavirus-1 (WCBLV-1)]; and one member of phylogroup IV [Ikoma lyssavirus (Ikoma)].

[0516] Because several of these glycoproteins do not produce sufficiently infectious pseudoviruses for neutralization assays in our hands or express well as soluble glycoprotein ectodomains, we assayed antibody binding against full-length glycoproteins expressed on 293T cells (data not shown). To aid the glycoproteins to reach the cell surface and folded correctly, they were tested in a fusion assay with a green fluorescent reporter as the output for successful fusion (data not shown). Two glycoproteins, Eastern Bat Lyssavirus 1-G and Mokola-G, failed to induce cell-cell fusion in this assay, but were both recognized by antibodies against conformational and quaternary epitopes (RVC68 against both glycoproteins and two different group III mAbs - 4C12 for Eastern Bat Lyssavirus- 1-G and 10H5 for Mokola-G), indicating that these glycoproteins did reach the cell surface in a properly folded form.

[0517] Of the antibodies in the present panel, site VI antibody RVC68 was by far the most broadly cross-reactive, in keeping with previous reports2. In the present assay, RVC68 bound to glycoproteins from Duvenhage, Eastern Bat Lyssavirus-1 , Irkut, Mokola, and Ikoma lyssaviruses (data not shown). The broad cross-reactivity we and others have observed for RVC68 almost certainly results from the broad sequence conservation of its epitope across lyssaviruses (Figures 9A-9D). The only glycoproteins that RVC68 did not recognize in the present assay were Australian Bat Lyssavirus-G and West Caucasian Bat Lyssavirus-G. Previous reports indicate that Australian Bat Lyssavirus neutralization was strain specific2, and while the RVC68 contacts are conserved in Australian Bat Lyssavirus-G, a lysine residue in the footprint at position 96 appears likely to disrupt binding. For West Caucasian Bat Lyssavirus-G, in contrast, the RVC68 binding site is much less conserved. Based on sequence comparison, the most disruptive non-conserved residue is likely at W100 (R in West Caucasian Bat Lyssavirus-G), which has also been shown to disrupt rabies neutralization in a deep mutational scanning assay.

[0518] The second most cross-reactive antibody in our panel, site V antibody 4C12, recognized glycoproteins from Eastern bat lyssavirus 1 , Duvenhage, and Irkut lyssaviruses (data not shown). The 4C12 binding footprint is largely conserved among rabies and these

[0519] 45803765 96 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US lyssaviruses (Figure 9B), with only two residues that interact with 4C12 substantially differing from rabies (residues 318 and 355, at the edge of the binding footprint). Similar conservation of binding sites was observed for site IL / IV antibodies 8C5 and 4H3, which recognize glycoproteins from Eastern bat lyssavirus 1 and Duvcnhagc viruses, respectively. Of the glycoprotein residues interacting with 8C5, only two substantially differ between RABV-G and Eastern Bat Lyssavirus 1-G (residues 194 and 242; N and A in RABV-G, and T and S in Eastern Bat Lyssavirus 1-G). A larger number of differences between RABV-G and Duvenhage-G exist in the 4H3 binding footprint, but only one residue that interacts with 4H3 substantially differs between the glycoproteins (residue 194; N in RABV-G and R in Duvenhage-G).

[0520] Site III antibody 10H5, finally, is notable because only 10C5 and RVC68 recognize Mokola glycoprotein. Possibly, sequence similarity between RABV-G and Mokola-G in the antibody binding site drives cross-reactivity.

[0521] Overall, it was found that antibodies with overlapping epitopes vary in their capacity for broad lyssavirus recognition. No single antigenic site yielded antibodies that are universally broad, highlighting the importance of individual antibody / glycoprotein contacts in binding and neutralization. Also, the two antibodies at new antigenic sites V and VI were found to have the broadest cross-reactivity, whereas antibodies recognizing sites II / I V and III are largely rabies-specific. Because the residues present in sites V and VI are well conserved among lyssaviruses, it is believed that other antibodies that bind these sites will show similar cross-reactivity.

[0522] All three murine antibodies, A2, A4, and Al l were specific for RABV-G, binding to none of the other lyssavirus glycoproteins in the panel. Of the six human antibodies, however, 4H3, 4C12, 7E8, and 8C5 each recognized DUVV and EBLV-1 glycoproteins, with 4H3 and 4C12 also recognizing Irkut. Human antibody 10H5 recognized Mokola G, but not any of the other lyssavirus glycoproteins. Antibody RVC68, the sole FD-binder, recognized all of the glycoproteins tested except for WCBLV-L as previously reported.

[0523] Antibody binding footprints were next compared and contacts to identify residues that allowed for broad neutralization.

[0524] Discussion

[0525] Here, structures of nine new RABV-G / antibody complexes were solved and assigned physical 3D surface footprints to previous mutation-based and competition-based epitope naming conventions. It was found that rather than targeting individual antigenic sites from mutagenesis-based assignments, rabies antibodies map more broadly to glycoprotein

[0526] 45803765 97 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US domains, often spanning more than one of the traditional antigenic sites. Based on binding footprints, broader antibody binding sites can be defined at the upper and lower PHD, the upper and lower CD, and the FD. This work maps the rabies antibody binding landscape and serves as a foundation for future structure-guided vaccine and therapeutic antibody design.

[0527] Five of the antibodies bound to overlapping sites on the PHD, bridging the residues of the point-mutation-assigned sites lla / b and IV. These five structures now join those of the previously mapped mAbs RVC20 and 17C7, allowing an in-depth analysis of residues that determine lyssavirus cross-reactivity on the PHD. Overall, PHD-binding mAbs exhibit little or no pH-dependence in binding, consistent with an epitope present in both pre- and postfusion forms, and are poor at blocking uptake. The results indicate that instead, these antibodies neutralize primarily through fusion inhibition. It was hypothesized that antibody binding sterically inhibits the formation of fusion intermediates, as the PHD-binders clash RABV-G, as modeled using a crystal structure of a RABV-G monomer in a near post-fusion conformation.

[0528] Two antibodies, A4 and 10H5, bind to overlapping sites on the upper CD, previously linked to antigenic sites III and ‘a’, and now join the previous structure of antibody RVA122. Binding of both A4 and 10H5 is pH dependent, with preferred binding at neutral pH and progressive loss of binding at lower pH. Furthermore, three of the upper CD-binding antibodies exclusively bind the pre-fusion conformation, with contacts stretching into the PHD to bridge domains and recognize a combined surface unavailable in the post-fusion conformation. Another of antibodies binds to the lower CD, an antigenic site not previously visualized in any structure or identified via mutation-based screening. Like the upper CD binding antibodies, its binding is pH-dependent and it bridges the CD and PHD, recognizing an epitope only present in the pre-fusion conformation. mAh RVC68 recognizes the fusion domain (FD), a new antigenic site not previously shown to elicit antibodies. Unlike the other antibodies that bind the PHD or CD, RVC68 exhibits preferential binding at low pH, and while it does not bridge domains, it does block fusion. The current modeling indicate that binding of RVC68 may sterically inhibit insertion of the fusion loops into a target membrane, with whole IgGs creating greater steric hindrance and thereby more potently inhibiting fusion. Preferential binding at low pH may also be driven by sterics, with greater availability of the binding site at low pH. RVC68 points down, towards viral or cell membranes, and association of pre-fusion RABV-G trimers with large cellular membranes (co-purified with RABV-G) would cause Fab and IgG Fc domains to clash with membranes. The cryo-EM data supports this conclusion, as the only membranes or 45803765 9g ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US detergent micelles that was observed is RABV-G / RVC68 complexes in contact with are too small to clash with the complex.

[0529] Overall, it was found that pH-dependence of binding and mechanism of neutralization arc linked to epitope, but potency of neutralization and cross-rcactivity among lyssaviruscs instead varies according to individual antibody binding interactions. In particular, crossreactivity requires binding to conserved residues on the glycoprotein. The identification of two new antigenic sites here, at the FD (RVC68) and the lower CD (4C12), indicates that more antigenic sites have yet to be discovered. That these previously unrecognized sites have not been previously identified in mutagenesis-based screens and are both human in origin indicates that antibodies targeting these sites could be more rare and / or require multiple rabies immunizations over a long period to elicit.

[0530] A mAb cocktail against different, complementary epitopes, and composed of members who each have broad specificity, may provide a mutation-resistant option for treatment, more potent, better characterized and more broadly reactive than rabies-specific human polyclonal antibody. Indeed, the polyclonal preparation is 1,000-fold less potent in neutralization than mAbs against RABV. Recent work has proposed a two-antibody cocktail to replace HRIG, consisting of RVC20, which binds to the PHD, and RVC68, which here is shown to bind to the FD. RVC20 has been shown to potently neutralize phy logroup I lyssaviruses, and RVC68 to broadly recognize lyssaviruses across three phylogroups, although not always with high neutralization potency. The current work on rabies epitope mapping indicates that there is room for a third antibody to target the CD in this and other cocktails. Including a combination of PHD, CD and FD-binding antibodies could allow for greater breadth of neutralization through targeting different sets of conserved residues for the four lyssavirus phylogroups at each domain. For example, a cocktail consisting of a broadly reactive PHD-binding antibody, a broadly reactive FD-binding antibody (such as RVC68) and also a broadly reactive CD-binding antibody such as 10115, which recognizes Mokola G, or mAb 4C12, which recognizes DUVV, EBLV-1, and Irkut glycoproteins, would reduce the likelihood of escape mutations and increase the number of complementary mechanisms of lyssavirus neutralization..

[0531] Together, these results set forth the antigenic landscape of RABV-G, and form a foundation for the improved rabies and pan-lyssavirus vaccines and rational design of rabies monoclonal antibody cocktails to replace HRIG. The antibody- antigen cryoEM structures presented here also provide templates for guidance and interpretation of rational or combinatorial improvements of the individual antibodies themselves for use as therapeutic

[0532] 45803765 99 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US agents. These combined results also provide guidance for vaccine development. For some viruses, especially those with class I glycoproteins which undergo an irreversible pre- to postfusion transition, antibodies specific for pre-fusion conformations are more likely to be neutralizing. However, rabies and other lyssaviruscs arc class III and reversibly transition between pre and post-fusion conformations. It is identified here that antibodies specific for the prefusion conformation (all three anti-CD antibodies) are no more or less potent in neutralization than the antibodies that bind equally well to both pre- and post-fusion conformations (all five anti-PHD antibodies). This finding indicates that a rabies virus vaccine does not necessarily need to present only pre-fusion structures to be effective. Analysis and engineering of these domains, guided by the epitopes of broadly recognizing antibodies against the PHD, CD and FD will facilitate development of broadly protective immunogens.

[0533] The five antibodies targeting overlapping antigenic site I I / I V showed little or no pH- dependence in binding, consistent with an epitope present in both pre- and post-fusion glycoprotein conformations. The three antibodies that bound to the RABV-G central domain, however, were pH-dependent, consistent with epitopes available only in the pre-fusion conformation of the glycoprotein. RVC68, which bound to a unique epitope on the fusion domain, also showed pH-dependence, but favored more acidic pH. While this epitope remains intact in both the pre- and post- fusion conformations of the glycoprotein, the antibody binding angle may result in steric clashes between the Fab and large liposomes or cellular membranes associated with RABV-G in the pre-fusion conformation.

[0534] A possible explanation for the difference in binding specificity between human and murine antibodies is the immunogen and number of vaccinations. To produce murine IgGs, mice were immunized twice with a DNA vaccine encoding full-length, wild-type RABV-G. Human antibodies, in contrast, were produced by immunization with commercial rabies vaccines, and either were collected (de Benedicts 2016) or were likely collected from individuals who had received multiple rabies vaccines over a long period of time.

[0535] The analysis of antibody breadth indicates that an ideal three-mAb combination could be the anti-PHD 4H3, anti-CD 4C12, and anti-FD RVC68 together or the anti-PHD 4H3 combined with a previous RVA58 and RVC68 pairing. 4H3 is the most broadly reactive of the PHD epitope classes and 4C12 the most broadly reactive member of the anti-CD class described here. 4H3 and 4C12 each recognize RABV, DUVV, EBLV, and Irkut viruses. They do not compete for binding, and indeed one of the cry EM structures is of the pair in complex. Anti-FD RVC68 is the most broadly reactive of all mAbs tested in this study,

[0536] 45803765 100 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US against any epitope, recognizing RABV, DUVV, EBLV and Irkut viruses as well as Mokola and Ikoma viruses and others previously described. A mAB cocktail against different, complementary epitopes, and composed of members who each have broad specificity, may provide a mutation-resistant option for treatment, more potent and better characterized, and more broadly reactive, than rabies-specific human polyclonal antibody treatment. The polyclonal preparation is 1,000-fold less potent in neutralization than mABs against RABV.

[0537] Based on the number of antibodies mapped to each antigenic site, the immunodominant epitopes on RABV-G are antigenic sites II / IV and III. However, residues contained in sites II and IV are poorly conserved among rabies -related lyssaviruses, and the site III antibodies in this study, 10H5 and A4, proved similarly rabies-specific. While some site II / IV and III binding antibodies have shown broader recognition of phylogroup I lyssaviruses, binding is also antibody and strain specific.

[0538] In contrast to the immunodominant epitopes, new antigenic site V and VI antibodies were rarer, but substantially more broadly reactive. Site VI antibody RVC68, in particular, recognized an extremely well-conserved binding site: likely with a W100R point mutation, which is only in West Caucasian Bat Lyssavirus glycoprotein, preventing recognition of one of the lyssaviruscs most distantly related to rabies. Although not quite as broadly reactive, site V antibody 4C12 recognized three of four phylogroup I lyssaviruses we tested (those more closely related to rabies) and had good sequence conservation across its binding site. While site V and VI antibodies are rarer than those recognizing immunodominant epitopes, their potential for broader neutralization makes them desirable in a vaccine response or therapeutic cocktail.

[0539] It was observed here that fusion inhibition is a strong determinant of antibody neutralization. Site III monoclonal antibodies and site V antibody 4C12 recognize epitopes that are only present in the pre-fusion conformation and presumably block fusion by inhibiting the transition to the post- fusion conformation. Interestingly, site II / IV antibodies and site VI antibody RVC68 recognize epitopes present in both pre- and post- fusion RABV- G and inhibit fusion even as Fabs. These antibodies bind well at acidic pH, but as their epitopes likely preclude intra-spike binding of the post-fusion trimer, it is suggested that they block fusion by either preventing post-fusion trimers from forming or by inter-spike crosslinking.

[0540] Angle of attachment is likely to be important for fusion inhibition, as within site II / IV, the less potently inhibiting antibodies A2 and Al l bind to the glycoprotein at a 90° angle compared to the better inhibiting antibodies 8C5, 4H3 and 7E8. Antibody binding angles that 45803765 J QI ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US support bivalent IgG binding, strengthening binding through avidity and inhibiting fusion either sterically or through glycoprotein crosslinking, may also compensate for less potently inhibiting Fabs.

[0541] It may be a good strategy to engineer an antibody for improved fusion inhibition and more potent viral neutralization would first require selecting an antibody that is either prefusion specific or binds at a site and angle that prevents formation of post-fusion trimers. Point mutations to that antibody could then introduce bonds to increase binding affinity, breadth of neutralization, and the efficacy of escape mutations. The current standard of care in rabies post-exposure antibody prophylaxis is polyclonal human serum (human rabies immunoglobulin - HRIG) to neutralize virus at the wound site. A mAb cocktail against different, complementary epitopes, and composed of members who each have broad specificity, will provide a more potent, better characterized, more reproducible, and more broadly reactive option for treatment than current HRIG. Indeed, for rabies virus, monoclonal antibodies have been observed to be more potent and broadly reactive than polyclonal sera2. Recent work has proposed a two-antibody cocktail to replace HRIG, consisting of RVC20, which binds at antigenic sites II / IV and RVC682, which as shown here binds to a previously undiscovered antigenic site VI in the fusion domain. RVC20 has been shown to potently neutralize phylogroup I lyssaviruses, and RVC68 to broadly recognize lyssaviruses across three phylogroups, although not always with high neutralization potency. The present work on rabies epitope mapping and mechanisms of antibody neutralization show that there is physical space for a third antibody bridging the central and fusion domains, either at site III or the more conserved site V, in this and other cocktails. Including a combination of Pleckstrin Homology Domain (PHD), Central Domain (CD) and Fusion Domain (FD)- binding antibodies could allow for greater breadth of neutralization through targeting different sets of conserved residues for the four lyssavirus phylogroups at each domain. For example, a cocktail consisting of a broadly reactive PHD-binding antibody such as RVC202,13, a broadly reactive FD-binding antibody such as RVC68 and also a broadly reactive CD- binding antibody such as 10H5, which recognizes Mokola G, or mAb 4C12, which recognizes Duvenhage, Eastern Bat Lyssavirus 1, and Irkut glycoproteins, would reduce the likelihood of escape mutations and increase the number of complementary mechanisms of lyssavirus neutralization.

[0542] Several of the antibodies with overlapping epitopes vary in their ability to bind to glycoproteins from other lyssaviruses (Figure 5), allowing identification of residue contacts that control broad recognition. No single antigenic site yielded antibodies that are universally 45803765 102 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US broad, highlighting the importance of individual and specific residue contacts in binding and neutralization.

[0543] Together, these results set forth the antigenic landscape of RABV-G, and form a foundation for the improved rabies and pan- lyssavirus vaccines and rational design of rabies monoclonal antibody cocktails to replace HRIG. The antibody- antigen cryo-EM structures presented here also provide templates for guidance and interpretation of rational or combinatorial improvements of the individual antibodies themselves for use as therapeutic agents, and for vaccine development as well

[0544] For some viruses, especially those with class I glycoproteins which undergo an irreversible pre- to post-fusion transition, antibodies specific for pre-fusion conformations are more likely to be neutralizing. However, rabies and other lyssaviruses are class III and reversibly transition between pre and post-fusion conformations. It was found here that antibodies specific for the prefusion conformation (all three anti-CD antibodies) are no more or less potent in neutralization than the antibodies that bind equally well to both pre- and post-fusion conformations (all five anti-PHD antibodies). This finding suggests that a rabies virus vaccine does not necessarily need to present only pre-fusion structures to be effective. References:

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[0551] AC, Grais R, Diaz J, Lane HC, Muyembe-Tamfum JJ, Group PW, Sivahera B, Camara M, Kojan R, Walker R, Dighero-Kemp B, Cao H, Mukumbayi P, Mbala-Kingebeni P, Ahuka S, Albert S, Bonnett T, Crozier I, Duvenhage M, Proffitt C, Teitelbaum M, Moench T, Aboulhab J, Barrett K, Cahill K, Cone K, Eckcs R, Hensley L, Hcrpin B, Higgs E, Ledgerwood J, Pierson J, Smolskis M, Sow Y, Tierney J, Sivapalasingam S, Holman W, Gettinger N, Vallee D, Nordwall J, Team PCS. A Randomized, Controlled Trial of Ebola Virus Disease Therapeutics. N Engl J Med. 2019;381(24):2293-303. Epub 20191127. doi: 10.1056 / NEJMoal910993. PubMed PMID: 31774950; PMCID: PMC10680050.

[0552] 5. Cross RW, Hastie KM, Mire CE, Robinson JE, Geisbert TW, Branco LM, Ollmann Saphire E, Garry RF. Antibody therapy for Lassa fever. Curr Opin Virol. 2019;37:97-104. Epub 20190808. doi: 10.1016 / j.coviro.2019.07.003. PubMed PMID: 31401518.

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[0556] 8. Gaudin Y, Raux H, Flamand A, Ruigrok RW. Identification of amino acids controlling the low-pH-induced conformational change of rabies vims glycoprotein. J Virol. 1996;70(l l):7371-8. doi: 10.1128 / JVI.70.11.7371-7378.1996. PubMed PMID: 8892855; PMCID: PMC 190804.

[0557] 9. Gaudin Y, Tuffereau C, Durrer P, Flamand A, Ruigrok RW. Biological function of the low-pH, fusion-inactive conformation of rabies vims glycoprotein (G): G is transported in a fusion-inactive state-like conformation. J Virol. 1995;69(9):5528-34. doi: 10.1128 / JVI.69.9.5528-5534.1995. PubMed PMID: 7543584; PMCID: PMC189404.

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[0562] 13. Hellert J, Buchrieser J, Larrous F, Minola A, de Melo GD, Soriaga L, England P, Haouz A, Telenti A, Schwartz O, Corti D, Bourhy H, Rey FA. Structure of the prefusionlocking broadly neutralizing antibody RVC20 bound to the rabies virus glycoprotein. Nat Commun. 2020;l 1(1):596. Epub 20200130. doi: 10.1038 / s41467-020-14398-7. PubMed PMID: 32001700; PMCID: PMC6992781.

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[0570] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention 45803765 105 ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0571] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0572] 45803765 106

Claims

ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO USCLAIMSWe claim:

1. A molecule or antibody comprising an antigen binding region of an antibody comprising six complementarity determining regions (CDRs), that immunospccifically binds to a surface unit or a transmembrane unit of a rabies virus glycoprotein (RABV-G).

2. The molecule or antibody of claim 1, wherein the CDRs include at least one CDR of the CDRs of anti-RABV-G antibody A2, A4, Al 1, or a variant thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto; with all remaining CDRs independently selected from anti- RABV-G antibodies A2, A4, Al l, and variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

3. The molecule or antibody of claim 2, wherein the CDRs include three heavy chain variable region CDRs independently selected from the heavy chain variable region CDRs of anti-RABV-G antibodies A2, A4, Al l, and variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto in combination with three light chain variable region CDRs independently selected from the light chain variable region CDRs of anti- RABV-G antibodies A2, A4, Al l, and variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

4. The molecule or antibody of claim 3, wherein the CDRs include the three heavy chain variable region CDRs of anti-RABV-G antibody A2, A4, Al 1, or variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto in combination with the three light chain variable region CDRs for anti- RABV-G antibody A2, A4, Al 1 or variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

5. The molecule or antibody of claim 4, wherein the six CDRs are(A) the three heavy chain variable region CDRs and the three light chain variable region CDRs of anti-RABV-G antibody A2;(B) the three heavy chain variable region CDRs and the three light chain variable region CDRs of anti-RABV-G antibody A4; or(C) the three heavy chain variable region CDRs and the three light chain variable region CDRs of anti-RABV-G antibody Al 1.

6. The molecule or antibody of claim 5, wherein the six CDRs are oriented and in the same orientation as in the anti-RABV-G antibody from which they were selected.

7. The molecule or antibody of claim 6, wherein the heavy chain variable regions and light chain variable regions of antibody A2, A4, and Al 1 are the heavy chain variable regions of A2, A4, or Al 1 according to Tables 2 or variants thereof with at least 60, 70, 75, 45803765 107ATTORNEY DOCKET NO. LII 2024-112-01 PRO US80, 85, 90, or 95% sequence identity thereto, and the light chain variable regions of A2, A4, and Al l according to Table 4 or variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

8. The molecule or antibody of claim 7, wherein the six CDRs arc the CDRs according to:Table 1: Heavy Chain Variable Region CDR SequencesTable 3: Light Chain Variable Region CDR Sequencesor variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

9. A molecule or antibody comprising a heavy chain variable region comprising three CDRs and a light chain variable region comprising three CDRs, wherein(A) the three heavy chain variable region CDRs comprise GYTFTNYG (SEQ ID NO:1), 1NTYTGEP (SEQ ID NO:2), RGDYEGTKYYEDY (SEQ ID NO:3), respectively, and the three light chain variable region CDRs comprise QDVSTA (SEQ ID NO:6), SAS, QQHYNTPT (SEQ ID NO:7) respectively;(B) the three heavy chain variable region CDRs comprise GYSFTDYI (SEQ ID NOTO), INPYYGTT (SEQ ID NO: 11), RADGPDYFDY (SEQ ID NO: 12), respectively, and45803765 108ATTORNEY DOCKET NO. LII 2024-1 12-01 PRO US the three light chain variable region CDRs comprise QDVNTA (SEQ ID NO: 15), SAS, CQQHYNTPPT (SEQ ID NO: 16), respectively;(C) the three heavy chain variable region CDRs comprise GYTFTDYA (SEQ ID NO:19), ITTYSGDA (SEQ ID NO:20), ARPYYYGNSWFAY (SEQ ID NO:21), respectively, and the three light chain variable region CDRs comprise QNVGTN (SEQ ID NO:24), SAS, QQYHTYPLT (SEQ ID NO:25), respectively;10. The molecule or antibody of claim 9, wherein the antigen binding region comprises the heavy chain variable region of A2, A4, Al 1, or a variant thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

11. The molecule or antibody of claim 10, wherein the antigen binding region comprises the light chain variable region of A2, A4, Al 1, or a variant thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

12. The molecule or antibody of claim 11, wherein the antigen binding region comprises the heavy chain variable region and light chain variable region of A2, A4, Al 1, or variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

13. The molecule or antibody of claim 12, wherein the antigen binding region comprises the heavy chain variable region and / or light chain variable region according to Table 2 and / or Table 4, or variants thereof with at least 60, 70, 75, 80, 85, 90, or 95% sequence identity thereto.

14. The molecule or antibody of claim 6, wherein the molecule or antibody is an antibody.

15. The molecule or antibody of claim 14, wherein the antibody is an intact antibody and functional antibody fragment or fusion protein.

16. The molecule or antibody of claim 15, wherein the functional fragment or fusion protein is selected from Fab fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments optionally single chain variable fragments (scFv), and single region antibodies optionally selected from sdAb, sdFv, and nanobody fragments.

17. The molecule or antibody of claim 14, wherein the antibody is selected from intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, and multispecific antibodies optionally selected bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, and tandem tri-scFv.

18. The molecule or antibody of claim 14 wherein the antibody is an IgM, IgE, IgA, IgD, or IgG optionally an IgGl, IgG2, IgG3, or IgG4.45803765 109ATTORNEY DOCKET NO. LJI 2024-1 12-01 PRO US19. The molecule or antibody of claim 6, wherein the molecule or antibody is delectably labeled or comprises a conjugated toxin, drug, receptor, enzyme, receptor ligand.

20. A fusion protein comprising the molecule or antibody of claim 6 and a heterologous amino acid sequence.

21. A chimeric antigen receptor (CAR) polypeptide comprising the molecule or antibody of claim 6.

22. The chimeric antigen receptor of claim 21 comprising an extracellular antigen binding region, a spacer region, a transmembrane region, and intracellular signaling region.

23. The chimeric antigen receptor of claim 22 comprising a co- stimulatory domain.

24. A nucleic acid or acids encoding the molecule or antibody of claim 6.

25. The nucleic acid or acids of claim 24 comprising an expression control sequence operably linked thereto, optionally wherein the expression control sequence comprises a promoter.

26. A vector comprising the nucleic acid or acids of claim 25.

27. A host cell comprising the nucleic acid or acids(s) of claim 25.

28. A CAR immune cell comprising the CAR of claim 21, optionally wherein the immune cell is a T cell.

29. A pharmaceutical composition comprising the molecule or antibody of any one of claims 1-19 optionally wherein the molecule or antibody comprises a drag conjugated thereto and / or has antibody-dependent cell-mediated cytotoxicity (ADCC) activity, or complement-dependent cytotoxicity activity (CDC), and / or is a bispecific immune cell engager.

30. A method of treating a subject in need thereof comprising administering the subject an effective amount of pharmaceutical composition of claim 29.

31. The method of claim 30, wherein the subject has a disease or disorder caused by or characterized by increased presence of RABV-G or a fragment thereof.

32. The method of claim 30, wherein the subject has a rabies infection, is at risk for developing symptoms of rabies infection, or complications caused by rabies infection.

33. The method of claim 32, wherein the subject has rabies infection and RABV- G or fragment thereof is an antigen of the rabies infection.

34. A method of detecting RABV-G or fragment thereof comprising contacting a biological sample with the pharmaceutical composition of claim 29, and detecting binding between the molecule(s) or antibod(ies) and the RABV-G or fragment thereof.45803765 | | ()ATTORNEY DOCKET NO. LII 2024-1 12-01 PRO US35. The method of claim 34 further comprising determining that the sample includes increased RABV-G or fragment thereof if the level of detected binding is higher in the biological sample than in a control.

36. The method of claim 34, wherein binding is detected by an immunoassay, immunohistochemistry, Western blotting, surface plasmon resonance, flow cytometry (FACS) analysis, or a biochip.

37. The method of claim 36, wherein the immunoassay is selected from an enzyme immunoassay (EIA), radioimmunoassay (RIA), fluoroimmunoassay (FIA), chemiluminescent immunoassay (CTIA) and counting immunoassay (CIA), homogeneous enzyme-multiplied immunoassays (“EMIT”), apoenzyme reactivation immunoassay (“ARIS”), dipstick immunoassays, or immuno-chromatography assays.

38. The method of claim 34, wherein the biological sample is cells or a cell lysate or a fraction thereof or a fluid optionally blood, saliva, or urine.

39. The method of claim 38, wherein the cells or cell lysate or fraction thereof are derived from a biopsy from a subject.

40. The method of claim 39, wherein the biopsy sample contains, or is suspected to contain, rabies infection.

41. A method of diagnosing a subject with a RAB V-G-related disease or disorder comprising detecting RABV-G or fragment thereof according to the method of claim 34.

42. The method of claim 41 , wherein the RABV-G-related disease or disorder is a rabies infection, is a risk for developing symptoms of rabies infection, or complications caused by rabies infection.

43. The method of claim 41 further comprising treating the subject.

44. The method of claim 43, wherein the treatment comprises a therapy effective for treating a RABV-G-related diseases and disorders.

45. The method of claim 44, wherein the treatment comprises administering the subject an effective amount of the pharmaceutical composition of claim 29 and / or an antiviral.

46. A method of treating a subject in need thereof comprising administering the subject an effective amount of a pharmaceutical composition comprising the molecule or antibody of claim 6.

47. The method of claim 46, wherein the subject has a disease or disorder caused by or characterized by increased presence of RABV-G or a fragment thereof.45803765 111ATTORNEY DOCKET NO. EJI 2024-1 12-01 PRO US48. The method of claim 47, wherein the subject has a rabies infection, is at risk for developing symptoms of rabies infection, or complications caused by rabies infection.

49. The method of claim 48, wherein the subject has rabies infection and RABV- G or fragment thereof is an antigen of the rabies infection.

50. A method of detecting RABV-G or fragment thereof comprising contacting a biological sample with one or more molecules or antibodies of claim 6, and detecting binding between the molecule(s) or antibod(ies) and the RABV-G or fragment thereof.

51. The method of claim 50 further comprising determining that the sample includes increased RABV-G or fragment thereof if the level of detected binding is higher in the biological sample than in a control.

52. The method of claim 51 , wherein binding is detected by an immunoassay, immunohistochemistry, Western blotting, surface plasmon resonance, flow cytometry (TAGS) analysis, or a biochip.

53. The method of claim 52, wherein the immunoassay is selected from an enzyme immunoassay (EIA), radioimmunoassay (RIA), fluoroimmunoassay (FIA), chemiluminescent immunoassay (CTIA) and counting immunoassay (CIA), homogeneous enzyme-multiplied immunoassays (“EMIT”), apoenzyme reactivation immunoassay (“ARIS”), dipstick immunoassays, or immuno-chromatography assays.

54. The method of claim 50, wherein the biological sample is cells or a cell lysate or a fraction thereof or a fluid optionally blood, saliva, or urine.

55. The method of claim 54, wherein the cells or cell lysate or fraction thereof are derived from a biopsy from a subject.

56. The method of claim 55, wherein the biopsy sample contains, or is suspected to contain, rabies infection.

57. A method of diagnosing a subject with a RABV-G-related disease or disorder comprising detecting RABV-G or fragment thereof according to the method of claim 56.

58. The method of claim 57, wherein the RABV-G-related disease or disorder is a rabies infection, is a risk for developing symptoms of rabies infection, or complications caused by rabies infection.

59. The method of claim 58 further comprising treating the subject.45803765 112

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