Receptor-mediated endocytosis for targeted delivery of therapeutic agents with low off-target effects

Abstract

Disclosed are fusion protein-drug conjugates (FPDC) that bind to a protein of interest (POI) on a cell surface via a POI binder (POIB), and to transferrin receptor (TR) via a transferrin receptor binder (TRB). In embodiments, a FPDC features a TRB with low binding affinity to TR relative to binding between the POI and POIB, such that the FPDC selectively binds cells expressing POI on the cell surface.

Description

DOCKET NO: 91016-434826RECEPTOR-MEDIATED ENDOCYTOSIS FOR TARGETED DELIVERY OF THERAPEUTIC AGENTS WITH LOW OFF-TARGET EFFECTSRELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 749416, filed January 24, 2025, of which are incorporated by reference in their entirety herein.

[0002] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.FIELD

[0004] Aspects of the invention are drawn to compositions and methods for internalizing and delivering a therapeutic agent or therapeutic agent by targeting specific cells.SEQUENCE LISTING

[0005] The sequence listing associated with this application is provided in XML format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the XML file containing the sequence listing is “91016-434826SequenceListing.xml”. The XML file is 200,622 bytes, and was created on January 22, 2026, and is being submitted electronically, concurrent with the filing of this application.BACKGROUND

[0006] Antibody-drug conjugates (ADCs) are a class of drugs typically composed of a monoclonal antibody conjugated to a therapeutic agent via a chemical linker. ADCs canDOCKET NO: 91016-434826selectively target and effectively deliver a payload to a cell or tissue of interest by using specificity of antibodies to deliver a therapeutic agent to cells to which the antibody binds. ADCs typically depend on natural receptor internalization and lysosomal trafficking for drug release and are highly dependent on abundance of antigens and high potency of payloads.

[0007] Traditionally, ADC success has been limited by toxicity and side effects as well as low or no efficacy. Examples of problems with traditional ADCs include the ability to timely and effectively internalize and clear ADCs from the target cell, affinity and selectivity for the target cell or molecule, and ADC linkers that are either ineffective at payload release or are too weakly bound to release without affecting unintended target cells. Another problem associated with traditional ADCs include off-target affects, such as those that occur when the drug payload conjugated to the antibody inadvertently affects healthy cells that do not express the target antigen, thus causing damage to these unintended cells. Conventional ADCs are limited by the number of targetable antigens and payload options. Of the 6,000-7,000 integral membrane proteins, only 11 have been successfully targeted by approved ADC therapies, and many of these are cell lineage markers. Further, approved ADC payloads cover only three cytotoxic mechanisms of action, including microtubule inhibitors (MTIs), DNA-damaging agents, and topoisomerase I (TOPI) inhibitors.

[0008] Thus, a desirable ADC would limit toxicity and side effects while demonstrating efficacy for the intended condition. Examples of desirable solutions that reduce toxicity and increase efficacy include the ability to effectively internalize in and clear the ADCs from the target cell, the demonstration of affinity and selectivity for the target cell or molecule, and an ADC linkage with sufficiently tight binding to avoid early, unintended, and off-target release with the ability to efficiently release the payload in the target cell.SUMMARY

[0009] In one embodiment described herein is a fusion protein-drug conjugate of the formula:(Fl -Fl)- (D)nwherein:D is a therapeutic moiety;n is 1 to 8;Fl is a protein of the formula:DOCKET NO: 91016-434826R1 -R2 -R3:wherein:R1 is a protein of interest (POI) binder (POIB);R2 is a complementary IgG Fc region; andR3 is a transferrin receptor (TfR) binder (TRB), wherein the TRB to TfR binding affinity (KD) is about 10 nM to about 100 pM; andF1-F1 form a homodimer.

[0010] A fusion protein-drug conjugate of the formula:(Fl -Fl)- (D)nwherein:D is a therapeutic moiety;n is 1 to 8;Fl is a protein of the formula:R1– R2 – R3:wherein:R1 is a means for binding a protein of interest (POI);R2 is a complementary IgG Fc region; andR3 is a means for binding a transferrin receptor (TfR), wherein the means for binding the TB to TfR has a binding affinity (KD) of of about 10 nM to about 100 pM; andF1-F1 form a homodimer.

[0011] In one embodiment described herein is a fusion protein-drug conjugate of the formula:(Fl -F2)-(D)nwherein:D is a therapeutic moiety;n is 1 to 8;Fl is a protein of the formula:R1 -R2 orRl -R2’:wherein:R1 is a protein of interest (POI) binder (POIB); andR2 is an IgG Fc region complementary to R2’;F2 is a protein of the formula:DOCKET NO: 91016-434826R3 -R2’ orR3 -R2:wherein:R2’ is an IgG Fc region complementary to R2; andR3 is a transferrin receptor (TfR) binder (TRB), wherein the TRB to TfR binding affinity (KD) is about 10 nM to about 100 pM; andF1-F2 form a heterodimer.

[0012] A fusion protein-drug conjugate of the formula:(Fl -F2)- (D)nwherein:D is a therapeutic moiety;n is 1 to 8;Fl is a protein of the formula:R1-R2 orRl -R2’:wherein:R1 is a means for binding a protein of interest (POI); andR2 is an IgG Fc region complementary to R2’;F2 is a protein of the formula:R3 - R2’ or R3 - R2:wherein:R2’ is an IgG Fc region complementary to R2; andR3 is a means for binding a transferrin receptor (TfR), wherein the means for binding the TfR has a binding affinity (KD) to TB of about 10 nM to about 100 pM; andF1-F2 form a heterodimer.

[0013] In one embodiment of the fusion protein-drug conjugate described herein the R2 and R2’ have complementary knob and hole structures.

[0014] In another embodiment of the fusion protein-drug conjugate described herein, D is conjugated to Fl at Rl. In another embodiment, the fusion protein-drug conjugate described herein, D is conjugated to Fl at R2. In another embodiment of the fusion protein-drug conjugate described herein, D is conjugated to Fl at R3. In yet another embodiment of the fusion proteindrug conjugate described herein, D is conjugated to Fl at Rl, and to Fl at R3.DOCKET NO: 91016-434826

[0015] In another embodiment of the fusion-protein drug conjugate described herein, the TRB is an scFv or a heavy chain variable region (VHH).

[0016] In another embodiment of the fusion-protein drug conjugate described herein the TRB comprises:a. a VH CDR1 comprising the amino acid sequence of GTDFSINF (SEQ ID NO:70), a VH CDR2 comprising the amino acid sequence of ITATGNT (SEQ ID NO:73), and a VH CDR3 comprising the amino acid sequence of YMLDK (SEQ ID NO: 72) [[VHHA-12]; orb. a VH CDR1 comprising the amino acid sequence of GTDFSINF (SEQ ID NO:70), a VH CDR2 comprising the amino acid sequence of ITATGNT (SEQ ID NO: 73), and a VH CDR3 comprising the amino acid sequence of AMLDK (SEQ ID NO: 74) [[VHHA-12+5]; orc. a VH CDR1 comprising the amino acid sequence of GTDFSINF (SEQ ID NO:70), a VH CDR2 comprising the amino acid sequence of ITATGNT (SEQ ID NO: 73), and a VH CDR3 comprising the amino acid sequence of YMADK (SEQ ID NO: 75)-[[VHHA-12+7],

[0017] In another embodiment of the fusion-protein drug conjugate described herein the POIB is an antibody or antibody fragment.

[0018] In another embodiment of the fusion-protein drug conjugate described herein fusion protein-drug conjugate binds the POI with an affinity (KD) of between 0.1 nM - 10 nM.

[0019] In another embodiment of the fusion-protein drug conjugate described herein the fusion protein-drug conjugate binds the TfR and the POI with a ratio of affinities (TfR KD / POI KD) of between about 0.01-5000. In another embodiment of the fusion-protein drug conjugate described herein the fusion protein-drug conjugate binds TfR with an affinity (KD) of about 10 nM to about 10 μM.

[0020] In another embodiment of the fusion-protein drug conjugate described herein the therapeutic moiety comprises a microtubule inhibitor, a DNA-damaging agent, a topoisomerase inhibitor, a cell death inducer, or a combination thereof.

[0021] In another embodiment of the fusion-protein drug conjugate described herein the POI is selected from the group consisting of EGFR, CD20, CCR6, MCT1, ICAM1, BST2, PVR, LAT1, and Trop-2. In another embodiment of the fusion-protein drug conjugate described herein theDOCKET NO: 91016-434826POI is selected from the group consisting of HER2, CD30, CD22, CD33, CD79b, Nectin-4, BCMA, CD 19, HERB, and CD25.

[0022] In another embodiment of the fusion-protein drug conjugate described herein is a nucleic acid encoding the fusion- protein drug conjugate described herein.

[0023] In another embodiment of the fusion-protein drug conjugate described herein a pharmaceutical composition comprising the fusion protein-drug conjugate and a pharmaceutically acceptable carrier, diluent, or excipient.

[0024] Another embodiment described herein is a method of administering a therapeutic moiety to a subject, comprising administering an effective amount of the fusion protein-drug conjugate described herein or the pharmaceutical composition described herein to the subject.

[0025] Another embodiment described herein is a method of treating a subject afflicted with a neurogenerative disease, an autoimmune disease or a cancer, comprising administering an effective amount of the fusion protein-drug conjugate described herein, or the pharmaceutical composition.

[0026] Another embodiment described herein is a method of treating a symptom of a neurogenerative disease, an autoimmune disease or a cancer, comprising administering to a subject in need thereof an effective amount of the fusion protein-drug conjugate described herein, or the pharmaceutical composition described herein.

[0027] In one embodiment described herein is a fusion protein-drug conjugate as described herein, wherein the TRB comprises a serine at position 7 of SEQ ID NO: 2, 3, or 4 substituted for a cysteine, a serine at position 84 of SEQ ID NO: 2, 3, or 4 substituted for a cysteine, or a combination thereof. In another embodiment described herein is a fusion protein-drug conjugate as described herein, wherein the TRB comprising SEQ ID NO: 2, 3, or 4 is humanized.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Certain illustrations, charts, or flow charts are provided to allow for a better understanding for the present invention. It is to be noted, however, that the drawings illustrate only selected embodiments of the inventions and are therefore not to be considered limiting of scope. Additional and equally effective embodiments and applications of the present invention exist.DOCKET NO: 91016-434826

[0029] FIGURE 1 shows a schematic diagram of exemplary fusion protein-drug conjugates disclosed herein. R1 is a protein of interest (POI) binder (POIB). R2 and R2’ are IgG Fc regions. R3 is a transferrin receptor (TR) binder (TRB). D is a therapeutic moiety.

[0030] FIGURE 2 shows a schematic diagram of an example FPDC disclosed herein interacting with the cell surface of a normal, noncancerous cell (left) and a target cell (right). The cancerous cell expresses a POI to which the POIB of the FPDC can bind. FPDC binding to the cell is dependent on POI on the cell surface.

[0031] FIGURES 3A-3C show binding to Jurkat cells with FPDCs with TRBs having different affinities to TRB on the surface of the cells. FIG. 3A shows example data for an FPDC having a TRB with a binding affinity of 1.073 x 10’1nM [VHHA, SEQ ID NO: 1], FIG. 3B and FIG. 3C show example data for FPDCs having TRBs with binding affinities of 1.242 x 102nM [VHHA-12; SEQ ID NO: 2] and 2.588 x 10 nM [VHHA-7], respectively. The relative affinities of the TRBs for transferrin receptor in these studies is A > C > B. The FPDCs used in this binding study did not include a therapeutic moiety. The protein of interest binder is an antibody specific for CCR6. The dimer includes a knob and hole configuration. In the graphs, cells having a POI to which the POIB of the FPDC can bind are shown with a red line. Cells not having a POI to which the POIB of the FPDC can bind are shown with a black line.

[0032] FIGURE 4A-4C show binding to Jurkat cells with FPDCs. The FPDCs used in Figure 4 A and 4B have transferrin receptor binders having a binding affinity of <1.0 x 10-3nM [VHHA, SEQ ID NO: 1] compared to transferrin receptor binder having a binding affinity of 2.814 x 10 nM [VHHA-12; SEQ ID NO: 2], >2 x 103nM [VHHA-12+5; SEQ ID NO: 3] and >2 x 103nM [VHHA- 12+7; SEQ ID NO: 4], The relative binding affinities of the TRBs for transferrin in these studies is VHHA > VHHA-12 > (VHHA 12+7 « VHHA 12+5). This binding study did not include a therapeutic moiety. The protein of interest binder is a CCR6 antibody. The FPDCs are homodimers of the configuration in Figure 5C. The cells either have Protein of Interest on the surface (POI+) or do not have the Protein of Interest on the surface (POI-). FIG.4A shows binding of 1 nM of each embodiment to cells. FIG. 4B shows binding of 10 nM of each embodiment to cells. FIG. 4C

[0033] FIGURES 5A-5B depict cell viability assays comparing the anti-tumor activity of fusion protein drug conjugates (red, EGFRFPDC2(MMAF*6); SEQ ID NO: 82; TRB heavy chain, SEQ ID NO: 85; POIB light chain; TRB heavy chain, SEQ ID NO:82; POIB light chain,DOCKET NO: 91016-434826SEQ ID NO: 83), traditional antibody drug conjugates (EGFR-TgG Fc-MC-MMAF in green), and TfRl-only binder-MC-MMAF in blue. FIG. 5A shows a triple-negative breast cancer cell line MDA-MB-231, and FIG. 5B depicts the assay in Hs578T cells. The protein of interest is EGFR. The therapeutic moiety (D) is monomethyl auristatin F (MMAF) with a non-cleavable linker for conjugation. Fusion protein drug conjugates exhibit significantly enhanced activity compared to traditional antibody drug conjugates. In contrast, the TfRl-only binder (which is the same as FPDC except R1 is deleted) did not demonstrate activity, indicating that the efficacy of the fusion protein drug conjugates depends on binding to the specific cell surface POI.

[0034] FIGURES 6A-6B show cell viability assay results. FIG. 6A depicts a western blot assay showing different cancer cell lines expressing heterogenous levels of EGFR. FIG. 6B shows cell viability assays comparing the anti-tumor activity of fusion protein drug conjugates (EGFR FPDC2(MMAF*6); SEQ ID NO: 82; R1 is an EGFR antibody (SEQ ID NO: 84); R2 is IgG Fc (SEQ ID NO: 81); R3 is SEQ ID NO: 85; D is MC MMAF; n- 6 in blue), traditional antibody-drug conjugates (EGFR-IgG Fc-MC-MMAF in green), TfRl-only binder-MC-MMAF in red, and unconjugated controls in triple-negative breast cancer cell lines (FIG. 6B) MDA-MB-231, KRAS G12S lung cancer A549 cell line, EGFR-driven lung cancer PC9 Dell9 cells, and EGFR / cMET driven lung cancer HCC827GR6 cells. These molecules target EGFR as the cell surface antigen (POI) and utilize MMAF as the payload with a non-cleavable linker for conjugation. Fusion protein drug conjugates exhibit significantly enhanced activity compared to traditional antibody drug conjugates across all four cell lines despite varying EGFR expression levels. The FPDC is R1 is SEQ ID NO: 84; R2 is IgG Fc of SEQ ID NO: 81; R3 is SEQ ID NO: 85; D is MC MMAF; n is 6).

[0035] FIGURES 7A-7B show cell viability assays when the POI is ICAM1. The data compare the anti -turn or activity of fusion protein drug conjugate in red (ICAM FPDC2(MMAF*6), FPDC wherein R1 is SEQ ID NO: 90; R2 is IgG Fc of SEQ ID NO: 81; R3 is SEQ ID NO: 85; D is MC MMAF; n is 6; TRB heavy chain, SEQ ID NO: 88; POIB light chain, SEQ ID NO: 89); traditional antibody drug conjugates (IgG Fc-MC-MMAF in green), and TfRl-only binder-MC-MMAF in blue in triple-negative breast cancer cell lines MDA-MB-231 as shown in FIG. 7A and Hs578T in FIG. 7B. These molecules target ICAM1 as the cell surface antigen (POI) and utilize MMAF as the payload with a non-cleavable linker for conjugation. Fusion protein drug conjugates of the present invention exhibit significantly enhanced activityDOCKET NO: 91016-434826compared to traditional antibody drug conjugates. In contrast, TfRl-only binder-MC-MMAF (which is the same as FPDC except R1 is deleted) did not demonstrate activity, indicating that the efficacy of the fusion protein conjugates depends on binding to the specific cell surface antigen.

[0036] FIGURES 8A-8B depict cell viability assays when the POI is PVR. The data compare the anti-tumor activity of fusion protein drug conjugate in red (PVR FPDC2(MMAF*6) FPDC wherein R1 is SEQ ID NO: 95; R2 is IgG Fc of SEQ ID NO: 81; R3 is SEQ ID NO: 85; D is MC MMAF; n is 6; TRB heavy chain, SEQ ID NO: 93; POIB light chain, SEQ ID NO: 94); traditional antibody drug conjugates (IgG Fc-MC-MMAF in green), and TfRl-only binder-MC-MMAF in blue in triple-negative breast cancer cell lines MDA-MB-231 as shown in FIG. 8A and Hs578T as shown in FIG. 8B. These molecules target PVR as the cell surface antigen (POI) and utilize MMAF as the payload with a non-cleavable linker for conjugation. Fusion protein drug conjugates of the present invention exhibit significantly enhanced activity compared to traditional antibody drug conjugates. In contrast, TfRl-only binder-MC-MMAF (which is the same as FPDC except R1 is deleted) did not demonstrate activity, indicating that the efficacy of the fusion protein conjugates depends on binding to the specific cell surface antigen. FIGURES 9A-9B depict cell viability assays when the POI is MCT1. The data compare the anti-tumor activity of fusion protein drug conjugate in red (MCT1 FPDC2(MMAF*6) FPDC wherein R1 is SEQ ID NO: 100; R2 is IgG Fc of SEQ ID NO: 81; R3 is SEQ ID NO: 85; D is MC MMAF; n is 6; TRB heavy chain, SEQ ID NO: 98; POIB light chain, SEQ ID NO: 99); traditional antibody drug conjugates (IgG Fc-MC-MMAF in green), and TfRl-only binder-MC-MMAF in blue in triple-negative breast cancer cell lines MDA-MB-231 as shown in FIG. 9A and Hs578T as in FIG. 9B. These molecules target MCT1 as the cell surface antigen (POI) and utilize MMAF as the payload with a non-cleavable linker for conjugation. Fusion protein drug conjugates of the present invention exhibit significantly enhanced activity compared to traditional antibody drug conjugates. In contrast, TfRl-only binder-MC-MMAF (which is the same as FPDC except R1 is deleted) did not demonstrate activity, indicating that the efficacy of the fusion protein conjugates depends on binding to the specific cell surface antigen.

[0037] FIGURES 10A-10B depict cell viability assays when the POI is LAT1. The data compare the anti-tumor activity of fusion protein drug conjugate in red (LAT1 FPDC2(MMAF*6) FPDC wherein R1 is SEQ ID NO: 105; R2 is IgGFc of SEQ ID NO: 81; R3DOCKET NO: 91016-434826is SEQ ID NO: 85; D is MC MMAF; n is 6; TRB heavy chain, SEQ ID NO: 103; POIB light chain, SEQ ID NO: 104); traditional antibody drug conjugates (IgG Fc-MC-MMAF in green), and TfRl-only binder-MC-MMAF in blue in triple-negative breast cancer cell lines MDA-MB-231 as shown in FIG. 10A and Hs578T as shown in FIG. 10B. These molecules target LAT1 as the cell surface antigen (POI) and utilize MMAF as the payload with a non-cleavable linker for conjugation. Fusion protein drug conjugates of the present invention exhibit significantly enhanced activity compared to traditional antibody drug conjugates. In contrast, TfRl-only binder-MC-MMAF (which is the same as FPDC except R1 is deleted) did not demonstrate activity, indicating that the efficacy of the fusion protein conjugates depends on binding to the specific cell surface antigen.

[0038] FIGURES 11A-11B depict cell viability assays when the POI is BST2. The data compare the anti -turn or activity of fusion protein drug conjugate in red (BST2 FPDC2(MMAF*6) FPDC wherein R1 is SEQ ID NO: 110; R2 is IgG Fc of SEQ ID NO: 81; R3 is SEQ ID NO: 85; D is MC MMAF; n is 6; TRB heavy chain, SEQ ID NO: 108; POIB light chain, SEQ ID NO: 109); traditional antibody drug conjugates (IgG Fc-MC-MMAF in green), and TfRl-only binder-MC-MMAF in blue in triple-negative breast cancer cell lines MDA-MB-231 as shown in FIG. 11A and Hs578T as shown in FIG. 11B. These molecules target BST2 as the cell surface antigen (POI) and utilize MMAF as the payload with a non-cleavable linker for conjugation. Fusion protein drug conjugates of the present invention exhibit significantly enhanced activity compared to traditional antibody drug conjugates. In contrast, TfRl-only binder-MC-MMAF (which is the same as FPDC except R1 is deleted) did not demonstrate activity, indicating that the efficacy of the fusion protein conjugates depends on binding to the specific cell surface antigen.

[0039] FIGURES 12 shows a cell viability assay showing efficacy with alternative therapeutic moieties (D). The data compare the anti-tumor activity of fusion protein drug conjugate in red (EGFR FPDC2(DOX*6) in left panel or EGFR FPDC2(RSL3*6) in right panel; FPDC wherein R1 is SEQ ID NO: 84; R2 is IgG Fc of SEQ ID NO: 81; R3 is SEQ ID NO: 85; D is MC-caproyl-hydrazone-doxorubicin or MC-RSL3; n is 6, in red); traditional antibody drug conjugates (IgGFc- MC-caproyl-hydrazone-doxorubicin (left panel) or MC-RSL3 (right panel) in green), and TfRl-only binder- MC-caproyl-hydrazone-doxorubicin or MC-RSL3 in black in triple-negative breast cancer cell lines MDA-MB-231. These molecules target EGFR as the cellDOCKET NO: 91016-434826surface antigen (POI) and vary the payload (D) with a non-cleavable linker for conjugation. Fusion protein drug conjugates of the present invention exhibit significantly enhanced activity compared to traditional antibody drug conjugates and the TfR1-only binder indicating that the efficacy of the fusion protein conjugates is achieved with multiple therapeutic moieties.

[0040] FIGURES 13A-13B depicts comparisons of non-cleavable and cleavable linkers showing little difference in anti-tumor activity. Cell viability assay comparing the anti-tumor activity of fusion protein drug conjugates (FPDC wherein R1 is SEQ ID NO: 84; R2 is IgG Fc of SEQ ID NO: 81; R3 is SEQ ID NO: 85; D is MMAE that is non-cleavable MC or cleavable MC-VC; n is 6; in red), in lung cancer A549 cells as shown in FIG. 13A and PC9 Del 19 cells as in FIG. 13B. These molecules target EGFR as the cell surface antigen and utilize Monomethyl auristatin E (MMAE) as the payload with either a non-cleavable linker or a cleavable linker (VC) for conjugation. Fusion protein drug conjugates exhibit significantly enhanced activity compared to traditional antibody-drug conjugates. TfR1-only binder did not demonstrate activity, indicating that the efficacy of the fusion protein conjugates depends on binding to the specific cell surface antigen. No significant differences in anti-tumor activity were observed between the two linker types, indicating that the fusion protein drug conjugates can use different linker types.

[0041] FIGURE 14 shows a pHrodo internalization assay in Raji wildtype (WT) and CD20 knockout (KO) Cells. A pHrodo internalization assay was conducted using Raji wildtype (WT) cells (CD20 positive) and Raji CD20 knockout (KO) cells to demonstrate antigen-dependent internalization of fusion protein conjugates of the present invention. An anti-CD20 antibody (black), fusion protein conjugates containing a TfR1 binder VHHA, in red, and fusion protein conjugates (CD20 FP4; R1 is POIB of SEQ ID NO: 124; R2 is TRB0comprising SEQ ID NO: 1 (binding affinity about 1.2-1.3 x 102nM) without payload, D) were labeled with pHrodo, a pH-sensitive fluorescent dye. Raji WT cells and CD20 KO Raji cells were treated with 5 nM of each test protein for 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, or 10 h. Fluorescence was measured using a flow cytometer to assess the internalization of the molecules. Only the low binding affinity CD20 Fc-TfR1 protein conjugates (CD20 FP2; R1 is POIB of SEQ ID NO: 124; R2 is TRB’ comprising SEQ ID NO: 4) exhibited antigen (CD20)-dependent internalization. Fusion protein conjugates containing VHHA showed internalization in both CD20 positive and CD20 negative cells. The anti-CD20 antibody-mediated internalization (black line) was significantly lower than that of the fusion protein conjugates.DOCKET NO: 91016-434826

[0042] FIGURE 15 illustrates fusion protein conjugates having low binding affinity. TfR1 binder -125 (binding affinity for 12+5; CD20 FP3) and TfR1 binder-127 (CD20 FP2) show minimal on-cell binding on antigen-negative cells, including both wildtype Jurkat cells (left) and Raji CD20 knockout cells (right). These molecules target CD20 as the cell surface antigen (POI).

[0043] FIGURES 16 demonstrates CD20-targeted fusion proteins exhibit reduced on-cell binding in CD20-negative cells. CD20-targeted fusion proteins, CD20 FP1 and CD20 FP2 demonstrate significantly lower on-cell binding compared to CD20 FP4 across various concentrations on Jurkat cells (CD20-negative).

[0044] FIGURES 17A-17C shows schematics (FIG. 17A), quantification (FIG. 17B), and examples of the percentage of cells in co-culture of Raji WT cells and Raji CD20 knockout cells treated with traditional drug conjugates or fusion protein drug conjugates (FIG. 17C; FPDC wherein R1 is SEQ ID NO: X; R2 is IgG Fc of SEQ ID NO X; R3 is SEQ ID NO: X (CD20 is POI / antigen); D is MMAF. The results show that the fusion protein drug conjugates selectively kill the antigen positive, Raji WT cells, and do not kill the Raji CD20 knockout cells, demonstrating the antigen selectivity of these molecules. The traditional ADC shows no observable killing effects of either cell type. These molecules target CD20 as the cell surface antigen and utilize MMAF as the payload with a non-cleavable linker for conjugation.

[0045] FIGURES 18A-18C depict cell viability assays comparing the anti-tumor activity of fusion protein drug conjugates (FPDC) containing variable number of payload (D). Data are provided for n=2 (CD20 FPDC2(MMAF*2)) and n=6 (CD20 FPDC2(MMAF*6) by conjugating to cysteines. These molecules target CD20 as the cell surface antigen (POI) and utilize MMAF as the payload with a non-cleavable linker for conjugation. In Raji cells as shown in FIG. 18A, fusion protein drug conjugates exhibit significantly enhanced activity compared to traditional antibody drug conjugates. Fusion protein drug conjugates containing the additional drug conjugate in the TfR1 binding domains show higher activity than Fusion protein drug conjugates not containing the additional cysteines. The TRB’ control did not demonstrate activity, indicating that the efficacy of the fusion protein conjugates depends on binding to the specific cell surface antigen. In Raji CD20 knock-out cells as shown in FIG. 18B, these molecules target CD20 as the cell surface antigen and utilize MMAF as the payload with a non-cleavable linker for conjugation. No molecules show cell killing effects, indicating their activities depend onDOCKET NO: 91016-434826presence of the specific cell surface antigen (POI). FIG. 18C illustrates the conjugation of the therapeutic moiety (D).

[0046] FIGURE 19 shows a cell viability study with fusion protein drug conjugates with a TfR1 H7 TRB (CD20 FPDC4(MMAF*2)). The cell viability assays compare the anti-tumor activity of fusion protein drug conjugates containing the TfR1 H7 binder on CD20-positive and CD20-negative cells. These molecules target CD20 as the cell surface antigen (POI) and utilize MMAF as the payload with a non-cleavable linker for conjugation. Unlike fusion protein drug conjugates of the present invention, those with the TfR1 H7 binder kill both CD20-positive cells (Raji and CD20-overexpressing Jurkat cells) and CD20-negative cells (Raji CD20 KO and wildtype Jurkat cells), lacking the necessary antigen specificity for cancer-specific killing.

[0047] FIGURE 20 shows a cell viability assays in primary human T cells demonstrating that a CD20-targeted fusion protein drug conjugate (FPDC) does not induce cytotoxicity in T cells, highlighting antigen selectivity in a primary cell setting. The T cells were either inactivated (left panel) or activated (right panel).

[0048] FIGURES 21A-21B show an embodiment comparing protease-cleavable and nonprotease cleavable linkers in FPDCs provided herein. In this embodiment, cathepsin b-cleavable amino acid sequence and non-cleavable amino acid sequence are compared in TransTAC showing similar anti-tumor activities. A cell viability assay is provided comparing the anti-tumor activity of fusion protein drug conjugates (grey), fusion protein (black) in Raji cells FIG.21A and structure of two fusion proteins as shown in FIG. 21B. In this embodiment, CD20 is targeted as the cell surface antigen, and MMAF is the payload with or without a cathepsin b-cleavable amino acid sequence (EVR) for conjugation. Both of the fusion protein drug conjugates exhibit significantly enhanced activity. No significant differences in anti-tumor activity were observed between the two linker types, indicating that the fusion protein drug conjugates can use different linker types between the POI binding domain and Fc.

[0049] FIGURE 22 depicts a schematic of a Trop2 ADC (datopotamab deruxtecan; DXd; DAR = 4) and Trop2 TransTAC ADCs with varying TfR1 binding affinities and valencies.

[0050] FIGURE 23 shows BLI binding curves for TfRl binders’ formats used in vl.l, vl.2 and vl.3.

[0051] FIGURES 24A-24B depict dose-response cytotoxicity of Trop2 ADC (datopotamab deruxtecan) and Trop2 TransTAC ADCs (vl.l-vl.3) in non-malignant MCF10A and TNBCDOCKET NO: 91016-434826lines, as in FIG. 24A. FIG. 24B shows a summary of IC50values and fold improvement compared to datopotamab deruxtecan.

[0052] FIGURES 25A-25B depict relative cell-surface expression of Trop2 and TfR1 across epithelial and fibroblast lines (MCF10A, BEAS-2B, NK-TerT, HS27, and IMR-90) and TNBC lines, as shown in FIG. 25A. and cytotoxicity of Trop2 ADCs versus Trop2 TransTAC ADCs vl.l-vl.3 (deruxtecan; DAR = 4) in non-malignant cells as shown in FIG. 25B.

[0053] FIGURES 26A-26B depict, as shown in FIG.26A, a xenograft study design (subcutaneous implantation; intravenous single-dose ADC injection 2 weeks later at -100 mm3 tumor size), and shown in FIG.26B, an antitumor activity of datopotamab deruxtecan versus Trop2 TransTAC ADC vl.l at 1 or 5 mg kg'1, shown as tumor growth, representative tumors, and terminal tumor weights, scale bar 1cm.

[0054] FIGURES 27A-27B, as shown in FIG. 27A, mice CBC hematology was assessed before sacrificed, RBC (red blood cell count), Hb (hemoglobin), HCT (hematocrit), MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin), MCHC (mean corpuscular hemoglobin concentration), RDW (red cell distribution width), and WBC (white blood cell count) assess hematologic toxicity. FIG.27B depicts kidney and liver toxicity was assessed before sacrificed. ALT (alanine aminotransferase) and AST (aspartate aminotransferase) assess liver toxicity. Creatinine and BUN (blood urea nitrogen) assess kidney toxicity.

[0055] FIGURE 28 shows the surfaceome-guided prioritization and antibody screening workflow for identifying TAA candidates.

[0056] FIGURE 29 depicts on-cell binding of the cloned antibodies to candidate targets measured by flow cytometry.

[0057] FIGURE 30 shows relative cell-surface expression of EGFR, PVR, ICAM1, and TfR1 in MCF10A and TNBC cell lines (BT549, MDA-231, MDA-468, Hs578T, and MDA-436).

[0058] FIGURE 31 shows cytotoxicity of EGFR ADCs versus EGFR TransTAC ADCs (MC-MMAF, DAR = 2) in MCF10A and TNBC cell lines.

[0059] FIGURE 32 shows the cytotoxicity of PVR ADCs versus PVR TransTAC ADCs (MC-MMAF, DAR = 2) in MCF10A and TNBC cell lines.

[0060] FIGURES 33A-33B depict cytotoxicity of ICAM1 ADCs versus PVR TransTAC ADCs (MC-MMAF, DAR = 2) in MCF10A and TNBC cell lines, as shown in FIG.33A and in FIG. 33B a summary of IC50values for ICAM1 ADCs and ICAM1 TransTAC ADCs.DOCKET NO: 91016-434826

[0061] FIGURES 34A-34B depict relative ICAM1 cell-surface expression across non-malignant lines and ICAM1 -positive high MDA-468 TNBC cells, as shown in FIG.34A, and the cytotoxicity of ICAM1 ADCs and ICAM1 TransTAC ADCs (vl.l-vl.3; MC-MMAF, DAR = 2) in non-malignant cells in FIG. 34B.

[0062] FIGURE 35 shows cytotoxicity of an earlier ICAM1 TransTAC ADC format (vl. O, KD <0.1nM; MC-MMAF, DAR = 2) across non-malignant cells.

[0063] FIGURE 36 shows dose-response curves for binding of CCR6 targeting compositions DJI (VHHA), LS13 (VHHA-12), LS15 (VHHA-5), and LS16 (VHHA-7) to CCR6A+ (Cells expressing CCR6) and CCR6- (cells that do not express CCR6) cells. Developed emobidments of the CCR6-targeting compositions provided herein minimally bind CCR6-negative cells and do not reduce cell-surface TfRl, mitigating potential safety concerns.

[0064] FIGURE 37 shows the therapeutic rationale and correlation between the binding affinity of the anti-TfR1 moiety and the binding affinity of anti-POI moiety for the TransTAC embodiments, i.e. the ratio of the TfR1 KDand the POI KD(TfR1 KD / POI KD).

[0065] FIGURE 38 depicts the various geometries of the TransTAC embodiments of the disclosure.

[0066] FIGURE 39 depicts a example binding affinities measured as KDfor the example vl.l-vl.3 embodiments depicted in FIG. 37.DETAILED DESCRIPTIONOverview

[0067] Disclosed herein are alternatives to antibody-drug conjugates (ADCs), referred to herein as fusion protein-drug conjugates (FPDCs). In embodiments, FPDCs herein limit toxicity and side effects, while demonstrating efficacy for delivering the conjugated drug to cells expressing a protein of interest (POI). In embodiments, FPDCs herein exhibit reduced toxicity and increased efficacy by, for example, effectively internalizing and clearing the FPDCs from target cells; exhibiting affinity and selectivity for a target cell or molecule; and including a drug conjugate linkage with sufficiently tight binding to avoid early, unintended, and off-target release as well as efficiently releasing the payload in the target cell.DOCKET NO: 91016-434826

[0068] Fusion protein-drug conjugates (FPDCs) disclosed herein have a protein of interest (POI) binder (POIB) and a transferrin receptor (TfR) binder (TRB). These FPDCs, when added to cells, bind a POI (e.g., an extracellular domain of an integral membrane protein) on the cell surface via the POIB, and bind transferrin receptor (TfR) on the cell surface via the TRB. Once an FPDC is bound, TfR, which is an internalizing receptor, is internalized along with the FPDC, as well as the bound cell-surface POI, which is bound by the POIB of the FPDC (e.g., WO 2024 / 064756 Al, published 28 March 2024;). Herein, TfR can refer to transferrin receptor 1 (TfR1) or transferrin receptor 2 (TfR2). In embodiments, TfR refers to transferrin receptor 1. The conjugated drug (also referred to as a therapeutic moiety) is internalized by TfR-dependent endocytosis. The endocytosed FPDC is transported to endosomes and lysosomes, where the therapeutic moiety is released.

[0069] Herein, compositions, methods, and improvements to FPDC are disclosed. In embodiments, off-target effects (e.g., delivery of a therapeutic agent to cells that do not express the cell-surface POI) are decreased and cell specificity of delivery is improved. In embodiments, potency is increased (e.g., relative to conventional ADCs). In embodiments, the binding affinity of the TRB to cell-surface TfR relative to the binding affinity of the POIB to the POI is modified (e.g., via mutating amino acids in the TRB sequence) to achieve the desired effects.

[0070] In embodiments, fusion protein-drug conjugates disclosed herein have TRBs and POIBs that have different relative binding affinities for their targets, TfR and POI, respectively. In embodiments, the affinity of the TRB for binding to TfR (e.g., on a cell surface) is less than the binding affinity of the POIB for binding to the POI (e.g., on a cell surface). In embodiments, the affinity of a modified TRB (e.g., via mutated amino acids that contribute to binding in the TRB sequence, such as amino acids in CDRs where the TRB is an antibody or antibody fragment) for binding to TfR (e.g., on a cell surface) is less than the binding affinity of an unmodified TRB (i.e., a TRB without mutated amino acids that contribute to binding in the TRB sequence, such as amino acids in CDRs where the TRB is an antibody or antibody fragment). In embodiments, the fusion protein-drug conjugates do not bind to a cell that expresses TfR but does not express a POI to which the POIB of the molecule binds. In embodiments, binding of the FPDCs described herein, which have a POIB, TRB and conjugated therapeutic moiety, to cells that express TfR depends on the binding between a cell-surface POI and the POIB of the FPDC.DOCKET NO: 91016-434826

[0071] In embodiments, binding affinity of the TRB in disclosed fusion protein-drug conjugates can contribute to few off-target effects. In embodiments, TRBs used herein can bind to TfR with low affinity (e.g., via mutated amino acids that contribute to binding in the TRB sequence, such as amino acids in CDRs where the TRB is an antibody or antibody fragment; e.g., the TRB has a dissociation constant from TfR of about 10 nM to about 100 pM or about 100 nM to 100 uM; i.e., relatively higher dissociation constant). This binding reduces off-target effects relative to other TRBs that bind TfR with relatively higher affinity (e.g., TRBs that have a KD lower than about 10 nM to about 100 pM or about 100 nM to 100 uM; i.e., relatively lower dissociation constant). In embodiments, the TRBs can be VHHA-12 (SEQ ID NO: 2), VHHA-12+7 (SEQ ID NO: 4), or VHHA-12+5 (SEQ ID NO: 3).

[0072] In embodiments, the particular FPDC formula for a low-affinity TRB disclosed herein also contributes to generating fewer off-target effects at high potency. In embodiments, the FPDC described herein can be homodimers or heterodimers. In embodiments, a homodimer FPDC can have two TRBs with lower affinity than a single TRB in a heterodimer FPDC and exhibit comparatively few off-target effects.

[0073] In embodiments, the FPDC TRB binding affinity to TfR relative to the POIB binding affinity to POI contributes to generating fewer off-target effects at high potency. For example, in embodiments, an FPDC TRB binds to TfR with 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, 1,000, 10,000, 100,000 -fold lower affinity, and the like, compared with the POIB binding affinity to the POI. Without being bound by theory, the activity of an FPDC (e.g., as measured by IC50) with a higher relative POIB / POI binding affinity to TRB / TfR depends on target recognition through the POIB / POI interaction, which limits, for example, off target effects from TRB binding and facilitates higher potency by enhancing safety.

[0074] Also disclosed herein are cysteine amino acids in the FPDCs to which therapeutic moieties can be conjugated. In embodiments, the cysteine amino acids substitute for amino acids present in polypeptides (e.g., TRB or POIB) comprising FPDCs. In embodiments, the cysteinesubstituted amino acids are present in an FPDC TRB. In embodiments, FPDCs have more therapeutic moieties conjugated thereto than therapeutic moieties conjugated to corresponding conventional ADC molecules.DOCKET NO: 91016-434826

[0075] Thus, the FPDCs disclosed herein have limited toxicity, limited side effects and increased efficacy, for example, compared to conventional ADCs and to molecules disclosed in WO 2024 / 064756 Al, published 28 March 2024.Terms

[0076] Detailed descriptions of one or more embodiments are provided herein. It is to be understood, however, that the present invention may be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner.

[0077] The singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0078] Wherever any of the phrases “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. Similarly, “an example,” “exemplary” and the like are understood to be nonlimiting.

[0079] The term “substantially” allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word “substantially” is not explicitly recited.

[0080] The terms “comprising” and “including” and “having” and “involving” (and similarly “comprises”, “includes,” “has,” and “involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open term meaning “at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, “a process involving steps a, b, and c” means that the process includes at least steps a, b and c. Wherever the terms “a” or “an” are used, “one or more” is understood, unless such interpretation is nonsensical in context.

[0081] As used herein, the term “about” can refer to approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. The termDOCKET NO: 91016-434826“about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).

[0082] Herein, means plus function claims under 35 U. S. C. § 112(f) that, for example, recite a “a protein of interest binder means” or “a transferrin receptor binding means” are equivalent to and are to be interpreted as “means for binding a protein of interest” or “means for binding transferrin receptor”, respectively.

[0083] The dash notation, reflects standard linkers (also known as linkages or linking means or means for linking) understood in the field. In examples, the dash notation occurs between polypeptides (‘R’) in a fusion protein. In this example, the dash notation between ‘R’s refers to one or more amino acids (e.g., natural or unnatural amino acids) conventionally used to link regions of a fusion protein (i.e., a means for linking polypeptides; e.g., a glycine-rich linker), forming a covalent bond between two polypeptides and generating a single, contiguous polypeptide. In another example, the dash notation occurs between a fusion protein dimer (‘F’ -‘F’) and a drug (‘D’; also known as a therapeutic moiety). In this example, the dash notation between (‘F’ - ‘F’) and ‘D’ refers to a direct covalent bond or small molecule-mediated covalent bond conventionally used to link drugs to an amino acid in a polypeptide, such as conventionally used linkages between antibodies or antibody fragments of an ADC and conjugated drug.

[0084] Linkers direct the cytotoxic preparation release instrument of an ADC and also assist in various ADC biochemical features. Commonly, linkages between antibodies or antibody fragments and conjugated drugs in ADCs are cleavable linkers, non-cleavable linkers, and enzyme cleavable linkers. For example, conjugation of a therapeutic moiety to an antibody or fragment thereof can use a chemical reaction(s). In embodiments, the conjugation can use bioconjugation. Bioconjugation reactions can be of different types. Common types of bioconjugation reactions can include coupling of cysteine, lysine, methionine, and tyrosine as well as unnatural amino acids. Common types of bioconjugation reactions can include modification of tryptophan amino acids and of the N- and C-terminus of a protein. The skilled artisan can utilize other linker chemistry known in the art (see, e.g., Jain et al., Current ADC Chemistry, Pharm Res. 2015 Nov;32(l 1 ):3526-40). For example, a strategy can be used to attach a specific site of a therapeutic moiety to a specific site on a POIB (such as an antibody or fragment thereof). In embodiments, a unique functional group can be introduced onto a protein portion and use a bioorthogonal reaction to couple the therapeutic moiety to the POIB. InDOCKET NO: 91016-434826embodiments, these reactions can use modification of ketone and aldehydes, Staudinger ligation with organic azides, copper-catalyzed Huisgen cycloaddition of azides, or strain promoted Huisgen cycloaddition of azides. In embodiments, the bioconjugation reaction can be a maleimide-based cysteine reaction.

[0085] In embodiments, a linker is an amino acid sequence that links two polypeptides into one contiguous polypeptide. In embodiments, a linker is an amino acid sequence linking an IgG Fc domain (e.g., an IgGl Fc domain) to a protein of interest binder (POIB). In embodiments, a linker is an amino acid sequence linking an IgG Fc domain (e.g., an IgGl Fc domain) to a transferrin receptor binder (TRB) (e.g., a TfR binding antibody or antibody fragment, such as a VHH). In embodiments, a linker is an amino acid sequence linking a TRB to a TRB, a TRB to a POIB, a POIB to a POIB, and the like. In embodiments, the linker does not include a protease-cleavable amino acid sequence. A person of ordinary skill in the art understands that peptide linkers are typically about one to 100 amino acids in length (e.g., 1, 2, 3, 4, 5, 10, 12, 1-10, 1-12, 1-20, or 1-100, or more amino acids) containing various types of amino acids (e.g., US Patent No. 11,041,023).

[0086] In embodiments, the linkage can be a glycine-rich linker (“GS” linker). In embodiments, a “GS” linker can be a combination of glycine and serine amino acids. In embodiments, the GS linker can be GSSGGSGGSGGS (SEQ ID NO: 113). Other sequences are possible. In embodiments, the GS linker can be GGSGSGGSGSG (SEQ ID NO: 114), SGGGG (SEQ ID NO: 115), SGGGSGGG (SEQ ID NO: 116), GSSGGSGGSGGS (SEQ ID NO: 117), GSGS (SEQ ID NO: 118), GSGGS (SEQ ID NO: 119), GSSGSS (SEQ ID NO: 120), GSSSSSS (SEQ ID NO: 121), and the like. In embodiments, a GS linker can have at least 4 amino acids that are glycine and / or serine. In embodiments, other amino acids can be part of a GS linker, as long as glycine and serine are in the majority.

[0087] Optionally, in embodiments, a linker or linking means includes a protease-cleavable amino acid sequence (also known as a protease-sensitive linking means). Optionally, in embodiments, the protease-sensitive linking means can be an amino acid sequence that can be cleaved by a protease. Optionally, in embodiments, the protease can be a protease in an endosome or lysosome. Optionally, in embodiments, the protease can be a cathepsin (e.g., cathepsin B), and the protease-sensitive linking means can be a cathepsin-cleavable peptide. Optionally, in embodiments, the linker includes a cathepsin b-cleavable amino acid sequence.DOCKET NO: 91016-434826

[0088] Traditional ADCs utilize a monoclonal antibody, a chemical linker, and a therapeutic payload to selectively deliver highly potent pharmaceutical agents to specific cell types. For example, hydrophobic Valine-Citrulline linkers (cleaved by CatB) can be used. In embodiments, a linker can include a protease-cleavable amino acid sequence (also known as a proteasesensitive linking means). In embodiments, the protease-sensitive linking means can be an amino acid sequence that can be cleaved by a protease. In embodiments, the protease can be a protease in an endosome or lysosome. In embodiments, the protease can be a cathepsin (e.g., cathepsin B), and the protease- sensitive linking means can be a cathepsin-cleavable peptide. In embodiments, the linker includes a cathepsin b-cleavable amino acid sequence. In embodiments, the protease-sensitive linking means or linker has an amino acid sequence that can be recognized by a cathepsin protease. The cathepsins can be cathepsin A, B, C, D, E, F, G, H, K, L1, L2, O, S, W or Z. Some examples of protease-sensitive linking means or linkers are described herein. For example, the protease-sensitive linking means or linker can be GGFLGGVRGVDG (SEQ ID NO: 122) or GSGSGGEVRGVDG (SEQ ID NO: 123). Sequences of other protease-sensitive linking means or linkers are disclosed herein. Functional alternative amino acid sequences that can be cleaved by a protease (e g., cathepsin) are known in the art.

[0089] Use of other protease-sensitive linking means or linkers are contemplated. Use of other non-cleavable linking means or linkers are also contemplated. For example, non-cleavable linkers such as thioether linkers and maleimido acproyl linkers known in the art can also be used (see Sheyi et al. Linkers: An Assurance for Controlled Delivery of Antibody-Drug Conjugate. Pharmaceutics. 2022 Feb 11; 14(2):396).

[0090] In some embodiment, these peptide sequences are sensitive to cathepsin cleavage: GRLVGFD (SEQ ID NO: 196), GRLVGFG (SEQ ID NO: 197), RMLVGFV (SEQ ID NO: 198), RRLYAFL (SEQ ID NO: 199), VFRLLMF (SEQ ID NO: 200), LVGVLLF (SEQ ID NO: 201), VKLYGLG (SEQ ID NO: 202), TWRVDLY (SEQ ID NO: 203), EQLYLYA (SEQ ID NO: 204), KLFLMIF (SEQ ID NO: 205), NFVIILF (SEQ ID NO: 206), MSLLIGV (SEQ ID NO: 207), VRLLSLQ (SEQ ID NO: 208), STLMWNV (SEQ ID NO: 209), VRFLAAA (SEQ ID NO: 210), HGWSFHE (SEQ ID NO: 211), ENLYFQG (SEQ ID NO: 212), VVMMFLH (SEQ ID NO: 213), VFRLLMF (SEQ ID NO: 214), or VGALVWL (SEQ ID NO: 215).

[0091] Herein, “binding” (or “binds”) refers to the interaction between a protein or polypeptide (e.g., an antibody or antibody fragment) and a target protein or polypeptide. BindingDOCKET NO: 91016-434826can be measured in a variety of ways (see, e.g., Antibody Engineering: Methods and Protocols (Damien Nevoltris and Patrick Chames eds., 3d ed. 2018)). In embodiments, binding is measured by kinetic assays, such as Octet (Biolayer Interferometry, BLI) or similar assays known to those skilled in the art, such as BIAcore or radioligand binding assays. “Specifically binds” or “has specificity to” can also refer to binding that entails some complementarity, for example between a ligand and its cognate receptor or an antibody or antibody fragment and its epitope. For example, an antibody or antibody fragments is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it would bind to a random, unrelated epitope.

[0092] Herein, “complementary” refers to polypeptides that form cognate pairs. In embodiments, the complementary polypeptides can be a part of a larger structure. For example, Fc regions (e.g., IgGl Fc regions), which are part of a fusion protein-drug conjugate protein or subunit, can form cognate pairs and are, therefore, complementary polypeptides. The complementary polypeptides can be symmetric or asymmetric, in which forming cognate pairs produces a homodimer or heterodimer, respectively. For example, a symmetric Fc region (e.g., IgGl Fc region) naturally forms a cognate pair with another Fc region (e.g., IgGl Fc region) through a variety of interactions, including hydrophobic interactions and disulfide bonding. In another example, asymmetric Fc regions (e.g., IgGl Fc regions) can be engineered to form a cognate pair with another Fc region (e.g., IgGl Fc region), such as through introducing mutations that produce variants in the amino acid sequence that form cognate pairs (e.g., knob in hole mutations or charge-pair mutations, Gunasekaran et al., J Biol Chem. 2010 Apr 16;285(25): 19637-19646. doi: 10.1074 / jbc.M110.117382; Xu et al., MAbs. 2015 Jan-Feb; 7(1): 231-242).

[0093] Herein, “off-target effects,” with respect to the fusion protein-drug conjugates disclosed herein, refers to effects (e.g., side effects) caused by drugs or biologies binding to and / or internalizing into cells, or tissues containing cells, that are not the intended targets of the drug or biologic. These effects can be caused by nonspecific binding of the drugs or biologies to cells that are not the intended targets for the molecules. In embodiments, fusion protein-drug conjugates disclosed herein that contain TRBs that are low-affinity binders to TfR have low or no nonspecific binding and / or produce few or no off-target effects.DOCKET NO: 91016-434826

[0094] Herein, “low affinity” (e.g., with respect to TRB of FPDC provided herein) refers to binding affinities measured as a KD (dissociation constant; e.g., using a kinetic assay described herein), in which affinity is has an inverse relationship with the KD value (i.e., a higher KD indicates a lower affinity; e.g., 1 x 103nM is a lower affinity than 1 nM). Thus, herein “low affinity” refers to KD of at or greater than 10 nM, KD of at or greater than 100 nM, KD of at or greater than 1 x 103nM, or KD of at or greater than 2 x 103nM, in either an FPDC heterodimer or an FPDC homodimer provided herein. In embodiments, the TRB is in a heterodimer FPDC provided herein (i.e., an FPDC with a single TRB), and “low affinity” refers to KD of 1 X 102nM to 1 x 105nM, such as a KD of 1 x 102nM to 1 x 103nM, KD of 1 x 102nM to 1 x 104nM, KD of 1 x 103nM to 1 x 104nM, KD of 1 X 103nM to 1 x 105nM, KD of 1.2-1.3 x 102nM, or KD of greater than 2 x 103nM. In embodiments, the TRB is in a homodimer FPDC provided herein (i.e., an FPDC with two TRB), and “low affinity” refers to KD of 1 X 101nM to 1 x 105nM, such as a KD of 1 x 10 nM to 1 x 102nM, KD of 1 X 10 nM to 1 x 103nM, KD of 1 x 10 nM to 1 x 104nM, KD of 1 x 102nM to 1 x 105nM, KD of 1 x 102nM to 1 x 103nM, KD of 1 x 102nM to 1 x 104nM, KD of 2-3 x 10 nM, or KD of greater than 2 x 103nM. In embodiments, “low affinity” refers to a homodimer FPDC provided herein (i.e., an FPDC with two TRB), wherein the TRB is VHHA 12+7 (e.g., SEQ ID NO: 4) or VHHA 12+5 (e.g., SEQ ID NO: 3). In embodiments, “low affinity” refers to a heterodimer FPDC provided herein (i.e., an FPDC with a single TRB), wherein the TRB is VHHA 12 (e.g., SEQ ID NO: 2), VHHA 12+7 (e.g., SEQ ID NO: 4), or VHHA 12+5 (e g., SEQ ID NO: 3).

[0095] Herein, “non-competitive” or “non-competitive binding” refers to a protein or polypeptide that binds at a first target protein site that is separate from a second protein or polypeptide binding site on the same target protein. In embodiments, a transferrin receptor binder (TRB) binds transferrin receptor non-competitively relative to the cognate transferrin. In other embodiments, a TRB binds transferrin receptor competitively relative to transferrin. In the competitive binding situation, the TRB may bind to transferrin receptor at a site that is the same, or overlapping with, a site on the transferrin receptor to which transferrin binds.

[0096] Herein, “epitope” refers to discrete sites of a molecule (e.g., protein) recognized by the disclosed antibodies and fragments thereof (e.g., POIBs and / or TRBs). Epitopes may be linear or three-dimensional. An antibody binds to a target protein when the interaction has a KDDOCKET NO: 91016-434826of less than 10’6molar, such as less than 10'7molar, less than 10'8molar, less than 10'9molar, or less than IO'10molar.

[0097] Herein, an “Fc region” (fragment crystallizable region) refers to the constant region fragment of an antibody, which is separated from two fragment antigen-binding regions when an antibody is digested by the enzyme papain. The constant region defines the isotype of an antibody. In embodiments, Fc regions of the present disclosure include IgGl Fc regions. In embodiments the Fc regions can be from IgG2, IgG3 or IgG4 subclasses of IgG. In embodiments, the antibodies of the present disclosure have, optionally, one or more conventional modifications in the Fc region, as described herein. Such modifications can reduce effector function of the antibodies and / or affect half-life of the antibodies, for example.Fusion Protein-Drug Conjugate Compositions

[0098] Provided herein are fusion-protein drug conjugates of the formula:(F1 – F1) – D(n)wherein:D is a therapeutic moiety;n is 1 to 6;Fl is a protein of the formula:R1 – R2 – R3:wherein:R1 is a protein of interest (POI) binder (POIB);R2 is complementary IgG Fc region; andR3 is a transferrin receptor (TfR) binder (TRB), wherein the TRB to TfR binding affinity is 10 nM to 100 pM; andF1-F1 form a homodimer.

[0099] D can be conjugated to (Fl - Fl) at a variety of positions and in various amounts. In embodiments, D is conjugated as described herein to (Fl -Fl) at Rl. In embodiments, one, two, or three D are conjugated to (F 1 - F 1) at Rl. In embodiments, one D is conjugated to (F 1 - Fl) at Rl. In embodiments, D is conjugated as described herein to (Fl - Fl) at R3. In embodiments,DOCKET NO: 91016-434826one, two, or three D are conjugated to (Fl - Fl) at R3. In embodiments, two D are conjugated to (F1 – F1) at R3. In embodiments, D is conjugated to (Fl - Fl) at R1 and to (Fl - Fl) at R3. In embodiments, one D is conjugated to (Fl - Fl) at Rl; and two D are conjugated to (Fl - Fl) at R3.

[0100] R3 can comprise a variety of amino acid sequences that bind transferrin receptor with an affinity of KD of 1 x 10 nM to 1 x 105nM. In embodiments, R3 comprises an amino acid sequence that binds transferrin receptor with an affinity of KD of 2-3 x 10 nM. In embodiments, R3 comprises an amino acid sequence that binds transferrin receptor with an affinity of KD of greater than 2 x 103nM. In embodiments, R3 comprises SEQ ID NO: 70, SEQ ID NO: 73, and SEQ ID NO: 72. In embodiments, R3 comprises SEQ ID NO: 2. In embodiments, R3 consists essentially of SEQ ID NO: 2. In embodiments, R3 consists of SEQ ID NO: 2. In embodiments, R3 comprises SEQ ID NO: 70, SEQ ID NO: 73, and SEQ ID NO: 74. In embodiments, R3 comprises SEQ ID NO: 3. In embodiments, R3 consists essentially of SEQ ID NO: 3. In embodiments, R3 consists of SEQ ID NO: 3. In embodiments, R3 comprises SEQ ID NO: 70, SEQ ID NO: 73, and SEQ ID NO: 75. In embodiments, R3 comprises SEQ ID NO: 4. In embodiments, R3 consists essentially of SEQ ID NO: 4. In embodiments, R3 consists of SEQ ID NO: 4.

[0101] In embodiments, R3 is an antibody fragment having a heavy chain variable region. In embodiments, R3 comprises:a VH CDR1 comprising the amino acid sequence of GTDFSINF (SEQ ID NO: 70), a VH CDR2 comprising the amino acid sequence of ITATGNT (SEQ ID NO:73), and a VH CDR3 comprising the amino acid sequence of YMLDK (SEQ ID NO: 72) [[VHHA-12]; ora VH CDR1 comprising the amino acid sequence of GTDFSINF (SEQ ID NO: 70), a VH CDR2 comprising the amino acid sequence of ITATGNT (SEQ ID NO: 73), and a VH CDR3 comprising the amino acid sequence of AMLDK (SEQ ID NO: 74) [[VHHA-12+5]; ora VH CDR1 comprising the amino acid sequence of GTDFSINF (SEQ ID NO: 70), a VH CDR2 comprising the amino acid sequence of ITATGNT (SEQ ID NO: 73), and a VH CDR3 comprising the amino acid sequence of YMADK (SEQ ID NO: 75)-[[VHHA-12+7],

[0102] R2 can comprise a variety of IgG Fc regions as described herein. In embodiments, R2 comprises SEQ ID NO: 80, SEQ ID NO: 81. In embodiments, R2 consists essentially of SEQ IDDOCKET NO: 91016-434826NO: 80, SEQ ID NO: 81. In embodiments, R2 consists of SEQ ID NO: 80, SEQ ID NO: 81. In embodiments, the POIB to POI binding affinity is higher than the TRB to TfR binding affinity.[001031

[0104] Provided herein are fusion-protein drug conjugates of the formula:(Fl -Fl)- (D)nwherein:D is a therapeutic moiety;n is 1 to 6;Fl is a protein of the formula:R1– R2 – R3:wherein:R1 is a means for binding a protein of interest (POI);R2 is a complementary IgG Fc region; andR3 is a means for binding a transferrin receptor (TfR), wherein the means for binding the TfR has a binding affinity to TfR of 10 nM to 100 pM; andF1-F1 form a homodimer.

[0105] D can be conjugated to at a variety of positions and in various amounts. In embodiments, D is conjugated as described herein to (Fl - Fl) at Rl. In embodiments, one, two, or three D are conjugated to (Fl - Fl) at Rl. In embodiments, one D is conjugated to (Fl - Fl) at Rl. In embodiments, D is conjugated as described herein to Fl at R3. In embodiments, one, two, or three D are conjugated to (Fl - Fl) at R3. In embodiments, two D are conjugated to (Fl - Fl) at R3. In embodiments, D is conjugated to (Fl - Fl) at Rl and to (Fl - Fl) at R3. In embodiments, one D is conjugated to (Fl - Fl) at Rl; and two D are conjugated to (Fl - Fl) at R3.

[0106] R3 can comprise a variety of amino acid sequences that bind transferrin receptor with an affinity of KD of 1 x 10 nM to 1 x 105nM. In embodiments, R3 comprises an amino acid sequence that binds transferrin receptor with an affinity of KD of 2-3 x 10 nM. In embodiments, R3 comprises an amino acid sequence that binds transferrin receptor with an affinity of KD of greater than 2 x 103nM. In embodiments, R3 comprises SEQ ID NO: 70, SEQ ID NO: 73, and SEQ ID NO: 72. In embodiments, R3 comprises SEQ ID NO: 2. In embodiments, R3 consistsDOCKET NO: 91016-434826essentially of SEQ ID NO: 2. In embodiments, R3 consists of SEQ ID NO: 2. In embodiments, R3 comprises SEQ ID NO: 70, SEQ ID NO: 73, and SEQ ID NO: 74. In embodiments, R3 comprises SEQ ID NO: 3. In embodiments, R3 consists essentially of SEQ ID NO: 3. In embodiments, R3 consists of SEQ ID NO: 3. In embodiments, R3 comprises SEQ ID NO: 70, SEQ ID NO: 73, and SEQ ID NO: 75. In embodiments, R3 comprises SEQ ID NO: 4. In embodiments, R3 consists essentially of SEQ ID NO: 4. In embodiments, R3 consists of SEQ ID NO: 4.

[0107] In embodiments, R3 is an antibody fragment having a heavy chain variable region. In embodiments, R3 comprises:a VH CDR1 comprising the amino acid sequence of GTDFSINF (SEQ ID NO: 70), a VH CDR2 comprising the amino acid sequence of ITATGNT (SEQ ID NO:73), and a VH CDR3 comprising the amino acid sequence of YMLDK (SEQ ID NO: 72) [[VHHA-12]; ora VH CDR1 comprising the amino acid sequence of GTDFSINF (SEQ ID NO: 70), a VH CDR2 comprising the amino acid sequence of ITATGNT (SEQ ID NO: 73), and a VH CDR3 comprising the amino acid sequence of AMLDK (SEQ ID NO: 74) [[VHHA-12+5]; ora VH CDR1 comprising the amino acid sequence of GTDFSINF (SEQ ID NO: 70), a VH CDR2 comprising the amino acid sequence of ITATGNT (SEQ ID NO: 73), and a VH CDR3 comprising the amino acid sequence of YMADK (SEQ ID NO: 75)-[[VHHA-12+7],

[0108] R2 can comprise a variety of IgG Fc regions as described herein. In embodiments, R2 comprises SEQ ID NO: 80, SEQ ID NO: 81. In embodiments, R2 consists essentially of SEQ ID NO: 80, SEQ ID NO: 81. In embodiments, R2 consists of SEQ ID NO: 80, SEQ ID NO: 81.

[0109] In embodiments, the POIB to POI binding affinity is higher than the TRB to TfR binding affinity.

[0110] Provided herein are fusion-protein drug conjugates of the formula:(Fl -F2)- (D)nwherein:D is a therapeutic moiety;n is 1 to 6;Fl is a protein of the formula:DOCKET NO: 91016-434826Rl -R2 orRl -R2’:wherein:R1 is a protein of interest (POI) binder (POIB); andR2 is an IgG Fc region complementary to R2’;F2 is a protein of the formula:R2’ -R3 orR2 -R3:wherein:R2’ is an IgG Fc region complementary to R2; andR3 is a transferrin receptor (TfR) binder (TRB), wherein the TRB to TfR binding affinity is 100 nM to 100 pM; andF1-F2 form a heterodimer.

[0111] D can be conjugated to at a variety of positions and in various amounts. In embodiments, D is conjugated as described herein to (Fl -F2) at Rl. In embodiments, one, two, or three D are conjugated to (F 1 - F2) at Rl. In embodiments, one D is conjugated to (F 1 - F2) at Rl. In embodiments, D is conjugated as described herein to (Fl - F2) at R3. In embodiments, one, two, or three D are conjugated to (Fl - F2) at R3. In embodiments, two D are conjugated to (Fl - F2) at R3. In embodiments, D is conjugated to (Fl - F2) at Rl and to (Fl - F2) at R3. In embodiments, one D is conjugated to (Fl - F2) at Rl; and two D are conjugated to (Fl - F2) at R3.

[0112] R3 can comprise a variety of amino acid sequences that bind transferrin receptor with an affinity of KD of 1 x 102nM to 1 x 105nM. In embodiments, R3 comprises an amino acid sequence that bind transferrin receptor with an affinity of KD of 1-2 X 102nM. In embodiments, R3 comprises an amino acid sequence that bind transferrin receptor with an affinity of KD of greater than 2 x 103nM. In embodiments, R3 comprises SEQ ID NO: 70, SEQ ID NO: 73, and SEQ ID NO: 72. In embodiments, R3 comprises SEQ ID NO: 2. In embodiments, R3 consists essentially of SEQ ID NO: 2. In embodiments, R3 consists of SEQ ID NO: 2. In embodiments, R3 comprises SEQ ID NO: 70, SEQ ID NO: 73, and SEQ ID NO: 74. In embodiments, R3 comprises SEQ ID NO: 3. In embodiments, R3 consists essentially of SEQ ID NO: 3. In embodiments, R3 consists of SEQ ID NO: 3. In embodiments, R3 comprises SEQ ID NO: 70, SEQ ID NO: 73, and SEQ ID NO: 75. In embodiments, R3 comprises SEQ ID NO: 4. InDOCKET NO: 91016-434826embodiments, R3 consists essentially of SEQ ID NO: 4. In embodiments, R3 consists of SEQ ID NO: 4.

[0113] In embodiments, R3 is an antibody fragment having a heavy chain variable region. In embodiments, R3 comprises:a VH CDR1 comprising the amino acid sequence of GTDFSINF (SEQ ID NO: 70), a VH CDR2 comprising the amino acid sequence of ITATGNT (SEQ ID NO:73), and a VH CDR3 comprising the amino acid sequence of YMLDK (SEQ ID NO: 72) [[VHHA-12]; ora VH CDR1 comprising the amino acid sequence of GTDFSINF (SEQ ID NO: 70), a VH CDR2 comprising the amino acid sequence of ITATGNT (SEQ ID NO: 73), and a VH CDR3 comprising the amino acid sequence of AMLDK (SEQ ID NO: 74) [[VHHA-12+5]; ora VH CDR1 comprising the amino acid sequence of GTDFSINF (SEQ ID NO: 70), a VH CDR2 comprising the amino acid sequence of ITATGNT (SEQ ID NO: 73), and a VH CDR3 comprising the amino acid sequence of YMADK (SEQ ID NO: 75)-[[VHHA-12+7],

[0114] R2 and R2’ can comprise a variety of modified and unmodified IgG Fc regions as described herein. In embodiments, R2 and R2’ are modified to form a heterodimer. Various engineering methods can be used to modify R2 and R2’ so that they form a heterodimer (e.g., knob-in-hole mutations or charge-pair mutations, Gunasekaran et al., J Biol Chem. 2010 Apr 16;285(25): 19637-19646. doi: 10.1074 / jbc.M110.117382; Xu et al., MAbs. 2015 Jan-Feb; 7(1): 231-242). Various methods can be used to separate and collect heterodimers (e.g., to separate (Fl - F2) heterodimers from homodimers formed by Fl and Fl or F2 and F2) formed through natural interactions (e.g., hydrophobic interactions or disulfide bonds) between unmodified R2 and R2’ Fc regions (e.g., Matsuda Y. Current approaches for the purification of antibody-drug conjugates. J Sep Sci. 2022; 45: 27-37. https: / / doi-org.ezp-prodl.hul.harvard.edu / 10.1002 / jssc.202100575). In embodiments, R2 and R2’ are modified via knob-in-hole mutations through mutating the R2 Fc region (e.g., at a CH3 domain) to comprise the ‘knob’ variants and mutating the R2’ Fc region (e.g., at a CH3 domain) to comprise the ‘hole’ variants; or through mutating the R2 Fc region (e.g., at a CH3 domain) to comprise the ‘hole’ variants and mutating the R2’ Fc region (e.g., at a CH3 domain) to comprise the ‘knob’ variants.

[0115] In embodiments, a residue that can be engineered for a “knob” configuration is substituting T for W at amino acid position 156 of SEQ ID NO: 80 or 81. In embodiments,DOCKET NO: 91016-434826residues that can be engineered for a “hole” configuration is substituting T for S at amino acid position 156 of SEQ ID NO: 80 or 81; substituting L for A at amino acid position 158 of SEQ ID NO: 80 or 81; substituting Y for V at amino acid position 197 of SEQ ID NO: 80 or 81.

[0116] In embodiments, the POIB to POI binding affinity is higher than the TRB to TfR binding affinity.

[0117] Provided herein are fusion-protein drug conjugates of the formula:(Fl -F2)- (D)nwherein:D is a therapeutic moiety;n is 1 to 6;Fl is a protein of the formula:R1-R2 orRl -R2’:wherein:R1 is a means for binding a protein of interest (POI); andR2 is an IgG Fc region complementary to R2’;F2 is a protein of the formula:R2’ -R3 orR2 -R3:wherein:R2’ is an IgG Fc region complementary to R2; andR3 is a means for binding a transferrin receptor (TfR), wherein the means for binding the TfR has a binding affinity to TfR of about 100 nM to about 100 pM; andF1-F2 form a heterodimer.

[0118] D can be conjugated to at a variety of positions and in various amounts. In embodiments, D is conjugated as described herein to (F 1 - F2) at Rl. In embodiments, one, two, or three D are conjugated to (F 1 - F2) at R1. In embodiments, one D is conjugated to (F 1 - F2) at Rl. In embodiments, D is conjugated as described herein to (Fl - F2) at R3. In embodiments, one, two, or three D are conjugated to (Fl - F2) at R3. In embodiments, two D are conjugated to (Fl - F2) at R3. In embodiments, D is conjugated to (Fl - F2) at Rl and to (Fl - F2) at R3. In embodiments, one D is conjugated to (Fl - F2) at Rl; and two D are conjugated to (Fl - F2) at R3.DOCKET NO: 91016-434826

[0119] R3 can comprise a variety of amino acid sequences that bind transferrin receptor with an affinity of KD of 1 x 102nM to 1 x 105nM. In embodiments, R3 comprises an amino acid sequence that bind transferrin receptor with an affinity of KD of 1-2 X 102nM. In embodiments, R3 comprises an amino acid sequence that bind transferrin receptor with an affinity of KD of greater than 2 x 103nM. In embodiments, R3 comprises SEQ ID NO: 70, SEQ ID NO: 73, and SEQ ID NO: 72. In embodiments, R3 comprises SEQ ID NO: 2. In embodiments, R3 consists essentially of SEQ ID NO: 2. In embodiments, R3 consists of SEQ ID NO: 2. In embodiments, R3 comprises SEQ ID NO: 70, SEQ ID NO: 73, and SEQ ID NO: 74. In embodiments, R3 comprises SEQ ID NO: 3. In embodiments, R3 consists essentially of SEQ ID NO: 3. In embodiments, R3 consists of SEQ ID NO: 3. In embodiments, R3 comprises SEQ ID NO: 70, SEQ ID NO: 73, and SEQ ID NO: 75. In embodiments, R3 comprises SEQ ID NO: 4. In embodiments, R3 consists essentially of SEQ ID NO: 4. In embodiments, consists of SEQ ID NO: 4.

[0120] In embodiments, R3 is an antibody fragment having a heavy chain variable region. In embodiments, R3 can have:a VH CDR1 comprising the amino acid sequence of GTDFSINF (SEQ ID NO: 70), a VH CDR2 comprising the amino acid sequence of ITATGNT (SEQ ID NO:73), and a VH CDR3 comprising the amino acid sequence of YMLDK (SEQ ID NO: 72) [[VHHA-12]; ora VH CDR1 comprising the amino acid sequence of GTDFSINF (SEQ ID NO: 70), a VH CDR2 comprising the amino acid sequence of ITATGNT (SEQ ID NO: 73), and a VH CDR3 comprising the amino acid sequence of AMLDK (SEQ ID NO: 74) [[VHHA-12+5]; ora VH CDR1 comprising the amino acid sequence of GTDFSINF (SEQ ID NO: 70), a VH CDR2 comprising the amino acid sequence of ITATGNT (SEQ ID NO: 73), and a VH CDR3 comprising the amino acid sequence of YMADK (SEQ ID NO: 75)-[[VHHA-12+7].R2 and R2’ can comprise a variety of modified and unmodified IgG Fc regions as described herein. In embodiments, R2 and R2’ are modified to form a heterodimer. Various engineering methods can be used to modify R2 and R2’ so that they form a heterodimer (e.g., knob-in-hole mutations or charge-pair mutations, Gunasekaran et al., J Biol Chem. 2010 Apr 16;285(25): 19637-19646. doi: 10.1074 / jbc.M110.117382; Xu et al., MAbs. 2015 Jan-Feb; 7(1): 231-242). Various methods can be used to separate and collect heterodimers (e.g., separate (Fl - F2) from homodimers formed by Fl and Fl or F2 and F2) formed through natural interactionsDOCKET NO: 91016-434826(e g., hydrophobic interactions or disulfide bonds) between unmodified R2 and R2’ Fc regions. In embodiments, R2 and R2’ are modified via knob-in-hole mutations through mutating the R2 Fc region (e.g., at a CH3 domain) to comprise the ‘knob’ variants at and mutating the R2’ Fc region (e.g., at a CH3 domain) to comprise the ‘hole’ variants; or through mutating the R2 Fc region (e.g., at a CH3 domain) to comprise the ‘hole’ variants and mutating the R2’ Fc region (e.g., at a CH3 domain) to comprise the ‘knob’ variants.

[0121] In embodiments, a residue that can be engineered for a “knob” configuration is substituting T for W at amino acid position 156 of SEQ ID NO: 80 or 81. In embodiments, residues that can be engineered for a “hole” configuration is substituting T for S at amino acid position 156 of SEQ ID NO: 80 or 81; substituting L for A at amino acid position 158 of SEQ ID NO: 80 or 81; substituting Y for V at amino acid position 197 of SEQ ID NO: 80 or 81.

[0122] Provided herein are fusion-protein drug conjugates of the formula:(Fl -Fl)- (D)nwherein:D is a therapeutic moiety;n is 1 to 6;Fl is a protein of the formula:R1 -R2 -R3:wherein:R1 is a protein of interest (POI) binder (POIB);R2 is a complementary IgG Fc region; andR3 is a transferrin receptor (TfR) binder (TRB), comprising:a. a VH CDR1 comprising the amino acid sequence of GTDFSINF (SEQ ID NO:70), a VH CDR2 comprising the amino acid sequence of ITATGNT (SEQ ID NO:73), and a VH CDR3 comprising the amino acid sequence of YMLDK (SEQ ID NO: 72) [[VHHA-12]; orb. a VH CDR1 comprising the amino acid sequence of GTDFSINF (SEQ ID NO:70), a VH CDR2 comprising the amino acid sequence of ITATGNT (SEQ ID NO: 73), and a VH CDR3 comprising the amino acid sequence of AMLDK (SEQ ID NO: 74) [[VHHA-12+5]; orDOCKET NO: 91016-434826c. a VH CDR1 comprising the amino acid sequence of GTDFSINF (SEQ ID NO:70), a VH CDR2 comprising the amino acid sequence of ITATGNT (SEQ ID NO: 73), and a VH CDR3 comprising the amino acid sequence of YMADK (SEQ ID NO: 75) [[VHHA-12+7]; andF1-F1 form a homodimer.

[0123] D can be conjugated to Fl at a variety of positions and in various amounts. In embodiments, D is conjugated as described herein to Fl at Rl. In embodiments, one, two, or three D are conjugated to Fl at Rl. In embodiments, one D is conjugated to Fl Rl. In embodiments, D is conjugated as described herein to Fl at R3. In embodiments, one, two, or three D are conjugated to Fl at R3. In embodiments, two D are conjugated to Fl at R3. In embodiments, D is conjugated to Fl at Rl and R3. In embodiments, one D is conjugated to Fl at Rl; and two D are conjugated to F 1 at R3.

[0124] R3 can comprise a variety of amino acid sequences that bind transferrin receptor with an affinity of KD of 1 x 10 nM to 1 x 105nM. In embodiments, R3 comprises an amino acid sequence that bind transferrin receptor with an affinity of KD of 1-2 X 102nM. In embodiments, R3 comprises an amino acid sequence that bind transferrin receptor with an affinity of KD of greater than 2 x 103nM. In embodiments, R3 comprises SEQ ID NO: 70, SEQ ID NO: 73, and SEQ ID NO: 72. In embodiments, R3 comprises SEQ ID NO: 2. In embodiments, R3 consists essentially of SEQ ID NO: 2. In embodiments, R3 consists of SEQ ID NO: 2. In embodiments, R3 comprises SEQ ID NO: 70, SEQ ID NO: 73, and SEQ ID NO: 74. In embodiments, R3 comprises SEQ ID NO: 3. In embodiments, R3 consists essentially of SEQ ID NO: 3. In embodiments, R3 consists of SEQ ID NO: 3. In embodiments, R3 comprises SEQ ID NO: 70, SEQ ID NO: 73, and SEQ ID NO: 75. In embodiments, R3 comprises SEQ ID NO: 4. In embodiments, R3 consists essentially of SEQ ID NO: 4. In embodiments, R3 consists of SEQ ID NO: 4.

[0125] R2 can comprise a variety of IgG Fc regions (e.g., an IgGl Fc region) as described herein.

[0126] In embodiments, the POIB to POI binding affinity is higher than the TRB to TfR binding affinity.DOCKET NO: 91016-434826

[0127] n can vary for the Formulas described herein. In embodiments, n ranges from 1-10, 1-8, 1-6, 2-6, or 2-4. In embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In embodiments, n is 1. In embodiments, n is 2. In embodiments, n is 3. In embodiments, n is 4. In embodiments, n is 5. In embodiments, n is 6. In embodiments, n is 7. In embodiments, n is 8. In embodiments, n is 9. In embodiments, n is 10. In embodiments, n is 11. In embodiments, n is 12.Table 1

[0128] Shows the components, and common names, for molecules used herein. These molecules are shown in FIGs. 5-20.Construct / Pieces POI Fc TRB (R3) Drug Common Name (Rl) (R2) for Labels EGFR-Fc-MC-MMAF EGFR Fc MC-MMAF POIB control (Ab- DC control) Fc-VHHA12+7-MC- Fc VHHA12+7 MC-MMAF TRB’ Control MMAF (Fusion Protein DC)EGFR TransTAC EGFR Fc VHHA12+7 MC-MMAF EGFR VHHA12+7-MC-MMAF FPDC2(MMAF*6) ICAM-Fc-MC-MMAF ICAM Fc MC-MMAF POIB control Fc-VHHA12+7-MC- Fc VHHA12+7 MC-MMAF TRB’ Control MMAF ICAM TransTAC ICAM Fc VHHA12+7 MC-MMAF ICAM VHHA12+7-MC-MMAF FPDC2(MMAF*6) PVR-Fc-MC-MMAF PVR Fc MC-MMAF POIB control Fc-VHHA12+7-MC- Fc VHHA12+7 MC-MMAF TRB’ Control MMAF PVR TransTAC PVR Fc VHHA12+7 MC-MMAF PVR VHHA12+7-MC-MMAF FPDC2(MMAF*6) MCTl-Fc-MC-MMAF MCT1 Fc MC-MMAF POIB control Fc-VHHA12+7-MC- Fc VHHA12+7 MC-MMAF TRB’ Control MMAF MCT1 TransTAC MCT1 Fc VHHA12+7 MC-MMAF MCT1 VHHA12+7-MC-MMAF FPDC2(MMAF*6) LATl-Fc-MC-MMAF LAT1 Fc MC-MMAF POIB control Fc-VHHA12+7-MC- Fc VHHA12+7 MC-MMAF TRB’ Control MMAF LAT1 TransTAC LAT1 Fc VHHA12+7 MC-MMAF LAT1 VHHA12+7-MC-MMAF FPDC2(MMAF*6)BST2-Fc-MC-MMAF BST2 Fc MC-MMAF POIB controlDOCKET NO: 91016-434826Fc-VHHA12+7-MC- Fc VHHA 12+7 MC-MMAF TRB’ Control MMAF BST2 TransTAC BST2 Fc VHHA 12+7 MC-MMAF BST2 VHHA12+7-MC-MMAF FPDC2(MMAF*6) EGFR-Fc-Dox EGFR Fc Doxorubicin POIB control Fc-VHHA12+7-C2-DOX Fc VHHA 12+7 Doxorubicin TRB’ Control EGFR TransTAC EGFR Fc VHHA 12+7 Doxorubicin EGFR VHHA12+7-C2-DOX FPDC2(DOX*6) EGFR-Fc-RSL3 EGFR Fc MC-RSL3 POIB control Fc-VHHA12+7-C2-DOX Fc VHHA 12+7 MC-RSL3 TRB’ Control EGFR TransTAC EGFR Fc VHHA 12+7 MC-RSL3 EGFR VHHA12+7-C2-DOX FPDC2(RSL3*6) EGFR-Fc-MC- VC- M M A E EGFR Fc MC-EC- POIB control MMAEFc-VHHA12+7 -C2-MC- Fc VHHA 12+7 MC-EC- TRB’ Control EC'-MMAE MMAEEGFR TransTAC EGFR Fc VHHA 12+7 MC-EC- EGFR VHHA12+7 -C2-MC-VC- MMAE FPDC2(EC- MMAE MMAE*6) EGFR-Fc-MC-MMAE EGFR Fc MC-MMAE POIB control Fc-VHHA12+7 -C2-MC- Fc VHHA 12+7 MC-MMAE TRB’ Control MMAE EGFR TransTAC EGFR Fc VHHA 12+7 MC-MMAE EGFR VHHA12+7 -C2-MC- FPDC2(MMAE*6) MMAE CD20-Fc CD20 Fc POIB control CD20-Fc-TfRl VHHA CD20 Fc VHHA CD20 FP4 CD20-Fc-TfRl VHHA CD20 Fc VHHA 12+7 CD20 FP2 12+7CD20-Fc-TfRl H7 CD20 Fc H7 POIB control CD20-Fc-TfRl VHHA5 CD20 Fc VHHA5 CD20 FP5 CD20-Fc-TfRl VHHA7 CD20 Fc VHHA7 CD20 FP6 CD20-Fc-TfRl VHHA 12 CD20 Fc VHHA12 CD20 FP1 CD20-Fc-TfRl VHHA CD20 Fc VHHA 12+5 CD20 FP3 12+5CD20 Fc-MC-MMAF CD20 Fc MC-MMAF POIB control Fc-VHHA12+7 -C2-MC- Fc VHHA 12+7 MC-MMAF TRB’ Control MMAF CD20 TransTAC CD20 Fc VHHA 12+7 MC-MMAF CD20 VHHA12+7-C2-MC- FPDC2(MMAF*6) MMAF CD20 TransTAC CD20 Fc VHHA 12+7 MC-MMAF CD20VHHA12+7-MC-MMAF FPDC2(MMAF*2)DOCKET NO: 91016-434826CD20 TransTAC H7-MC- CD20 Fc H7 MC-MMAF CD20MMAF FPDC4(MMAF*2) CD20-Fc CD20 Fc POIB control w / o drugFc-VHHA12+7 -C2 Fc VHHA 12+7 TRB’ Control (w / o drug)CD20 TransTAC CD20 Fc VHHA 12+7 CD20 FPDC2 VHHA12+7-C2CD20 Fc-MC-MMAF CD20 Fc MC-MMAF POIB control Fc-VHHA12+7 -C2-MC- Fc VHHA 12+7 MC-MMAF TRB’ Control MMAF CD20 TransTAC CD20 Fc VHHA 12+7 MC-MMAF CD20 VHHA12+7-C2-MC- FPDC2(MMAF*6)MMAFTable 1. Nomenclature for constructs and construct pieces described herein. Table Key: VHHA = TRB0; VHHA12 = TRB; VHHA12+7= TRB’; VHHA12+5=TRB”; VHHA5 = TRBa; VHHA7 = TRBb; H7 = TRBC. FPDC1 = Fusion Protein Drug Conjugate comprising VHHA12 (TRB comprising SEQ ID NO: 2); FPDC2 = Fusion Protein Drug Conjugate comprising VHHA12+7 (TRB’ comprising SEQ ID NO: 4); FPDC3 = Fusion Protein Drug Conjugate comprising VHHA12+5 (TRB” comprising SEQ ID NO: 3); FPDC4 = Fusion Protein Drug Conjugate comprising H7 (TRBc comprising SEQ ID NO: 9). FP1 = Fusion Protein comprising VHHA12 (TRB comprising SEQ ID NO: 2); FP2 = Fusion Protein comprising VHHA12+7 (TRB’ comprising SEQ ID NO: 4); FP3 = Fusion Protein comprising VHHA12+5 (TRB” comprising SEQ ID NO: 3); FP4 = Fusion Protein comprising VHHA (TRB0comprising SEQ ID NO: 1); FP5 = Fusion Protein comprising VHHA5 (TRBacomprising SEQ ID NO: 5); FP6 = Fusion Protein comprising VHHA7 (TRBbcomprising SEQ ID NO: 6); FP7 = Fusion Protein comprising H7 (TRBCcomprising SEQ ID NO: 9).Dimers

[0129] The FPDC provided herein form a homodimer or a heterodimer. A homodimer is a complex of two same or essentially the same proteins (also known as polypeptides, subunits, or protein subunits), formed through covalent or noncovalent interactions (e.g., hydrogen bonding or cysteine disulfide bonding). In embodiments, a homodimer is formed by protein subunits herein, wherein the amino acid sequences of each protein subunit are the same, and the posttranslational modifications are the same; or wherein the amino acid sequences are the same and the posttranslational modifications are different. A heterodimer is a complex of two different proteins (also known as polypeptides, subunits, or protein subunits), formed through covalent or noncovalent interactions (e.g., hydrogen bonding or cysteine disulfide bonding). In embodiments, a heterodimer is formed by protein subunits herein, wherein the amino acidDOCKET NO: 91016-434826sequences of each protein subunit are different, and the posttranslational modifications are different.[001301 In embodiments, the FPDC form a homodimer or a heterodimer through the Fc region of R2 or an R2’. In embodiments, Fl and Fl form a homodimer. In embodiments, Fl and Fl form a homodimer through the Fc region of R2 and R2. In embodiments, Fl and F2 form a heterodimer. In embodiments, Fl and F2 form a heterodimer through the Fc region of R2 and R2’. In embodiments, Fl and F2 form a heterodimer through a CH3 domain of the Fc region of R2 and R2’.

[0131] Binding affinity of a TRB to TfR can be affected by dimerization of the FPDCs. This is discussed elsewhere herein.Geometries

[0132] Additional geometry embodiments of fusion proteins are further described herein in Figure 38.

[0133] In one embodiment described herein is a fusion protein-drug conjugate of the formula:(Fl -F2)- (D)nwherein:D is a therapeutic moiety;n is 1 to 8;Fl is a protein of the formula:R1 -R2 -R3 orR1 – R2 – R3:wherein:R1 is a protein of interest (POI) binder (POIB); andR2 is an IgG Fc region complementary to R2’;R3 is a transferrin receptor (TfR) binder (TRB), wherein the TRB to TfR binding affinity (KD) is about 10 nM to about 100 pM;F2 is a protein of the formula:R2-R3 orR2’-R3:wherein:R2’ is an IgG Fc region complementary to R2; andDOCKET NO: 91016-434826R3 is a transferrin receptor (TfR) binder (TRB), wherein the TRB to TfR binding affinity (KD) is about 10 nM to about 100 pM; andF1-F2 form a heterodimer.

[0134] Another embodiment described herein is a fusion protein-drug conjugate of the formula:(Fl -F2)- (D)nwherein:D is a therapeutic moiety;n is 1 to 8;Fl is a protein of the formula:R1 -R2 -R3 orR1 – R2 – R3:wherein:R1 is a means for binding a protein of interest (POI); andR2 is an IgG Fc region complementary to R2’;R3 is a means for binding a transferrin receptor (TfR), a TfR binder (TRB), wherein the TRB to TfR binding affinity (KD) is about 10 nM to about 100 pM;F2 is a protein of the formula:R2-R3 orR2’-R3:wherein:R2’ is an IgG Fc region complementary to R2; andR3 is a means for binding a transferrin receptor (TfR), a TfR binder (TRB), wherein the TRB to TfR binding affinity (KD) is about 10 nM to about 100 pM; andF1-F2 form a heterodimer.

[0135] Another embodiment described herein is a fusion protein-drug conjugate of the formula:(Fl -F2)- (D)nwherein:D is a therapeutic moiety;n is 1 to 8;Fl is a protein of the formula:R1 -R2 -R3 orR1 – R2 – R3:DOCKET NO: 91016-434826wherein:R1 is a protein of interest (POI) binder (POIB); andR2 is an IgG Fc region complementary to R2’;R3 is a transferrin receptor (TfR) binder (TRB), wherein the TRB to TfR binding affinity (KD) is about 10 nM to about 100 pM;F2 is a protein of the formula:R2-R3 orR2’-R3:wherein:R2’ is an IgG Fc region complementary to R2; andR3 is a transferrin receptor (TfR) binder (TRB), wherein the TRB to TfR binding affinity (KD) is about 10 nM to about 100 pM; andF1-F2 form a heterodimer.Another embodiment described herein is a fusion protein-drug conjugate of the formula:(Fl -F2)- (D)nwherein:D is a therapeutic moiety;n is 1 to 8;Fl is a protein of the formula:R1 -R2 -R3 orR1 – R2 – R3:wherein:R1 is a means for binding a protein of interest (POI); andR2 is an IgG Fc region complementary to R2’;R3 is a means for binding a transferrin receptor (TfR), a TfR binder (TRB), wherein the TRB to TfR binding affinity (KD) is about 10 nM to about 100 pM;F2 is a protein of the formula:R2-R3 orR2’-R3:wherein:R2’ is an IgG Fc region complementary to R2; andR3 is a means for binding a transferrin receptor (TfR), a TfR binder (TRB), wherein the TRB to TfR binding affinity (KD) is about 10 nM to about 100 pM; andF1-F2 form a heterodimer.DOCKET NO: 91016-434826

[0136] Another embodiment described herein is a fusion protein-drug conjugate of the formula:(Fl -F2)-(D)nwherein:D is a therapeutic moiety;n is 1 to 8;Fl is a protein of the formula:R1 -R2 orRl -R2’:wherein:R1 is a protein of interest (POI) binder (POIB); andR2 is an IgG Fc region complementary to R2’;F2 is a protein of the formula:R1 - R2 - R3 or R1 - R2 - R3:wherein:R2’ is an IgG Fc region complementary to R2; andR3 is a transferrin receptor (TfR) binder (TRB), wherein the TRB to TfR binding affinity (KD) is about 10 nM to about 100 pM;F1-F2 form a heterodimer.

[0137] Another embodiment described herein is a fusion protein-drug conjugate of the formula:(Fl -F2)- (D)nwherein:D is a therapeutic moiety;n is 1 to 8;F l is a protein of the formula:R1 -R2 orRl -R2’:wherein:R1 is a means for binding a protein of interest (POI); andR2 is an IgG Fc region complementary to R2’;F2 is a protein of the formula:R1 - R2 - R3 or R1 - R2 - R3:DOCKET NO: 91016-434826wherein:R2’ is an IgG Fc region complementary to R2; andR3 is a means for binding a transferrin receptor (TfR), a TfR binder (TRB), wherein the TRB to TfR binding affinity (KD) is about 10 nM to about 100 pM;F 1 -F2 form a heterodimer.

[0138] A fusion protein-drug conjugate of the formula:(Fl -F2)- (D)nwherein:D is a therapeutic moiety;n is 1 to 8;Fl is a protein of the formula:R1 -R2 -R3 orR1 – R2 – R3:wherein:R1 is a protein of interest (POI) binder (POIB); andR2 is an IgG Fc region complementary to R2’;R3 is a transferrin receptor (TfR) binder (TRB), wherein the TRB to TfR binding affinity (KD) is about 10 nM to about 100 pM;F2 is a protein of the formula:R2 or R2’:wherein:R2’ is an IgG Fc region complementary to R2; and;F1-F2 form a heterodimer.

[0139] Another embodiment described herein is a fusion protein-drug conjugate of the formula:(Fl -F2)- (D)nwherein:D is a therapeutic moiety;n is 1 to 8;Fl is a protein of the formula:R1 -R2 -R3 orR1 – R2 – R3:wherein:DOCKET NO: 91016-434826R1 is a means for binding a protein of interest (POI); andR2 is an IgG Fc region complementary to R2’;R3 is a means for binding a transferrin receptor (TfR), a TfR binder (TRB), wherein the TRB to TfR binding affinity (KD) is about 10 nM to about 100 pM;F2 is a protein of the formula:R2 or R2’:wherein:R2’ is an IgG Fc region complementary to R2; and;F1-F2 form a heterodimer.

[0140] Another embodiment described herein is a fusion protein-drug conjugate of the formula:(Fl -F2)- (D)nwherein:D is a therapeutic moiety;n is 1 to 8;Fl is a protein of the formula:R1 -R2 orRl -R2’:wherein:R1 is a protein of interest (POI) binder (POIB); andR2 is an IgG Fc region complementary to R2’;F2 is a protein of the formula:R3 — R3 — R2 or R3 — R3 — R2:wherein:R2’ is an IgG Fc region complementary to R2; andR3 is a transferrin receptor (TfR) binder (TRB), wherein the TRB to TfR binding affinity (KD) is about 10 nM to about 100 pM;two copies of R3 are in tandem; andF1-F2 form a heterodimer.

[0141] Another embodiment described herein is a fusion protein-drug conjugate of the formula:DOCKET NO: 91016-434826(Fl -F2)- (D)nwherein:D is a therapeutic moiety;n is 1 to 8;Fl is a protein of the formula:R1 -R2 orRl -R2’:wherein:R1 is a means for binding a protein of interest (POI); andR2 is an IgG Fc region complementary to R2’;F2 is a protein of the formula:R3 — R3 — R2 or R3 — R3 — R2:wherein:R2’ is an IgG Fc region complementary to R2; andR3 is a means for binding a transferrin receptor (TfR), a TfR binder (TRB), wherein the TRB to TfR binding affinity (KD) is about 10 nM to about 100 pM;two copies of R3 are in tandem; andF1-F2 form a heterodimer.

[0142] Another embodiment described herein is a fusion protein-drug conjugate of tire foumula:(F 1 - F2) - (D)n (Formula N)wherein:D is a therapeutic moiety;n is 1 to 8;Fl is a protein of the formula:R1 - R2 or R1 - R2’ (formula O):wherein:R1 is a protein of interest (POI) binder (POH3); and R2 is an IgG Fc region complementary to R2’;F2 is a protein of the formula:R2 - R3 or R2’ - R3wherein:DOCKET NO: 91016-434826R2’ is an IgG Fc region complementary to R2; andR3 is a transferrin receptor (TfR) binder (TRB), wherein the TRB is a (Fab)m wherein m is 1 or 2 and the TRB binding affinity is about 10 nM to about 100 pM; andFl - F2 form a heterodimer.Sequences of Molecules Disclosed HereinTable 2.pLS15 Anti-CCR6_B368-G29A_HC-Knob_Fc-His (SEQ ID NO: 13) MRMQLLLLIALSLALVTNSTSEVQLVESGGGLVQPGGSLRLSCAASGFSFSDYYMYWVRQAP GKGLEWVSYITTGGRTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTSPLRGA WFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGGSG GSGTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDEL TKNQ VSL WCL VKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFL YS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSHHHHHH [SEQ ID NO: 13] [CCR6 (POI) Binder: SEQ ID NO: 14; Fc Region: SEQ ID NO: 15]pLS16-Anti-CCR6_B3G8-G29A_LC (SEQ ID NO: 16) _ MYRMQLLSCIALSLALVTNSDWMTQSPLSLPVTLGQPASISCRSSQSIVHSNANTYLEWYQ QRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGTYLPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO: 16][CCR6 (POI) Binder: SEQ ID NO: 17]pIKA5-VHHA-Hole (SEQ ID NO: 18) _M RMQLLLLIALSLALVTNSTSEVQLVESGGGWQPGGSLKLSCVASGTDFSINFIRWYRQAPGK QREFVAGFTATGNTNYADSMKGRFTISRDNTKNAVYLQIDSLKPEDTAVYYCYMLDKWGQGTQ\ / TVSSGTGGD^P\TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDEL TKNQ VSLSCA VKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDG SFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [SEQ ID NO: 18] [TfR1 Binder: SEQ ID NO: 19; Fc Region: SEQ ID NO: 20]DOCKET NO: 91016-434826 pLS21-Tf-Hole (SEQ ID NO: 21) MRMQLLLLIALSLALVTNSTSVPDKTVRWCAVSEHEATKCQSFRDHMKSVIPSDGPSVACVKKA SYLDCIRAIAANEADAVTLDAGLVYDAYLAPNNLKPVVAEFYGSKEDPQTFYYAVAVVKKDSGFQMNQLRGKKSCHTGLGRSAGWNIPIGLLYCDLPEPRKPLEKAVANFFSGSCAPCADGTDFPQLCQLCPGCGCSTLNQYFGYSGAFKCLKDGAGDVAFVKHSTIFENLANKADRDQYELLCLDNTRKPVDEYKDCHLAQVPSHTVVARSMGGKEDLIWELLNQAQEHFGKDKSKEFQLFSSPHGKDLLFKDSAHGFLKVPPRMDAKMYLGYEYVTAIRNLREGTCPEAPTDECKPVKWCALSHHERLKCDEWSVNSVGKIECVSAETTEDCIAKIMNGEADAMSLDGGFVYIAGKCGLVPVLAENYNKSDNCEDTPEAGYFAIAVVKKSASDLTWDNLKGKKSCHTAVGRTAGWNIPMGLLYNKINHCRFDEFFSEGCAPGSKKDSSLCKLCMGSGLNLCEPNNKEGYYGYTGAFRCLVEKGDVAFVKHQTVPQNTGGKNPDPWAKNLNEKDYELLCLDGTRKPVEEYANCHLARAPNHAVVTRKDKEACVHKILRQQQHLFGSNVTDCSGNFCLFRSETKDLLFRDDTVCLAKLHDRNTYEKYLGEEYVKAVGNLRKCSTSSLLEACTFRRPGTGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK[SEQ ID NO: 21] [Tf R1 Binder: SEQ ID NO: 22; Fc Region: SEQ ID NO: 23]pLS23-VHHA12-Hole (SEQ ID NO: 24)M RMQLLLLIALSLALVTNSTSEVQLVESGGGWQPGGSLKLSCVASGTDFSINFIRWYRQAPGK OREFVAGITATGNTNYADSMKGRFTISRDNTKNAVYLOIDSLKPEDTAVYYCYMLDKWGOGTOV TVSSGTGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK[SEQ ID NO: 24][Tf R1 Binder: SEQ ID NO: 25; Fc Region: SEQ ID NO: 26]pLS24-VHHA12+7-Hole (SEQ ID NO: 27)M RMQLLLLIALSLALVTNSTSEVQLVESGGGWQPGGSLKLSCVASGTDFSINFIRWYRQAPGK OREFVAGITATGNTNYADSMKGRFTISRDNTKNAVYLOIDSLKPEDTAVYYCYMADKWGOGTO\ / TYSSG1GGDYl\-\1CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE\ / TCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDEL TKNQ VSLSCA VKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDG SFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK[SEQ ID NO: 27][Tf R1 Binder: SEQ ID NO: 28; Fc Region: SEQ ID NO: 29]pLS25-VHHA5-Hole (SEQ ID NO: 30)DOCKET NO: 91016-434826M RM QLLLLIALSLALVTNSTSEVQLVESGGGWQPGGSLKLSCVASGTDFSINFIRWYRQAPGK OREFVAGFTATGNTNYADSMKGRFTISRDNTKNAVYLOIDSLKPEDTAVYYCAMLDKWGOGTOyiySSGTGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDEL TKNQ VSLSCA VKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDG SFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK[SEQ ID NO: 30][Tf R1 Binder: SEQ ID NO: 31; Fc Region: SEQ ID NO: 32]pLS26-VHHA7-Hole (SEQ ID NO: 33)M RM QLLLLI ALSLALVTNSTSEVOLVESGGGWOPGGSLKLSCVASGTDFSINFIRWYROAPGK OREFVAGFTATGNTNYADSMKGRFTISRDNTKNAVYLOIDSLKPEDTAVYYCYMADKWGOGTO\rPJSSGTGGDK1\-\TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDEL TKNQ VSLSCA VKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDG SFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK[SEQ ID NO: 33][Tf R1 Binder: SEQ ID NO: 34; Fc Region: SEQ ID NO: 35]pLS27-VHHA 12+5 -Hole (SEQ ID NO: 36)M RM QLLLLIALSLALVTNSTSEVOLVESGGGWQPGGSLKLSCVASGTDFSINFIRWYRQAPGK OREFVAGITATGNTNYADSMKGRFTISRDNTKNAVYLOIDSLKPEDTAVYYCAMLDKWGOGTO\ / TVSSGTGGDKT\-\TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDEL TKNQ VSLSCA VKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDG SFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK[SEQ ID NO: 36][Tf R1 Binder: SEQ ID NO: 37; Fc Region: SEQ ID NO: 38]pIKA5.1-G9-Hole (SEQ ID NO: 39)M RMQLLLLIALSLALVTNSTSSELTQDPAVSVALGOTVRITCOGDSLRSYYASWYOOKPGQAPV LVIYGKNNRPSGIPDRFSGSGSGNTASLTITGAOAEDEADYYCAAWDDSLSGPVFGGGTKVTVL GGGGGSGGGGSGGGGSOVOLVESGGGLVEPGGSLRLSCAASGFTFSNYAINWVROAPGKG LEWVANIHHDGNGKYYVDSVEGRFTISRDNAKNSLYLOMDSLRAEDTAIYYCARDGYGGYLDL WGOGTLVTVSSGTGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK[SEQ ID NO: 39]DOCKET NO: 91016-434826[Tf R 1 Binder: SEQ ID NO: 40; Fc Region: SEQ ID NO: 41 ]pIKA5.2-H7-Hole (SEQ ID NO: 42)M RM QLLLLIALSLALVTNSTSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPV LVMYGRNERPSGVPDRFSGSKSGTSASLAISGLQPEDEANYYCAGWDDSLTGPVFGGGTKLTVLGGGGGSGGGGSGGGGSQVQLQESGGGVVQPGRSLRLSCAASRFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLSGYGDYPDYWGQGTLVTVSSGTGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [SEQ ID NO: 42][Tf R1 Binder: SEQ ID NO: 43; Fc Region: SEQ ID NO: 44]pIKA5.3-N5-Hole (SEQ ID NO: 45)M RMQLLLLIALSLALVTNSTSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPV LVMYGRNERPSGVPDRFSGSKSGTSASLAISGLOPEDEANYYCAGWDDSLTGPVFGGGTKLTV LGGGGGSGGGGSGGGGGSQVQLQESGGGVVQPGRSLRLSCAASRFTFNNYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLSGYGDYPDYWGQGTLVTVSSGTGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLSCA VKGFYPSDIA VEWESNGQPENNYKT TPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK[SEQ ID NO: 45][TfR1 Binder: SEQ ID NO: 46; Fc Region: SEQ ID NO: 47]pLS32-CCR6_HC-Fc-TfRl VHHA (SEQ ID NO: 48) MRMQLLLLIALSLALVTNSTSEVQLVESGGGLVQPGGSLRLSCAASGFSFSDYYMYWVRQAP GKGLEWVSYITTGGRTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTSPLRGA WFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGGVD GDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGLYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGSGGSEVQLVESGGGWG PGGSLKLSCVASGTDFSINFIRWYROAPGKOREFVAGFTATGNTNYADSMKGRFTISRDNTKNA VYLQIDSLKPEDTAVYYCYMLDKWGQGTQVTVSS [SEQ ID NO: 48]DOCKET NO: 91016-434826[CCR6 (POI) Binder: SEQ ID NO: 49; Fc Region: SEQ ID NO: 50; TfR1 Binder: SEQ ID NO: 51]pLS35-CCR6 HC-Hole Fc-VHHA (SEQ ID NO: 52) MRMQLLLLIALSLALVTNSTSEVQLVESGGGLVQPGGSLRLSCAASGFSFSDYYMYWVRQAP GKGLEWVSYITTGGRTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTSPLRGA WFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGGVD GDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDEL TKNQVSLSCA VKGFYPSDIA VEWESNGQPENN YKTTPP VLDSDGSFFL VSKL TV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGSGGSEVQLVESGGGVVQ PGGSLKLSCVASGTDFSINFIRWYRQAPGKQREFVAGFTATGNTNYADSMKGRFTISRDNTKNA VYLQIDSLKPEDTAVYYCYMLDKWGQGTQVTVSrSEQ ID NO: 52][CCR6 (POI) Binder: SEQ ID NO: 53; Fc Region: SEQ ID NO: 54; TfR1 Binder: SEQ ID NO: 55] pLS38-CCR6_HC-Fc-TfRl VHHA12+5 (SEQ ID NO: 56) MRMQLLLLIALSLALVTNSTSEVQLVESGGGLVQPGGSLRLSCAASGFSFSDYYMYWVRQAP GKGLEWVSYITTGGRTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTSPLRGA WFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGGVD GDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGLYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGSGGSEVQLVESGGGVVQPGGSLKLSCVASGTDFSINFIRWYRQAPGKQREFVAGITATGNTNYADSMKGRFTISRDNTKNAVYLQIDSLKPEDTAVYYCAMLDKWGQGTQVTVSS [SEQ ID NO: 56][CCR6 (POI) Binder: SEQ ID NO: 57; Fc Region: SEQ ID NO: 58; TfR1 Binder: SEQ ID NO: 59] pLS39-CCR6_HC-Fc-TIRl VHHA12+7 (SEQ ID NO: 60) _ MRMQLLLLIALSLALVTNSTSEVQLVESGGGLVQPGGSLRLSCAASGFSFSDYYMYWVRQAP GKGLEWVSYITTGGRTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTSPLRGA WFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGGVD GDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDEL TKNQVSL TCL VKGL YPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFL YSKL TV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGSGGSEVQLVESGGGVVQDOCKET NO: 91016-434826PGGSLKLSCVASGTDFSINFIRWYRQAPGKQREFVAGITATGNTNYADSMKGRFTISRDNTKNAVYLQIDSLKPEDTAVYYCYMADKWGQGTQVTVSS [SEQ ID NO: 60][CCR6 (POI) Binder: SEQ ID NO: 61; Fc Region: SEQ ID NO: 62; TfR1 Binder: SEQ ID NO: 63] pJD47-VHHA-VHHA-Hole Fc (SEQ ID NO: 64)M RMQLLLLIALSLALVTNSTSEVQLVESGGGWQPGGSLKLSCVASGTDFSINFIRWYRQAPGK QREFVAGFTATGNTNYADSMKGRFTISRDNTKNAVYLQIDSLKPEDTAVYYCYMLDKWGQGTQVTVSSGGGGSSGGSGGGGSEVQLVESGGGVVQPGGSLKLSCVASGTDFSINFIRWYRQAPGKQREFVAGFTATGNTNYADSMKGRFTISRDNTKNAVYLQIDSLKPEDTAVYYCYMLDKWGQGTQVTVSSGTGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDEL TKNQ VSLSCA VKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDG SFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK[SEQ ID NO: 64][TfRI Binder: SEQ ID NO: 65; Fc Region: SEQ ID NO: 66]Anti-CCR6_B368-G29A_HC (SEQ ID NO: 67) EVQLVESGGGLVQPGGSLRLSCAASGFSFSDYYMYWVRQAPGKGLEWVSYITTGGR TYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTSPLRGAWFAYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQS SGLYSLS SVVTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCAnti-CCR6_B3G8-G29A_LC (SEQ ID NO: 68) DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNANTYLEWYQQRPGQSPRLLIYKVSNR FSGVPDRFSGSGSGTDFTLK1SRVEAEDVGVYYCFQGTYLPLTFGQGTKLE1KRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC VHHA (SEQ ID NO: 1) EVOLVESGGGVVOPGGSLKLSCVASGT / IFS’ / AFIRWYRQAPGKQREFVAGFFITGTVT NYADSMI< GRFTISRDNTI< NAVYLOIDSLI< PEDTAVYYC EWJIFWGOGTOVTVSSTf(SEQ IDNO: 7) VPDKTVRWCAVSEHEATKCQSFRDHMKSVIPSDGPSVACVKKASYLDCIRAIAANEA DAVTLDAGLVYDAYLAPNNLKPVVAEFYGSKEDPQTFYYAVAVVKKDSGFQMNQL RGKKSCHTGLGRSAGWNIPIGLLYCDLPEPRKPLEKAVANFFSGSCAPCADGTDFPQL CQLCPGCGCSTLNQYFGYSGAFKCLKDGAGDVAFVKHSTIFENLANKADRDQYELLC LDNTRKPVDEYKDCHLAQVPSHTVVARSMGGKEDLIWELLNQAQEHFGKDKSKEFQ LF S SPHGKDLLFKD S AHGFLKVPPRMD AKMYLGYEYVT A I RN LREGTC PE APTDECK PVKWCALSHHERLKCDEWSVNSVGKIECVSAETTEDCIAKIMNGEADAMSLDGGFV YIAGKCGLVPVLAENYNKSDNCEDTPEAGYFAIAVVKKSASDLTWDNLKGKKSCHTAVGRTAGWNIPMGLLYNKINHCRFDEFFSEGCAPGSKKDSSLCKLCMGSGLNLCEPNDOCKET NO: 91016-434826 NKEGYYGYTGAFRCLVEKGDVAFVKHQTVPQNTGGKNPDPWAKNLNEKDYELLCL DGTRKPVEEYANCHLARAPNHAVVTRKDKEACVHKILRQQQHLFGSNVTDCSGNFC LFRSETKDLLFRDDTVCLAKLHDRNTYEKYLGEEYVKAVGNLRKCSTSSLLEACTFR RP VHHA12 (SEQ ID NO:2) EVOLVESGGGVVOPGGSLI< LSC: VASGT / )FS7NFIRWYROAPGI< OREFVAG / F4TGAT NYADSMKGRFTISRDNTKNAVYLOIDSLKPEDTAVYYCFA / FDA'WGOGTQVTVSS VHHA5 (SEQ ID NO: 5) EVQLVESGGGVVQPGGSLKLSCVASGTDFSINFIRWYRQAPGKQREFVAGFTATGNTNYADSMKGRFTISRDNTKNAVYLQIDSLKPEDTAVYYCAMLDKWGQGTQVTVSS VHHA12+5 (SEQ ID NO: 3) EVQLVESGGGVVQPGGSLKLSCVASGTDFSINFIRWYRQAPGKQREFVAGITATGNTNYADSMKGRFTISRDNTKNAVYLQIDSLKPEDTAVYYCAMLDKWGQGTQVTVSSVHHA7 (SEQ ID NO: 6) EVQLVESGGGVVQPGGSLKLSCVASGTDFSINFIRWYRQAPGKQREFVAGFTATGNTNYADSMKGRFTISRDNTKNAVYLQIDSLKPEDTAVYYCYMADKWGQGTQVTVSS VHHA12+7 (SEQ ID NO: 4) EVQLVESGGGWQPGGSLKLSCVASGTDFSINFIRWYRQAPGKQREFVAG^TATGNTNYADSM KGRFTISRDNTKNAVYLQIDSLKPEDTAVYYCYMAD / < WGQGTQVTVSSG9 (SEQ ID NO: 8) SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPD RFSGSGSGNTASLTITGAQAEDEADYYCAAWDDSLSGPVFGGGTKVTVLGGGGGSG GGGSGGGGSQVQLVESGGGLVEPGGSLRLSCAASGFTFSNYAINWVRQAPGKGLEW VANIHHDGNGKYYVDSVEGRFTISRDNAKNSLYLQMDSLRAEDTAIYYCARDGYGG YLDLWGQGTLVT VS SH7 (SEQ ID NO: 9) SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVMYGRNERPSGV PDRFSGSKSGTSASLAISGLQPEDEANYYCAGWDDSLTGPVFGGGTKLTVLGGGGGS GGGGSGGGGSQVQLQESGGGVVQPGRSLRLSCAASRFTFSSYAMHWVRQAPGKGLE WVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLSG YGDYPD YWGQGTLVTVS SN5 (SEQ ID NO: 10)DOCKET NO: 91016-434826SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVMYGRNERPSGV PDRFSGSKSGTSASLAISGLQPEDEANYYCAGWDDSLTGPVFGGGTKLTVLGGGGGS GGGGSGGGGSQVQLQESGGGVVQPGRSLRLSCAASRFTFNNYAMHWVRQAPGKGL EWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLS GYGD YPD YWGQGTLVTVS SLinker (SEQ ID NO: 11)GGSGGSGTDKTHTCLinker (SEQ ID NO: 12)GTGGDKTHTCPPCEGFR conjugated with drug MC-MMAFHC: (Cetuximab Fab) MYRMQLLSCIALSLALVTNSQVQLKQSGPGLVQPSQSLSITCTVSGFSL7NYGVHWVRQSPG KG LE WLGVII / I / SGGA / TDYNTPFTS RLS 1 N KD N S KSQ VF F KM N SLQS N DTAI YYC ARAL7YYDY FFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGSVD GDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWOOGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGSGGSEVOl-VECGGGVVOPG GSLKLSCVASGTDFS / / VFIRWYROAPGKOREFVAG / TATGNTNYADSMKGRFTISRDNTKNAVYL QIDCLKPEDTAVYYCYMADKWGQGTQVTVSS (SEQ ID NO:82)LC: (Cetuximab + huIgG VLk constant region) MYRMQLLSCIALSLALVTNSDILLTQSPVILSVSPGERVSFSCRASQS / GTA / / HWYQQRTNGSP RLLIKYASES / SGIPSRFSGSGSGTDFTLSINSVESEDIADYYCOO / V / V / VI / VP7TFGAGTKLELKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWCVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 83)[EGFR (POI) Binder HC: SEQ ID NO: 84; Fc Region: SEQ ID NO: 81; TfR1 Binder: SEQ ID NO: 85] [EGFR (POI) Binder LC: SEQ ID NO: 86; LC Constant Region: SEQ ID NO: 87]ICAM1 conjugated with drug MC-MMAF (See WO2012022734A2; KD:0.98 nM)HC:MYRMQLLSCIALSLALVTNSQVQLQQSGPGLVKPSETLSLTCTVSGYSISSGYFVI / GWIRQPPGKGLEWIG SIYQSGSTYYA / PSZ. KSRVTISLDTSKNQFSLKLSSVTAADTAVYYCAROGYCSGGSCYPFOYWGQGTTVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCGGSGSGGSGSGVDGD / < THTCPPCPAPE£ / . RGPS\ / F1. F PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKARPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNDOCKET NO: 91016-434826GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSG G S G G SEVQLVECGGGVVQPGGSLKLSCVASGTDFS / A / FIRWYRQAPGKQREFVAG / TATG / VTNYADSMKG RFTISRDNTKNAVYLQIDCLKPEDTAVYYCYMADKWGQGTQVTVSS (SEQ ID NO: 88)LC: ( huIgG VLY) MYRMQLLSCIALSLALVTNSQSVLTQPPSASGTPGQRVTISCSGSSSA / / GSA / TW / WYQRLPGAAPQLLIY MVDQPPSGIPDRFSGSKSGTSGSLVISGLQSEDEADYYCASVI / DDSLA / GRI / FGGGTKLTVLGQPKANPTV TLFPPSSEELQANKATLVCLISDFYPGAVTVAWCADGSPVKAGVETTKPSKQSNNKYAA SSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 89)[ICAM1 (POI) Binder HC: SEQ ID NO: 90; Fc Region: SEQ ID NO: 81; TfR1 Binder: SEQ ID NO: 85] [ICAM1 (POI) Binder LC: SEQ ID NO: 91; LC Constant Region: SEQ ID NO: 92]PVR conjugated with drug MC-MMAF (See W02021070181A1; KD: 5.12nM)HC:MYRMQLLSCIALSLALVTNSQVQLVQSGAEVKKPGASVKVSCKATGYTFSA / YW / / FWVRQAPGQGLEWI GF / FPGSGR / / VFA / E / CF / CGRVTFTADTSISTTYMELSRLRSDDTAVYYCAR7K / YG / VSFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCGGSGSGGSGSGVDGD / CmrCPPCPAPELLRGPSV'FLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWL NGKEYKCKVSNKARPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGS GGSEVQLVECGGGVVQPGGSLKLSCVASGTDFS / / VFIRWYRQAPGKQREFVAG / 7ATG / VTNYADSMKGRF TISRDNTKNAVYLQIDCLKPEDTAVYYCY / VMDKWGQGTQVTVSS (SEQ ID NO: 93)LC: (hu VLk constant region) MYRMQLLSCIALSLALVTNSDIQMTQSPSSLSASVGDRVTITCRASQDVGTAVI / WYQQKPGKAPKLLIY VI / ASS / WEGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYSftYP / . TFGQGTKLEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWC VDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 94)[PVR (POI) Binder HC: SEQ ID NO: 95; Fc Region: SEQ ID NO: 81; TfR1 Binder: SEQ ID NO: 85] [PVR (POI) Binder LC: SEQ ID NO: 96; LC Constant Region: SEQ ID NO: 97]MCT1 conjugated with drug MC-MMAF (See W02019136300A2)HC:MYRMQLLSCIALSLALVTNSAVTLDESGGGLQTPGGGLSLVCKASGFSFSSRGMFWVRQAPGKGLEYVA G I DNDGGYPN YGSAVKG RATI SRDN RQSTVRLQLN N LRAD DTGTYYCAKGAYGGGVI / YAASS / DAWG H GTEVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCGGSGSGGSGSGVDGDKTHTCPPCPAPELLRG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKARPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEDOCKET NO: 91016-434826WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSS G GSGGSGGSEVQLVECGGGVVQPGGSLKLSCVASGTOFS / / VFIRWYRQAPGKQREFVAG / TATG / VTNYAD SMKGRFTISRDNTKNAVYLQIDCLKPEDTAVYYCY / VMDKWGQGTQVTVSS (SEQ ID NO: 98)LC: (huIgG VLY) MYRMQLLSCIALSLALVTNSALTQPSSVSANLGEAVKITCSGGVGQVI / YGWYQQKAPGSAPVTVIYO / V7K RPSNIPSRFSGSASGSTATLTITGVRAEDEAVYFCAA / TYSOGA / OAPFGAGTTLTVLGQPKANPTVTLFPPS SEELQANI< ATLVCL1SDFYPGAVTVAWCADGSPVKAGVETTI< PSKQSNNI< YAASSYLS LTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 99)[MCT1 (POI) Binder HC: SEQ ID NO: 100; Fc Region: SEQ ID NO: 81; TfR1 Binder: SEQ ID NO: 85] [MCT1 (POI) Binder LC: SEQ ID NO: 101; LC Constant Region: SEQ ID NO: 102]LAT1 conjugated with drug MC-MMAF (See US20150147278A1)HC:MYRMQLLSCIALSLALVTNSQLQLKESGPGLVQPSQTLSLTCTVSGFSLPTSSI / SWIRQPPGKGLEWMGV / I4 / S / VG / VTDYSSA / KSRLSISRDTSKSQVFLKMNSLQTEDTARYFCAR / VFR / VDPGI / / V7OAWGQGASVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCGGSGSGGSGSGVDGDKTHTCPPCPAPELLRGPSI / FLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWL NGKEYKCKVSNKARPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGS GGSEVQLVECGGGVVQPGGSLKLSCVASGTOFS / / VFIRWYRQAPGKQREFVAG / TATG / VTNYADSMKGRF TISRDNTKNAVYLQIDCLKPEDTAVYYCYMADKWGQGTQVTVSS (SEQ ID NO: 103)LC: (mouse IgG VLk) MYRMOLLSCIALSLALVTNSNIVMTOSPRSMSLSGGDRVTMNC / MAPAF(7A7VE4W YOORPGOSPKLLISE4, SA7? ATGVPGRFTGSGSGTDFTLTISSVOAEDAAFYYCOfl7Y AAPFTFGGGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWCI DGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKS FNR. NEC (SEQ ID NO: 104)[LAT1 (POI) Binder HC: SEQ ID NO: 105; Fc Region: SEQ ID NO: 81; TfR1 Binder: SEQ ID NO: 85] [LAT1 (POI) Binder LC: SEQ ID NO: 106; LC Constant Region: SEQ ID NO: 107]BST2 conjugated with drug MC-MMAF (See PM ID: 10444002)HC:MYRMQLLSCIALSLALVTNSQVQLQQSGAELARPGASVKLSCKASGYTFTPYWMQWVKQRPGQGLEW IGS / FPGOGDTRYSQKFKGKATLTADKSSSTAYMQLSILAFEDSAVYYCARGLRRGGYYFDYWGQGTTLTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCGGSGSGGSGSGVDGDKTHTCPPCPAPELLRGPSl / FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDDOCKET NO: 91016-434826WLNGKEYKCKVSNKARPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSG GSGG SEVQLVECGGGVVQPGGSLKLSCVASGT0F5 / / VFIRWYRQAPGKQREFVAG / TATGA / TNYADSMKG RFTISRDNTKNAVYLQIDCLKPEDTAVYYCYMAOKWGQGTQVTVSS (SEQ ID NO: 108)LC: (hu VLk constant region) MYRMQLLSCIALSLALVTNSDIVMTQSHKFMSTSVGDRVSITCKASQDVTVrAVAWYQQKPGQSPKLLIY SAS / yffYTGVPDRITGSGSGTDFTFTISSVQAEDLALYYCQQHmPFTFGSGTKLEIKRTVAAPSVFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWCVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 109)[BST2 (POI) Binder HC: SEQ ID NO: 110; Fc Region: SEQ ID NO: 81; TfR1 Binder: SEQ ID NO: 85] [BST2 (POI) Binder LC: SEQ ID NO: 111; LC Constant Region: SEQ ID NO: 112]CD20 Ab 1 sequenceHC MYRMQLLSCIALSLALVTNSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEW I G AIYPGNGDTSYNQKFKG K ATLTAD KSSSTAYM QLSSLTSE DSAVYYCARSTYYGGDWYFNVWG AGTT VTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCGGSGSGGSGSGVDGD / CTHTCPPCPAPELLRGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLH QDWLNGKEYKCKVSNKARPAPIEKTISKAKGQ. PREPQ. VYTLPPSREEMTKNQ. VSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 130)LC MYRMQLLSCIALSLALVTNSQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATS NLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWCVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 131)[CD20 (POI) Binder HC: SEQ ID NO: 124; Fc Region: SEQ ID NO: 81][CD20 (POI) Binder LC: SEQ ID NO: 125; LC Constant Region: SEQ ID NO: 112]EPHA4 (Ephrin Type-A Receptor 4) Antibody [W02016019280A1; Kd 83nM] - conjugated with UAMC3203, a ferroptosis inhibitorHC MYRMQLLSCIALSLALVTNSQVQLQQSGAEVKKPGSSVKVSCKASGYTFTGYYMHWVRQAPGQGLEW MGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCATAPMVCSSTSCYLRGFDYWGQ GTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLY TLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGC / CPC / CTV'PFV’SSI / F / FPPK'P / CDV'LT / F / . TP / d / TCVVVDISKDDPEVQFSWFVDDVEVHTAQ. TQPREEQ. FNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPADOCKET NO: 91016-434826PIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNT NGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 132)LC MYRMQLLSCIALSLALVTNSDVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQL LIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPITFGQGTRLEIKRADAAPTVSIFP PSSEQLTSGGASVVCFLNNFYPKDINVKWCIDGSERQNGVLNSWTDQDSKDSTYSMSS TLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 133)[EPHA4 (POI) Binder HC: SEQ ID NO: 126; Fc Region: SEQ ID NO: 127][EPHA4 (POI) Binder LC: SEQ ID NO: 128; LC Constant Region: SEQ ID NO: 104]CD90 (Thy-1 Cell Surface Antigen) Antibody [CD90 Abl from CN111320692B] - can be conjugated with UAMC3203, a ferroptosis inhibitor MYRMQLLSCIALSLALVTNSDIVMTQSPDSLAVSLGERATINCKSSQSVLSSSKNKNYLAWYQQKPGQPP KLLIYWASTRQSGVPARFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSIPVTFGQGTKVDIKAAAEQKG GGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISG SGGSTYYADSVKGRFTI SRD N SKNTLYLQM NSLRAEDTAVYYCARGARMDVWGQGTLVTVSSG SSG GS GGSGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQ. FNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTC MITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNH HTEKSLSHSPGK (SEQ ID NO: 134)[CD90 (POI) Binder HC: SEQ ID NO: 129; Fc Region: SEQ ID NO: 127]Tropl TransTAC vl.lHC QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQWVRQAPGQGLEWMGWINTHSGVPKYAEDFKG RVTISADTSTSTAYLQLSSLKSEDTAVYYCARSGFGSSYWYFDVWGQGTLVTVSSCSTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGSGGSEVQLVESGGGVVOPGGSLKLSCV ASGTDFSINFIRWYRQAPGKQREFVAGITATGNTNYADSMKGRFTISRDNTKNAVYLQ ID SLKPEDT AVYYC AMLDKWGQGTQ VT VS S(SEQ ID NO: 157)LC DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDF TLTISSLQPEDFAVYYCQQHYITPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWCVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 158)DOCKET NO: 91016-434826[TROP2 (POI) Binder HC: SEQ ID NO: 159; TfRl binder: SEQ ID NO: 3]Tropl TransTAC vl.2HC Knob QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQWVRQAPGQGLEWMGWINTHSGVPKYAEDFKG RVTISADTSTSTAYLQLSSLKSEDTAVYYCARSGFGSSYWYFDVWGQGTLVTVSSCSTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNAYTQKSLSLSPGKGSSHHHHHH (SEQ ID NO: 160)[TROP2 (POI) Binder HC: SEQ ID NO: 161]HC C QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQWVRQAPGQGLEWMGWINTHSGVPKYAEDFKG RVTISADTSTSTAYLQLSSLKSEDTAVYYCARSGFGSSYWYFDVWGQGTLVTVSSCSTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGSGGSEVQLVESGGGVVQPGGSLKLSC VASGTDFSINFIRWYRQAPGKQREFVAGITATGNTNYADSMKGRFTISRDNTKNAVYL QIDSLKPEDTAVYYCYMLDKWGQGTQVTVSS LC DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDF TLTISSLQPEDFAVYYCQQHYITPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWCVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 158)POI binder is in Bold. Fc Region is italicized. TfRl binder is underlined. Cl) Rs areunderlined, bolded and italicized. VLk or VLY. are in Times New Roman text.POIB

[0143] A “protein of interest (POI) binder (POIB)” is a protein (or peptide or polypeptide) or other molecule that a cell-surface protein of interest binds (e g., a part of a natural ligand for the POI or an antibody or antibody fragment (e.g., an scFv)). In embodiments, the POIB is an antibody or antibody fragment. In embodiments, the antibody fragment is an scFv, Fab, singledomain antibody, nanobody, monobody, DARPin or affibody. In embodiments, the POIB is anDOCKET NO: 91016-434826scFv. Antibody fragments and other molecules can be used. In embodiments, the POTB has the amino acid sequences as shown below.

[0144] A “POIB means” or “means for binding a protein of interest (POI)” is a protein (or peptide or polypeptide) or other molecule that the cell-surface protein of interest binds (e.g., a part of a natural ligand for the POI or an antibody or antibody fragment (e.g., an scFv)). In embodiments, the POIB means is an antibody or antibody fragment. In embodiments, the antibody fragment can be an scFv, Fab, single-domain antibody, nanobody, monobody, DARPin or affibody. In embodiments, the POIB means is an scFv. Antibody fragments and other molecules can be used.

[0145] The POIB can specifically bind the extracellular domain of an integral membrane protein on the surface of a target cell. Exemplary POI can be integral membrane proteins, such as EGFR, CD20, CCR6, MCT1, LAT1, ICAM1, BST2, and PVR. In embodiments, the POI can be integral membrane proteins, such as HER2, CD30, CD22, CD33, CD79b, Nectin-4, Trop-2, BCMA, CD19, HER3, and CD25. In embodiments, the POI is selected from the group consisting of: EGFR, CD20, CCR6, MCT1, ICAM1, BST2, PVR, HER2, CD30, CD22, CD33, CD79b, Nectin-4, Trop-2, BCMA, DE19, HER3, and CD25. These POIs have historically been deemed non-targetable, or undruggable, therapeutic proteins because they are considered difficult or nearly impossible to target with drugs. This has been attributed to their large, complex structure, function, and / or location within the cell, making it difficult to develop small molecule inhibitors or other therapeutic agents that can specifically interact with them and alter their activity using conventional drug design strategies. Thus, targeting such undruggable POIs according to the invention described herein is a welcomed opportunity for treatment of human disease.

[0146] POIs are described below.

[0147] For example, EGFR (epidermal growth factor receptor; Gene ID: 1956) comprises amino acids from NCBI Reference Sequence accession no. NP_005219 (1210 amino acids)(SEQ ID NO: 135):1 MRPSGTAGAA LLALLAALCP ASRALEEKKV CQGTSNKLTQ LGTFEDHFLS LQRMFNNCEV 61 VLGNLEITYV QRNYDLSFLK TIQEVAGYVL IALNTVERIP LENLQIIRGN MYYENSYALA 121 VLSNYDANKT GLKELPMRNL QEILHGAVRF SNNPALCNVE SIQWRDIVSS DFLSNMSMDF 181 QNHLGSCQKC DPSCPNGSCW GAGEENCQKL TKIICAQQCS GRCRGKSPSD CCHNQCAAGC 241 TGPRESDCLV CRKFRDEATC KDTCPPLMLY NPTTYQMDVN PEGKYSFGAT CVKKCPRNYV 301 VTDHGSCVRA CGADSYEMEE DGVRKCKKCE GPCRKVCNGI GIGEFKDSLS INATNIKHFK 361 NCTSTSGDLH ILPVAFRGDS FTHTPPLDPQ ELDILKTVKE ITGFLLIQAW PENRTDLHAF 421 ENLEIIRGRT KQHGQFSLAV VSLNITSLGL RSLKEISDGD VIISGNKNLC YANTINWKKL 481 FGTSGQKTKI ISNRGENSCK ATGQVCHALC SPEGCWGPEP RDCVSCRNVS RGRECVDKCNDOCKET NO: 91016-434826541 LLEGEPREFV ENSECIQCHP ECLPQAMNIT CTGRGPDNCI QCAHYIDGPH CVKTCPAGVM 601 GENNTLVWKY ADAGHVCHLC HPNCTYGCTG PGLEGCPTNG PKIPSIATGM VGALLLLLVV 661 ALGIGLFMRR RHIVRKRTLR RLLQERELVE PLTPSGEAPN QALLRILKET EFKKIKVLGS 721 GAFGTVYKGL WIPEGEKVKI PVAIKELREA TSPKANKEIL DEAYVMASVD NPHVCRLLGI 781 CLTSTVQLIT QLMPFGCLLD YVREHKDNIG SQYLLNWCVQ IAKGMNYLED RRLVHRDLAA 841 RNVLVKTPQH VKITDFGLAK LLGAEEKEYH AEGGKVPIKW MALESILHRI YTHQSDVWSY 901 GVTVWELMTF GSKPYDGIPA SEISSILEKG ERLPQPPICT IDVYMIMVKC WMIDADSRPK 961 FRELIIEFSK MARDPQRYLV IQGDERMHLP SPTDSNFYRA LMDEEDMDDV VDADEYLIPQ 1021 QGFFSSPSTS RTPLLSSLSA TSNNSTVACI DRNGLQSCPI KEDSFLQRYS SDPTGALTED 1081 SIDDTFLPVP EYINQSVPKR PAGSVQNPVY HNQPLNPAPS RDPHYQDPHS TAVGNPEYLN 1141 TVQPTCVNST FDSPAHWAQK GSHQISLDNP DYQQDFFPKE AKPNGIFKGS TAENAEYLRV 1201 APQSSEFIGA

[0148] For example, CD20 (also referred to as MS4A1 membrane spanning 4-domains Al; Gene ID: 931) comprises amino acids from NCBI Reference Sequence accession no. NP 068769 (297 amino acids) (SEQ ID NO: 136):1 MTTPRNSVNG TFPAEPMKGP IAMQSGPKPL FRRMSSLVGP TQSFFMRESK TLGAVQIMNG 61 LFHIALGGLL MIPAGIYAPI CVTVWYPLWG GIMYIISGSL LAATEKNSRK CLVKGKMIMN 121 SLSLFAAISG MILSIMDILN IKISHFLKME SLNFIRAHTP YINIYNCEPA NPSEKNSPST 181 QYCYSIQSLF LGILSVMLIF AFFQELVIAG IVENEWKRTC SRPKSNIVLL SAEEKKEQTI 241 EIKEEVVGLT ETSSQPKNEE DIEIIPIQEE EEEETETNFP EPPQDQESSP IENDSSP

[0149] For example, CCR6 (C-C motif chemokine receptor 6; Gene ID: 1235) comprises amino acids from NCBI Reference Sequence accession no. NP 001381511 (374 amino acids) (SEQ ID NO: 137):1 MSGESMNFSD VFDSSEDYFV SVNTSYYSVD SEMLLCSLQE VRQFSRLFVP IAYSLICVFG 61 LLGNILVVIT FAFYKKARSM TDVYLLNMAI ADILFVLTLP FWAVSHATGA WVFSNATCKL 121 LKGIYAINFN CGMLLLTCIS MDRYIAIVQA TKSFRLRSRT LPRSKIICLV VWGLSVIISS 181 STFVFNQKYN TQGSDVCEPK YQTVSEPIRW KLLMLGLELL FGFFIPLMFM IFCYTFIVKT 241 LVQAQNSKRH KAIRVIIAVV LVFLACQIPH NMVLLVTAAN LGKMNRSCQS EKLIGYTKTV 301 TEVLAFLHCC LNPVLYAFIG QKFRNYFLKI LKDLWCVRRK YKSSGFSCAG RYSENISRQT 361 SETADNDNAS SFTM

[0150] For example, MCT1 (monocarboxylate transporter 1, also referred to as SLC16A1, solute carrier family 16 member 1; Gene ID: 6566) comprises amino acids from NCBI Reference Sequence accession no. NP_001159968 (500 amino acids) (SEQ ID NO: 138):1 MPPAVGGPVG YTPPDGGWGW AVVIGAFISI GFSYAFPKSI TVFFKEIEGI FHATTSEVSW 61 ISSIMLAVMY GGGPISSILV NKYGSRIVMI VGGCLSGCGL IAASFCNTVQ QLYVCIGVIG 121 GLGLAFNLNP ALTMIGKYFY KRRPLANGLA MAGSPVFLCT LAPLNQVFFG IFGWRGSFLI 181 LGGLLLNCCV AGALMRPIGP KPTKAGKDKS KASLEKAGKS GVKKDLHDAN TDLIGRHPKQ 241 EKRSVFQTIN QFLDLTLFTH RGFLLYLSGN VIMFFGLFAP LVFLSSYGKS QHYSSEKSAF 301 LLSILAFVDM VARPSMGLVA NTKPIRPRIQ YFFAASVVAN GVCHMLAPLS TTYVGFCVYA 361 GFFGFAFGWL SSVLFETLMD LVGPQRFSSA VGLVTIVECC PVLLGPPLLG RLNDMYGDYK 421 YTYWACGVVL IISGIYLFIG MGINYRLLAK EQKANEQKKE SKEEETSIDV AGKPNEVTKA 481 AESPDQKDTD GGPKEEESPV

[0151] For example, ICAM1 (intercellular adhesion molecule 1; Gene ID: 3383) comprises amino acids from NCBI Reference Sequence accession no. NP_ 000192 (532 amino acids) (SEQ ID NO: 139):DOCKET NO: 91016-4348261 MAPSSPRPAL PALLVLLGAL FPGPGNAQTS VSPSKVILPR GGSVLVTCST SCDQPKLLGI 61 ETPLPKKELL LPGNNRKVYE LSNVQEDSQP MCYSNCPDGQ STAKTFLTVY WTPERVELAP 121 LPSWQPVGKN LTLRCQVEGG APRANLTVVL LRGEKELKRE PAVGEPAEVT TTVLVRRDHH 181 GANFSCRTEL DLRPQGLELF ENTSAPYQLQ TFVLPATPPQ LVSPRVLEVD TQGTVVCSLD 241 GLFPVSEAQV HLALGDQRLN PTVTYGNDSF SAKASVSVTA EDEGTQRLTC AVILGNQSQE 301 TLQTVTIYSF PAPNVILTKP EVSEGTEVTV KCEAHPRAKV TLNGVPAQPL GPRAQLLLKA 361 TPEDNGRSFS CSATLEVAGQ LIHKNQTREL RVLYGPRLDE RDCPGNWTWP ENSQQTPMCQ 421 AWGNPLPELK CLKDGTFPLP IGESVTVTRD LEGTYLCRAR STQGEVTRKV TVNVLSPRYE 481 IVIITVVAAA VIMGTAGLST YLYNRQRKIK KYRLQQAQKG TPMKPNTQAT PP

[0152] For example, BST2 (bone marrow stromal cell antigen 2; Gene ID: 684) comprises amino acids from NCBI Reference Sequence accession no. NP 004326 (180 amino acids) (SEQ ID NO: 140):1 MASTSYDYCR VPMEDGDKRC KLLLGIGILV LLIIVILGVP LIIFTIKANS EACRDGLRAV 61 ECRNVTHLL QQELTEAQKG FQDVEAQAAT CNHTVMALMA SLDAEKAQGQ KKVEELEGEI 121 TTLNHKLQDA SAEVERLRRE NQVLSVRIAD KKYYPSSQDS SSAAAPQLLI VLLGLSALLQ

[0153] For example, PVR (PVR cell adhesion molecule, also referred to as CD155; Gene ID: 5817) comprises amino acids from NCBI Reference Sequence accession no. NP_006496 (417 amino acids) (SEQ ID NO: 141):1 MARAMAAAWP LLLVALLVLS WPPPGTGDVV VQAPTQVPGF LGDSVTLPCY LQVPNMEVTH 61 VSQLTWARHG ESGSMAVFHQ TQGPSYSESK RLEFVAARLG AELRNASLRM FGLRVEDEGN 121 YTCLFVTFPQ GSRSVDIWLR VLAKPQNTAE VQKVQLTGEP VPMARCVSTG GRPPAQITWH 181 SDLGGMPNTS QVPGFLSGTV TVTSLWILVP SSQVDGKNVT CKVEHESFEK PQLLTVNLTV 241 YYPPEVSISG YDNNWYLGQN EATLTCDARS NPEPTGYNWS TTMGPLPPFA VAQGAQLLIR 301 PVDKPINTTL ICNVTNALGA RQAELTVQVK EGPPSEHSGM SRNAIIFLVL GILVFLILLG 361 IGIYFYWSKC SREVLWHCHL CPSSTEHASA SANGHVSYSA VSRENSSSQD PQTEGTR

[0154] For example, HER2 (Human Epidermal Growth Factor Receptor 2; also referred to as ERBB2, erb-b2 receptor tyrosine kinase 2; Gene ID: 2064) comprises amino acids from NCBI Reference Sequence accession no. NP_004439 (1255 amino acids); CD30 (also referred to as TNFRSF8, TNF receptor superfamily member 8; Gene ID: 943) comprises amino acids from NCBI Reference Sequence accession no. NP_001234 (595 amino acids); CD22 (B-cell receptor CD22 molecule; Gene ID: 933) comprises amino acids from NCBI Reference Sequence accession no. NP_001762 (847 amino acids); CD33 (myeloid cell surface antigen CD33 molecule; Gene ID: 945) comprises amino acids from NCBI Reference Sequence accession no. NP 001763 (364 amino acids); CD79b (also referred to as B-cell antigen receptor complex-associated protein beta chain; Gene ID: 974) comprises amino acids from NCBI Reference Sequence accession no. NP_001035022 (230 amino acids); Nectin-4 (nectin cell adhesion molecule 4; Gene ID: 81607) comprises amino acids from NCBI Reference Sequence accession no. NP_112178 (510 amino acids); Trop-2 (also referred to as TACSTD2 tumor associated calcium signal transducer 2; Gene ID: 4070) comprises amino acids from NCBI ReferenceDOCKET NO: 91016-434826Sequence accession no. NP_002344 (323 amino acids); BCMA (B-cell Maturation Antigen; also referred to as TNFRSF17, TNF receptor superfamily member 17; Gene ID: 608) comprises amino acids from NCBI Reference Sequence accession no. NP 001183 (184 amino acids); CD19 (B-lymphocyte antigen CD19; Gene ID: 930) comprises amino acids from NCBI Reference Sequence accession no. NP_001171569 (557 amino acids); HER3 (Human Epidermal Growth Factor Receptor 3; also referred to as ERBB3, erb-b2 receptor tyrosine kinase 3; Gene ID: 2065) comprises amino acids from NCBI Reference Sequence accession no.NP_001005915 (183 amino acids); and CD25 (also referred to as IL2RA, interleukin 2 receptor subunit alpha; Gene ID: 3559) comprises amino acids from NCBI Reference Sequence accession no. NP_000408 (272 amino acids).

[0155] The skilled artisan understands that integral membrane proteins described herein, which can be POIs according to the invention, can be targeted by POIBs commercially available, for example those listed in Table 3 herein:Table 3. POIBsTarget POIBCD30 Brentuximab vedotinHER2 Trastuzumab emtansineCD22 Inotuzumab ozogamicinCD33 Gemtuzumab ozogamicinCD79b Polatuzumab vedotin-piiqNectin-4 Enfortumab vedotin-ejfvSacituzumab govitecan-hziyTrop-2Datopotamab deruxtecanBCMA Belantamab mafodotinCD19 Loncastuximab tesirine-lpylHER3 Patritumab deruxtecanCD25 Camidanlumab tesirineCD20 rituximab-vcMMAETRB

[0156] A “transferrin receptor binder (TRB)” is a protein (or peptide or polypeptide) or other molecule that binds to TfR on a cell surface. Herein, TfR can refer to transferrin receptor 1 (TfRl) or transferrin receptor 2 (TfR2). In embodiments, TfR refers to transferrin receptor 1. In embodiments, the TRB is an antibody or antibody fragment that binds to transferrin receptor on a cell surface. In embodiments, the TRB is an antibody, antibody fragment or other molecule thatDOCKET NO: 91016-434826binds TfR Tn embodiments, the TRB means is an scFv, Fab, single-domain antibody, nanobody, monobody, DARPin or affibody. In embodiments TRB is non-competitive with endogenous TfR ligand (i.e., a non-competitive TfR binder). In embodiments TRB is competitive with endogenous TfR ligand (i.e., a competitive TfR binder). In embodiments, the TRB is a nanobody (also called a camelid antibody or single-domain antibody) that binds to TfR on a cell surface. In embodiments, the nanobody can be called a VHH. In embodiments, TRB has a variable heavy chain (VH) amino acid sequence (e.g., SEQ ID NO: 1), VHHA-12 (e.g., SEQ ID NO: 2), VHHA-12+5 (e.g., SEQ ID NO: 3), VHHA-12+7 (e.g., SEQ ID NO: 4), as shown in Table 2, or combinations thereof. In embodiments, the TRB means is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences shown in Table 2, for example, SEQ ID NO: 1, 2, 3, or 4.

[0157] A “transferrin receptor binder (TRB) means” or “means for binding TfR” (also called TfR herein) is a protein (or peptide or polypeptide) or other molecule that binds to TfR on a cell surface. In embodiments, the TRB means is an antibody or antibody fragment that binds to transferrin receptor on a cell surface. In embodiments, the TRB means is an antibody, antibody fragment or other molecule that can bind TfR. In embodiments, the TRB means is an scFv, Fab, single-domain antibody, nanobody, monobody, DARPin or affibody. In embodiments TRB means is non-competitive with endogenous TfR ligand (i.e., a non-competitive TfR binder means). In embodiments, the TRB means is competitive with endogenous TfR ligand (i.e., a competitive TfR binder means). In embodiments, the TRB means is a nanobody (also called a camelid antibody or single-domain antibody) that binds to TfR on a cell surface. In embodiments, the nanobody can be called a VHH. In embodiments, TRB means has a variable heavy chain (VH) amino acid sequence (e.g., SEQ ID NO: 1), VHHA-12 (e.g., SEQ ID NO: 2), VHHA-12+5 (e.g, SEQ ID NO: 3), VHHA-12+7 (e.g, SEQ ID NO: 4), as shown in Table 2, or combinations thereof. In embodiments, the TRB means is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences shown in Table 2, for example, SEQ ID NO: 1, 2, 3, or 4.

[0158] In embodiments, TRBs provided herein can also be referred to as “low-affinity”. In embodiments, the TRB is in a heterodimer FPDC provided herein (i.e, an FPDC with a single TRB) and has a KD of 1 x 102nM to 1 x 105nM, such as a KD of 1 X 102nM to 1 x 103nM, KD of 1 x 102nM to 1 x 104nM, KD of 1 X 103nM to 1 x 104nM, KD of 1 X 103nM to 1 x 1 O’ nM,DOCKET NO: 91016-434826KD of 1.2-1.3 x 102nM, or KD of greater than 2 x 103nM. In embodiments, the TRB is in a homodimer FPDC provided herein (i.e., an FPDC with two TRB) and has a KD of 1 x 10 nM to 1 x 105nM, such as a KD of 1 x 10 nM to 1 x 102nM, KD of 1 x 10 nM to 1 x 103nM, KD of 1 X 101nM to 1 x 104nM, KD of 1 x 102nM to 1 x 105nM, KD of 1 x 102nM to 1 x 103nM, KD of 1 x 102nM to 1 x 104nM, KD of 2-3 x 10 nM, or KD of greater than 2 x 103nM. In embodiments, a homodimer FPDC provided herein (i.e., an FPDC with two TRB) has a TRB comprising SEQ ID NO: 3 or SEQ ID NO: 4. In embodiments, a heterodimer FPDC provided herein (i.e., an FPDC with a single TRB) has a TRB comprising SEQ ID NO: 2; SEQ ID NO: 3; or SEQ ID NO:4.

[0159] In embodiments, low-affinity TRBs can be any molecule comprising VHHA-12+7 (SEQ ID NO: 4), VHHA-12+5 (SEQ ID NO: 3), and the like.

[0160] In embodiments, a TRB may not be a low-affinity TRB (e.g., is a high- or mediumaffinity TRB) but can be modified to reduce its binding affinity to TfR. For example, if the TRB is an antibody or antibody fragment, it may be possible to mutate specific regions of the antibody or antibody fragment (e.g., introduce an amino acid substitution within a CDR) to reduce binding affinity. In embodiments, the VHHA-12+7 (SEQ ID NO: 4) and VHHA-12+5 (SEQ ID NO: 3) TRBs are modified versions of VHHA.

[0161] In embodiments, the VHHA TRB, shown below, can be modified:EVOLVESGGGVVOPGGSLKLSCVAS E / NFIRWYROAPGKOREFVAGETHT VTN YAD SMKGRFTISRDNTKNA VYLQID SLKPEDTAVYYC MD WGQGTQ VT VS S * * * (111 amino acids; SEQ ID NO: 1). The italicized, bolded and underlined amino acid sections correspond to CDR1, CDR2, and CDR3, respectively. In embodiments, the shaded amino acids in CDR2 and CDR3 can be substituted for a neutral amino acid(s). In embodiments, the modifications can be one or more amino acid substitutions in CDR2. In embodiments, the modifications can be one or more amino acid substitutions in CDR3.

[0162] In embodiments, the nucleotide sequence that encodes VHHA, shown below, can be modified.

[0163] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTGCAGCCTGGGGGGTC TCTAAAACTCTCCTGCGTAGCCTCGGGAACGGACTTCAGTATCAATTTTATACGCTG GTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTCGTCGCAGGATTTACTGCGACTG GTAACACAAACTATGCAGACTCCATGAAGGGGCGATTCACCATCTCCAGAGACAAC ACCAAGAACGCGGTGTATCTGCAAATAGACAGCCTGAAACCTGAGGACACGGCCGTDOCKET NO: 91016-434826GTATTACTGCTATATGTTGGACAAGTGGGGCCAGGGGACCCAGGTCACAGTATCCTCC*** (SEQ ID NO: 69).

[0164] In embodiments, CDRs of the antibodies or antibody fragments of the TRB means can be modified. In embodiments, the following CDRs may be modified: GTDFSINF (SEQ ID NO: 70), FTATGNT (SEQ ID NO:71), YMLDK (SEQ ID NO: 72). In embodiments, the following CDRs can be a CDR1 comprising GTDFSINF (SEQ ID NO: 70), a CDR2 comprising ITATGNT (SEQ ID NO: 73), and a CDR3 comprising YMLDK (SEQ ID NO: 72). In embodiments, the following CDRs can be a CDR1 comprising GTDFSINF (SEQ ID NO: 70), a CDR2 comprising FTATGNT (SEQ ID NO: 71), and a CDR3 comprising AMLDK (SEQ ID NO: 74). In embodiments, the following CDRs can be a CDR1 comprising GTDFSINF (SEQ ID NO: 70), a CDR2 comprising FTATGNT (SEQ ID NO: 71), and a CDR3 comprising YMADK (SEQ ID NO: 75). In embodiments, these CDRs can be from a heavy chain. CDRs can be grafted to other antibody scaffold sequences to generate TRBs.

[0165] As shown herein, different TRBs have different binding affinities for transferrin receptor. In addition, as shown below in Table 4, dimerization, including the type of dimerization (e.g., homodimer, heterodimer) can affect binding affinity of a TRB within the context of a FPDC to transferrin receptor.

[0166] In embodiments, the TRB is in a heterodimer FPDC provided herein (i.e., an FPDC with a single TRB) and has a KD of 1 X 102nM to 1 x 1 O’ nM, such as a KD of 1 x 102nM to 1 x 103nM, KD of 1 x 102nM to 1 x 104nM, KD of 1 x 103nM to 1 x 104nM, KD of 1 x 103nM to 1 x 105nM, KD of 1.2-1.3 x 102nM, or KD of greater than 2 x 103nM. In embodiments, the TRB is in a homodimer FPDC provided herein (i.e., an FPDC with two TRB) and has a KD of 1 x 10 nM to 1 x 105nM, such as a KD of 1 x 10 nM to 1 x 102nM, KD of 1 x 10 nM to 1 x 103nM, KD of 1 x 10 nM to 1 x 104nM, KD of 1 X 102nM to 1 x 105nM, KD of 1 X 102nM to 1 x 103nM, KD of 1 x 102nM to 1 x 104nM, KD of 2-3 x 10 nM, or KD of greater than 2 x 103nM. In embodiments, a homodimer FPDC provided herein (i.e., an FPDC with two TRB) has a TRB comprising SEQ ID NO: 3or SEQ ID NO: 4. In embodiments, a heterodimer FPDC provided herein (i.e., an FPDC with a single TRB) has a TRB comprising SEQ ID NO: 2; SEQ ID NO: 3; or SEQ ID NO:4.

[0167] DOCKET NO: 91016-434826Table 4.

[0168] Shows KDfor various TRBs described hereinDimer format TRB KD(nM)Heterodimer (monovalent VHHA 1.073 x 10’1TRB) VHHA-5 2.697VHHA-7 2.588 x 10VHHA- 12 1.242 x 102Homodimer (divalent TRB) VHHA <1.0 x IO’3VHHA-5 <1.0 x IO’3VHHA-7 2.079VHHA- 12 2.814 x 10VHHA 12-7 >2 x 103VHHA12-5 >2 x 103Table 4. Various TRB binding af ’mities in different dimer formats provided herein (e.g., heterodimer format, which contains a single TRB, and homodimer format, which contains two TRB).

[0169] In some embodiments, the fusion-protein conjugate described herein binds a transferrin receptor (TfR) with an affinity (KD) of about 10 nM - 1000 nM, about 10 nM - 500 nM, about 20 nM - 500 nM, about 50 nM - 500 nM, about 50 nM - 300 nM, about 50 nM - 200 nM, about 80 nM - 200 nM, about 100 nM - 200 nM, about 100 nM - 180 nM, about 100 nM -150 nM, about 110 nM - 150 nM, or about 120 nM - 150 nM. In some embodiments, the TRB comprises an affinity (KD) of about 100 nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM. In one aspect, the fusion-protein conjugate is monovalent for the transferrin receptor binder (TRB).

[0170] In some embodiments, the fusion-protein conjugate described herein binds a transferrin receptor (TfR) with an affinity (KD) of about 10 nM - 2000 nM, about 10 nM - 1500 nM, about 20 nM - 60 nM, about 25 nM - 50 nM, about 30 nM - 45 nM, about 30 nM - 40 nM, about 35 nM - 40 nM, about 35.8 nM ± 5 nM, about 1000 nM - 2000 nM, about 1200 nM - 1800 nM, about 1300 nM - 1700 nM, about 1350 nM - 1500 nM, about 1400 nM - 1500 nM, about 1428 nM ± 150 nM, about 10 nM - 20 nM, about 10 nM - 15 nM, about 10 nM - 20 nM. In one aspect, the fusion-protein conjugate is divalent for the transferrin receptor binder (TRB).DOCKET NO: 91016-434826

[0171] In some embodiments, the fusion-protein conjugate described herein binds a protein of interest (POI) with an affinity (KD) of about 0.1 nM - 10 nM, about 0.25 nM - 10 nM, about 0.35 nM - 1.0 nM, about 0.4 nM - 1.0 nM, about 0.45 nM - 0.9 nM, about 0.35 nM - 0.5 nM, about 0.38 nM - 0.45 nM, about 0.4 nM ± 0.05 nM, about 0.45 nM - 0.65 nM, about 0.5 nM -0.6 nM, about 0.54 nM ± 0.05 nM, about 0.6 nM - 0.9 nM, about 0.7 nM - 0.85 nM, about 0.74 nM ± 0.05 nM, about 2 nM - 10 nM, about 3 nM - 7 nM, about 4 nM - 6 nM, about 5 nM ± 1 nM.

[0172] In some embodiments, the fusion-protein conjugate described herein binds a transferrin receptor (TfR) and a protein of interest (POI) with an affinity (KD) ratio (KD TfR / KD POI) of about 1 - 500, about 50 - 450, about 100 - 400, about 150 - 350, about 150 - 300, about 150 -250, about 150 - 275, about 150 -260, about 170 - 250, about 170 -240, about 175 -240, about 175 - 250. In one aspect, the fusion-protein conjugate is monovalent for the transferrin receptor binder (TRB).

[0173] In some embodiments, the fusion-protein conjugate described herein herein binds a transferrin receptor (TfR) and a protein of interest (POI) with an affinity (KD) ratio (KD TfR / KD POI) of about 0.01 - 5000, about 0.1 - 4000, about 0.1 - 3000, about 0.1 - 0.5, about 0.1 - 0.3, about 0.1 - 0.25, about 0.15 - 0.25, about 40 - 80, about 45 - 70, about 40 - 70, about 45 - 75, about 50 - 70, about 250 - 350, about 260 - 340, about 270 - 330, about 280 - 300, about 1800 - 3000, about 1700 - 2900, about 1900 - 2800. In one aspect, the fusion-protein conjugate is divalent for the transferrin receptor binder (TRB).Fc Regions

[0174] In embodiments, an Fc region that can be used as a component or module of the fusion protein-drug conjugates disclosed herein can be from IgG. In embodiments, an Fc region can be (SEQ ID NO: 80):DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ IDNO: 80).

[0175] In embodiments, an FC region can be (SEQ ID NO: 81):DOCKET NO: 91016-434826DKTHTCPPCPAPELLRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKARPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLiSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 81).

[0176] In embodiments, the Fc regions can dimerize, forming homodimers or heterodimer structures. In embodiments, the Fc regions can have, or can be modified to have, cysteine amino acids that are capable of forming disulfide bonds. In embodiments, dimers of polypeptides can form through disulfide bonds (1 or more, such as 2 disulfide bonds) between cysteine residues in Fc regions of separate fusion protein molecules.

[0177] In embodiments, the Fc region can be a variant comprising one or more amino acid substitutions that alter antigen-independent effector functions, like the circulating half-life of a molecule to which it is linked. Molecules linked to these Fc regions can exhibit either increased or decreased binding to FcRn compared to Fc regions lacking these substitutions and can have an increased or decreased half-life in serum, respectively. Fc variants with improved affinity for FcRn are anticipated to have longer serum half-lives, and such molecules have useful applications in methods in which long half-life of the linked molecule is desired. In contrast, Fc variants with decreased FcRn binding affinity are expected to have shorter half-lives, and such molecules are also useful, for example, where a shortened circulation time can be advantageous. In embodiments, Fc variants with decreased FcRn binding affinity are also less likely to cross the placenta. In addition, other applications in which reduced FcRn binding affinity can be desired include those applications in which localization to the brain, kidney, and / or liver is desired. In embodiments, the Fc variant-linked molecules can exhibit reduced transport across the epithelium of kidney glomeruli from the vasculature.

[0178] In embodiments, the Fc region can be a variant comprising one or more amino acid substitutions that alter Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC), antibodydependent cellular phagocytosis (ADCP), or complement activation activities, like the altered binding to FcyRs or Clqs. In embodiments, the Fc region can be a variant comprising one or more amino acid substitutions that improve thermal stability, reduce aggregation, or enhance expression yields. In embodiments, the Fc region can be a variant comprising one or more aminoDOCKET NO: 91016-434826acid substitutions that facilitate heterodimerization or multivalency to generate bispecific or multispecific antibodies.

[0179] In another embodiment, the Fc variant-linked molecules can exhibit reduced transport across the blood brain barrier (BBB), from the brain into the vascular space. In embodiments, an Fc region with altered FcRn binding comprises an Fc domain having one or more amino acid substitutions within the " FcRn binding loop" of an Fc domain. The FcRn binding loop is comprised of amino acid residues 280-299 (according to EU numbering). Exemplary amino acid substitutions with altered FcRn binding activity are disclosed in PCT Publication No.WO05 / 047327 which is incorporated by reference herein. In certain exemplary embodiments, the targeting compositions disclosed herein comprise an Fc domain having one or more of the following substitutions: V284E, H285E, N286D, K290E and S304D (EU numbering).

[0180] In embodiments, a composition provided herein can have an Fc variant comprising an amino acid substitution which alters glycosylation. For example, the Fc variant can have reduced glycosylation (e.g., N- or O-linked glycosylation). In embodiments, the Fc variant comprises reduced glycosylation of the N-linked glycan normally found at amino acid position 297 (EU numbering). In another embodiment, the molecules can have an amino acid substitution near or within a glycosylation motif, for example, an N-linked glycosylation motif that contains the amino acid sequence NXT or NXS. In a particular embodiment, the Fc variant can have amino acid substitution at amino acid position 228 or 299 (EU numbering). Exemplary amino acid substitutions which confer reduced or altered glycosylation are described in PCT Publication No., WO 2005 / 018572, which is incorporated by reference herein in its entirety.

[0181] In embodiments, a composition provided herein can be modified to eliminate glycosylation and can be referred to as "agly" molecules. Exemplary agly molecules, can have an aglycosylated Fc region of an IgG4 antibody which is devoid of Fc-effector function thereby eliminating the potential for Fc mediated toxicity to the normal vital tissues and cells. In yet other embodiments, the molecules disclosed herein can have an altered glycan. For example, there can be a reduced number of fucose residues on an N-glycan at Asn297 of the Fc region, i.e., is afucosylated. In embodiments, there can be an altered number of sialic acid residues on the N-glycan at Asn297 of the Fc region.

[0182] In embodiments, the CH2 or CH3 region of an Fc antibody domain can be truncated or modified to adjust the half-life of the molecule. In embodiments, an Fc truncation includes CH3DOCKET NO: 91016-434826or CH2 (e.g., Gehlsen, Kurt R, et al. " Pharmacokinetics of engineered human monomeric and dimeric CH2 domains." MAbs. Vol. 4. No. 4. Taylor & Francis, 2012; Ying, Tianlei, et al." Engineered soluble monomeric IgGl CH3 domain: generation, mechanisms of function, and implications for design of biological therapeutics." Journal of Biological Chemistry 288.35 (2013): 25154-25164).

[0183] In embodiments, a dimerization domain can be a component or module of a composition provided herein. The dimerization domain can be any region that can associate with another dimerization domain, through covalent or non-covalent bonds, to form a dimer (e g., a composition provided herein that is a homodimer or heterodimer).

[0184] There are many protein dimerization domains known in the art (e.g., see Dang, Dung Thanh. " Molecular Approaches to Protein Dimerization: Opportunities for Supramolecular Chemistry." Frontiers in Chemistry 10 (2022): 829312). An example dimerization domain can include zipper motifs, like a leucine zipper.

[0185] In embodiments, the dimerization can form between Fc regions of a composition provided herein. In embodiments, dimerization can form between regions of a composition provided herein that are not Fc regions.Therapeutic Moieties

[0186] In embodiments, a therapeutic agent or therapeutic moiety can be conjugated to (e.g., covalently attached) the fusion proteins described herein.

[0187] The fusion proteins can cause cell internalization of the drugs and release of active forms of the drug inside the cell. In embodiments, protease-sensitive linkers can be used to facilitate this process. In embodiments, ADCs are delivered to treat malignancies.

[0188] In embodiments, the therapeutic moiety can be conjugated to the part of the fusion protein that binds to a protein of interest (POI).

[0189] In embodiments, the therapeutic moiety can be conjugated to the part of the fusion protein that binds to the transferrin receptor binder (TRB).

[0190] In embodiments, conjugation of the therapeutic moiety to the fusion protein can use a chemical reaction(s). In embodiments, the conjugation can use bioconjugation. Bioconjugation reactions can be of different types. Common types of bioconjugation reactions can include coupling of cysteine, lysine and tyrosine amino acids. Common types of bioconjugationDOCKET NO: 91016-434826reactions can include modification of tryptophan amino acids and of the N- and C-terminus of a protein. In embodiments, the bioconjugation reaction can use a linker to connect the therapeutic moiety to the fusion protein.

[0191] In embodiments, a serine at position 7 of SEQ ID NO: 2, 3, or 4 can be substituted for a cysteine in order to facilitate a cysteine coupling bioconjugation. In embodiments, a serine at position 84 of SEQ ID NO: 2, 3, or 4 can be substituted for a cysteine in order to facilitate a cysteine coupling bioconjugation. In embodiments, a serine at position 7 of SEQ ID NO: 2, 3, or 4 and a serine at position 84 of SEQ ID NO: 2, 3, or 4 can be substituted for a cysteine to facilitate cysteine coupling bioconjugations. For example, the cysteine bioconjugation reaction for linking drug payloads to the fusion protein can be used to create the fusion protein drug conjugates described herein:Cysteine BioconjugationO OMaleimide

[0192] Furthermore, the skilled artisan can utilize other linker chemistry known in the art (see, e.g., Jain et al., Current ADC Chemistry, Pharm Res. 2015 Nov;32(l 1):3526-40).

[0193] In embodiments, a strategy is used to attach a specific site of a therapeutic moiety to a specific site on the fusion proteins. In embodiments, this can be done by installing a unique functional group onto a protein portion and use a bioorthogonal reaction to couple the therapeutic moiety to the fusion protein. In embodiments, these reactions can use modification of ketone and aldehydes, Staudinger ligation with organic azides, copper-catalyzed Huisgen cycloaddition of azides, or strain promoted Huisgen cycloaddition of azides. Other types of reactions can be used.

[0194] In embodiments, the bioconjugation reaction can be a maleimide-based cysteine reaction.DOCKET NO: 91016-434826

[0195] For example, the skilled artisan can utilize fragments of the POIBs listed in Table 5 to engineer POIBs conjugated with a therapeutic moiety according to bioconjugation reactions known in the art.Table 5.Shows characteristics of traditional ADC molecules.POI POIB Affinity, KD Indication(s) US Patent No.EGFR Cetuximab 0.2-0.4 nM colorectal cancer 6,217,866head and neck cancerPanitumumab 0.05 nM colorectal cancer 6,235,883 and 7,807,798CD20 Rituximab ~8 nM Non-Hodgkin's 6,652,852lymphomaChronic lymphocyticleukemia (CLL)Ofatumumab 0.12 nM B-cell acute leukemiaCLL CD30 Brentuximab 1.4-2.6 nM Hodgkin lymphoma, 7,659,241systemic anaplastic largecell lymphoma (sALCL),and primary cutaneousanaplastic large celllymphoma (pcALCL)HER2 Trastuzumab 0.1 nM breast cancer and gastric 6,627,196 and cancer 7,371,379CD22 Inotuzumab 235 pM non-Hodgkin’s B-cell 8153768lymphoma 874785788356119351986CD33 Gemtuzumab 0.07 to 0.08 acute myeloid leukemia 5,773,001nM (AML) 5,585,089CD79b Polatuzumab 1.6-1.8 nM B-cell lymphoma 8,088,378Nectin- Enfortumab 0.01nM urothelial cancer 11,274,1604Trop-2 Sacituzumab 0.3 nM triple-negative breast 7,999,083cancerBCMA Belantamab 0.7-1. O nM refractory multiple 9,273,141myelomaCD19 Loncastuximab 0.15 nM B-cell lymphoma 9,931,414HER3 Patritumab 10 nM non-small cell lung 10,383,878cancerCD25 Camidanlumab Hodgkin and nonHodgkin’s lymphomaDOCKET NO: 91016-434826

[0196] In embodiments, a therapeutic agent or therapeutic moiety can be conjugated to the fusion proteins with a drug-antibody ratio (DAR) of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In examples, the therapeutic agent or therapeutic moiety can be conjugated to the fusion proteins with an average DAR of 2, 4, 6, 8, 10 or 12. In examples the therapeutic agent or therapeutic moiety can be conjugated to the fusion proteins with an average DAR of 2. In examples, the therapeutic agent or therapeutic moiety can be conjugated to the fusion proteins with an average DAR of 4. In examples, the therapeutic agent or therapeutic moiety can be conjugated to the fusion proteins with an average DAR of 6. In examples, the therapeutic agent or therapeutic moiety can be conjugated to the fusion proteins with an average DAR of 8. In examples, the therapeutic agent or therapeutic moiety can be conjugated to the fusion proteins with an average DAR of 10. In examples, the therapeutic agent or therapeutic moiety can be conjugated to the hinge region of a fusion protein.

[0197] In embodiments, when a population of molecules (e.g., a pharmaceutical composition) is said to have n = 1, 2, 3, 4, 5, 6, 8, 10, or 12 in reference to the number of therapeutic moieties (D) that are conjugated to individual molecules, not every individual molecule will have that number of therapeutic moieties conjugated thereto. For example, if a fusion protein-drug conjugate disclosed herein is said to have n = 3, the majority of FPDCs in the composition have 3 drug moieties conjugated thereto. However, the population may have a minority of molecules with n = 1, 2, 4, 5, 6, 8, 10, 12 or greater. Some FPDCs in the population may not have a therapeutic moiety conjugated thereto (n = 0).

[0198] D can be conjugated to at a variety of positions and in various amounts. In embodiments, D is conjugated as described herein to (Fl -F2) at Rl. In embodiments, one, two, or three D are conjugated to (Fl - F2) at Rl. In embodiments, one D is conjugated to (Fl - F2) at Rl. In embodiments, D is conjugated as described herein to (Fl - F2) at R2. In embodiments, one, two, or three D are conjugated to (Fl - F2) at R2. In embodiments, one D is conjugated to (Fl - F2) at R2 (for example as described in US Pat. No. 11,938,194, incorporated herein by reference in its entirety on January 23, 2026). In embodiments, D is conjugated as described herein to (Fl - F2) at R3. In embodiments, one, two, or three D are conjugated to (Fl - F2) at R3. In embodiments, two D are conjugated to (Fl - F2) at R3. In embodiments, D is conjugatedDOCKET NO: 91016-434826to (Fl - F2) at R1 and to (Fl - F2) at R3. In embodiments, one D is conjugated to (F1 – F2) at R1; and two D are conjugated to (F1 – F2) at R3.

[0199] In embodiments, D is conjugated as described herein to (Fl - Fl) at Rl. In embodiments, one, two, or three D are conjugated to (Fl - Fl) at Rl. In embodiments, one D is conjugated to (Fl - Fl) at Rl. In embodiments, D is conjugated as described herein to (Fl - Fl) at R2. In embodiments, one, two, or three D are conjugated to (Fl - Fl) at R2. In embodiments, one D is conjugated to (Fl - Fl) at R2 (for example as described in US Pat. No. 11,938,194, incorporated herein by reference in its entirety on January 23, 2026). In embodiments, D is conjugated as described herein to (Fl - Fl) at R3. In embodiments, one, two, or three D are conjugated to (Fl - Fl) at R3. In embodiments, two D are conjugated to (Fl - Fl) at R3. In embodiments, D is conjugated to (Fl - Fl) at Rl and to (Fl - Fl) at R3. In embodiments, one D is conjugated to (Fl - Fl) at Rl; and two D are conjugated to (Fl - Fl) at R3.

[0200] Various types of therapeutic moieties can be used. In embodiments, the therapeutic moiety can be a small molecule (e.g., < 1,000 daltons).

[0201] In embodiments, the therapeutic moiety comprising the FPDCs of the invention can be any agent that can be used in the context of a conventional antibody-drug conjugate (ADC). For example, these conventional ADCs can be POIBs. In embodiments, the POIB that can be used includes, but is not limited to, gemtuzumab ozogamicin, brentuximab vedotin, tsastuzumab emtansine, inotuzumab ozogamicin, polatuzumab vedotin, enfortumab vedotin, trastuzumab deruxtecan, sacituzumab govitecan, belantamab mafodotin, moxetumomab pasudotox, loncastuximab tesirine, tisotumab vedotin-tftv, mirvetuximab soravtansine, datopotamab deruxtecan and the like.

[0202] In embodiments, the therapeutic moiety can be a cell death inducer, PROTACs (proteolysis-targeting chimeras) or molecular glue degrader, epigenetic or immune modulator, or DNA damage response (DDR) inhibitor.

[0203] In embodiments, the therapeutic moiety can be an anticancer agent or a glucocorticoid receptor modulator (GRM).

[0204] In embodiments, the anticancer agent can be a microtubulin inhibitor (e.g., monomethyl auristatin E or MMAE; monomethyl auristatin F or MMAF, mertansine and the like), a DNA binder (e.g., calicheamicin and the like), topoisomerase 1 inhibitors (e.g., SN-38, exatecan, deruxtecan and the like), or a ferroptosis inducer (e.g., alkyne -RSL3, propynoic acidDOCKET NO: 91016-434826carbamoyl methyl-amide-31 or PACMA 31, BCP-T. A, UAMC3203, and the like). In embodiments, the GRM can be dexamethasone, budesonide, and the like.

[0205] In embodiments, the therapeutic moiety that is conjugated to the FPDC can be classed as cleavable or non-cleavable. In embodiments, this classification refers to the ability of a therapeutic moiety conjugated to an FPDC to be cleaved from the FPDC. Cleavage of the therapeutic moiety from the FPDC occurs within a cell into which the FPDC has been internalized. In embodiments, cleavage of the therapeutic moiety from the FPDC can occur within the endosomal machinery of the cell. Cleavage of the therapeutic moiety from the FPDC can be facilitated by cleavable linkers (discussed elsewhere herein) that are part of the FPDC molecule. In embodiments, placement of cleavable linkers between a conjugated therapeutic moiety and the part of the FPDC from which the therapeutic moiety is to be disengaged can facilitate the separation. In embodiments, a cleavable therapeutic moiety that is conjugated to the FPDC can be valine-citrulline (VC) monomethyl auristatin E or VC-MMAE, VC-SN38, deruxtecan, MC-Val-Cit-PAB-Ispinesib, MC-caproyl-hydrazone doxorubicin, UAMC3203, and the like. In embodiments, a non-cleavable therapeutic moiety that is conjugated to the FPDC can be MMAF, MMAE, SN-38, (+)-JQl maleimide, alkyne -RSL3, PACMA 31, BCP-T. A, and the like.Antibodies

[0206] In embodiments, the TRBs and / or POIBs described herein are antibodies or antibody fragments.

[0207] In embodiments, antibodies or fragments can have amino acid substitutions. For example, a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in an immunoglobulin polypeptide is replaced with another amino acid residue from the same sideDOCKET NO: 91016-434826chain family. In another embodiment, a string of amino acids can be replaced with a structurally similar string that differs in order and / or composition of side chain family members.

[0208] Some embodiments also feature antibodies or fragments that have a specified percentage identity or similarity to the amino acid or nucleotide sequences of the antibodies described herein. For example, “homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence, which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. For example, the antibodies can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher amino acid sequence identity when compared to a specified region or the full length of any one of the antibodies described herein. For example, the antibodies can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher nucleic acid identity when compared to a specified region or the full length of any one of the antibodies described herein. Sequence identity or similarity to the nucleic acids and proteins of the present invention can be determined by sequence comparison and / or alignment by methods known in the art, for example, using software programs known in the art, such as those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. For example, sequence comparison algorithms (i.e., BLAST or BLAST 2.0), manual alignment or visual inspection can be utilized to determine percent sequence identity or similarity for the nucleic acids and proteins of the present invention.

[0209] The term “isolated” as used herein with respect to biological molecules, refers to molecules separated from other biological molecules that are present in the natural source of the macromolecule. The term “isolated” can also refer to a molecule that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. “Isolated” can also refer to cells or polypeptides which are isolated from other cellular proteins or tissues. Isolated polypeptides can include both purified and recombinant polypeptides.

[0210] As used herein, an “antibody” or “antigen-binding polypeptide” can refer to a polypeptide or a polypeptide complex that specifically recognizes and binds to an antigen. AnDOCKET NO: 91016-434826antibody can be a whole antibody and any antigen binding fragment or a single chain thereof. An antibody can be a fragment. For example, “antibody” can include any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule having biological activity of binding to the antigen. Non-limiting examples a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one portion of a binding protein. As used herein, the term "antibody" can refer to an immunoglobulin molecule and immunologically active portions of an immunoglobulin (Ig) molecule, i.e., a molecule that contains an antigen binding site that specifically binds (immunoreacts with) an antigen. " Specifically binds" or "immunoreacts with" can refer to the antibody reacting with one or more antigenic determinants of the desired antigen and does not react with other polypeptides.

[0211] The terms “antibody fragment” or “antigen-binding fragment”, as used herein, is a portion of an antibody such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. The term “antibody fragment” can include aptamers (such as spiegelmers), minibodies, and diabodies. The term “antibody fragment” can also include any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. Antibodies, antigen-binding polypeptides, variants, or derivatives described herein include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, dAb (domain antibody), minibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies.

[0212] A “single-chain variable fragment” or “scFv” refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins. A single chain Fv ("scFv") polypeptide molecule is a covalently linked VH: VL heterodimer, which can be expressed from a gene fusion including VH- and VL-encoding genes linked by a peptide-encoding linker. (See Huston et al. (1988) Proc Nat Acad Sci USA 85(16):5879-5883). In some aspects, the regions are connected with a short linker peptide of ten to about 25 amino acids. The linker can be rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-DOCKET NO: 91016-434826terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. A number of methods have been described to discern chemical structures for converting the naturally aggregated, but chemically separated, light and heavy polypeptide chains from an antibody V region into an scFv molecule, which will fold into a three-dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S. Patent No. 5,091,513; No. 5,892,019; No. 5,132,405; and No. 4,946,778, each of which are incorporated by reference in their entireties.

[0213] Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can be bound with either a kappa or lambda light chain. For example, the light and heavy chains are covalently bonded to each other, and the “tail” portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells, or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.

[0214] Both the light and heavy chains are divided into regions of structural and functional homology. The terms “constant” and “variable” are used functionally. The variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CHI, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. The term "antigen-binding site," or "binding portion" can refer to the part of the immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable (" V") regions of the heavy (" H") and light (" L") chains. Three highly divergent stretches within the V regions of the heavy and light chains, referred to as "hypervariable regions," are interposed between more conserved flanking stretches known as "framework regions," or " FRs". Thus, the term " FR" can refer to amino acid sequences which are naturally found between, and adjacent to, hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the threeDOCKET NO: 91016-434826hypervariable regions of each of the heavy and light chains are referred to as "complementaritydetermining regions," or " CDRs."

[0215] The six CDRs present in each antigen-binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain as the antibody assumes its three-dimensional configuration in an aqueous environment. The remainder of the amino acids in the antigen-binding domains, the FR regions, show less inter-molecular variability. The framework regions largely adopt a P-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the P-sheet structure. The framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions. The antigen-binding domain formed by the positioned CDRs provides a surface complementary to the epitope on the immunoreactive antigen, which promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids comprising the CDRs and the framework regions, respectively, can be readily identified for a heavy or light chain variable region by one of ordinary skill in the art, since they have been previously defined (See, “Sequences of Proteins of Immunological Interest,” Kabat, E., et al., U. S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol, 196:901-917 (1987)).

[0216] Where there are two or more definitions of a term which is used and / or accepted within the art, the definition of the term as used herein is intended to include all such meanings unless explicitly stated to the contrary. A specific example is the use of the term “complementarity determining region” (“CDR”) to describe the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. This region has been described by Kabat et al., U. S. Dept, of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and by Chothia et al., J. Mol.Biol. 196:901-917 (1987), which are incorporated herein by reference in their entireties. The CDR definitions according to Kabat and Chothia include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth in the table below as a comparison. The exact residue numbers which encompass a particular CDR will vary depending on theDOCKET NO: 91016-434826sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.Table 6. CDRsCDR Kabat Numbering Chothia Numbering VH CDR1 31-35 26-32VH CDR2 50-65 52-58VH CDR3 95-102 95-102VL CDR1 24-34 26-32VL CDR2 50-56 50-52VL CDR3 89-97 91-96

[0217] Kabat et al. defined a numbering system for variable domain sequences that is applicable to any antibody. The skilled artisan can unambiguously assign this system of “Kabat numbering” to any variable domain sequence, without reliance on any experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering system set forth by Kabat et al., U. S. Dept, of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983).

[0218] In addition to table above, the Kabat number system describes the CDR regions as follows: CDR-H1 begins at approximately amino acid 31 (i.e., approximately 9 residues after the first cysteine residue), includes approximately 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins at the fifteenth residue after the end of CDR-H1, includes approximately 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins at approximately the thirty third amino acid residue after the end of CDR-H2; includes 3-25 amino acids; and ends at the sequence W-G-X-G, where X is any amino acid. CDR-L1 begins at approximately residue 24 (i.e., following a cysteine residue); includes approximately 10-17 residues; and ends at the next tryptophan residue. CDR-L2 begins at approximately the sixteenth residue after the end of CDR-L1 and includes approximately 7 residues. CDR-L3 begins at approximately the thirty third residue after the end of CDR-L2 (i.e., following a cysteineDOCKET NO: 91016-434826residue); includes approximately 7-11 residues and ends at the sequence F or W-G-X-G, where X is any amino acid.

[0219] As used herein, the term "epitope" can include any protein determinant that can specifically bind to an immunoglobulin, a scFv, or a T-cell receptor. The variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. For example, the VL domain and VH domain, or subset of the complementarity determining regions (CDRs), of an antibody combine to form the variable region that defines a three-dimensional antigen-binding site. More specifically, the antigen-binding site is defined by three CDRs on each of the VH and VL chains (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3).

[0220] Antibodies can be prepared and purified by well-known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen, which is the target of the immunoglobulin sought, or an epitope thereof, can be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0221] The term “monoclonal antibody” or “mAb” or “Mab” or “monoclonal antibody composition”, as used herein, can refer to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. For example, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs contain an antigen binding site that can immunoreact with a specific epitope of the antigen characterized by a unique binding affinity for it.

[0222] In embodiments, the antibody is a humanized antibody, antibodies” A “humanized” antibody or antigen binding fragment, as used herein, can refer to a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) antibody or antigen binding fragment. Humanized antibodies can be prepared by grafting only non-human CDRs onto human framework and constant regions (Jones et al., Nature 321: 522-25 (1986);Verhoeyen et al., Science 239: 1534-1536 ( 1988)); Humanized antibodies can also be prepared by grafting entire non-human variable domains in order to retain their ligand binding properties via replacing exposed residues to reduce immunogenicity in order to generate a human-likeDOCKET NO: 91016-434826surface (Padlan, Molec. Immun. 28: 489-498 (1991); Padlan, Molec. Immun. 31(3): 169-217 (1994)). “Human antibodies”, or “fully human antibodies”, as used herein, can refer to antibody molecules in which the entire sequence of both the light chain and the heavy chain, including the CDRs, arise from human genes. In embodiments, humanized antibodies and fully human antibodies can interchangeably be used as a POIB according to the invention.

[0223] Human monoclonal antibodies can be prepared by using trioma technique; the human B cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72); and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In:MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 20262030) or by transforming human B cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 7796).

[0224] In addition, human antibodies can also be produced using additional techniques, including phage display libraries. (See Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U. S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;5,633,425; 5,661,016, and in Marks et al., Bio / Technology 10, 779783 (1992); Lonberg et al., Nature 368856 859 (1994); Morrison, Nature 368, 812 13 (1994); Fishwild et al, Nature Biotechnology 14, 845 51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); and Lonberg andHuszar, Intern. Rev. Immunol. 13 65 93 (1995).

[0225] Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal’s endogenous antibodies in response to challenge by an antigen. (See PCT publication W094 / 02602). The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host’s genome. The human genes are incorporated, for example, using yeastDOCKET NO: 91016-434826artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications. The preferred embodiment of such a nonhuman animal is a mouse, and is termed the XenomouseTM as disclosed in PCT publications WO 96 / 33735 and WO 96 / 34096. This animal produces B cells which secrete fully human immunoglobulins. The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv (scFv) moleculesOne method for producing an antibody of interest, such as a human antibody, is disclosed in U. S. Patent No. 5,916,771. This method includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy chain and the light chain.Nucleic Acids, Vectors and Cells Expressing Fusion Protein-Drug Conjugates

[0226] Also disclosed are nucleic acids encoding all or part of the compositions provided herein. Also disclosed are various vectors (e.g., plasmids, viral, and the like) that include the nucleic acids. Also disclosed are various cells (e.g., prokaryotic, eukaryotic) that contain nucleic acids or vectors that express compositions provided herein.

[0227] In embodiments, the molecules disclosed herein can be made using methods known in the art (see WO 2024 / 064756 Al, published 28 March 2024).Methods

[0228] Described herein are methods for administrating the fusion protein-drug conjugate (FPDC) described herein to a subject. In embodiments, the methods are used to treat a subject afflicted with cancer. " Treating" can refer to a therapeutic intervention that ameliorates a sign orDOCKET NO: 91016-434826symptom of a disease or pathological condition after it has begun to develop, such as a reduction in tumor burden or a decrease in the number or size of metastases. " Ameliorating" can refer to the reduction in the number or severity of signs or symptoms of a disease. For example, the methods are used to treat, prevent or alleviate a symptom of cancer. Non-limiting examples of cancer comprise lung cancer, pancreatic cancer, ovarian cancer, colorectal cancer, and gastric cancer.

[0229] In embodiments, the methods are used to treat a subject afflicted with a neurogenerative disease. For example, the neurodegenerative disease can comprise Amyotrophic Lateral Sclerosis (ALS). In some embodiments, the diseases treated can be Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and the like.

[0230] In embodiments, the methods are used to treat a subject afflicted with an autoimmune disease. For example, the autoimmune disease can comprise rheumatoid arthritis, systemic lupus erythematosus, psoriasis and the like.

[0231] In embodiments, the FPDCs can internalize membrane proteins of interest (e.g., cellular receptors or other membrane proteins) and selectively degrade and / or modulate these proteins.

[0232] In embodiments, the conjugated therapeutic moiety is removed from the FPDC such that it is active in the cell. In embodiments, the FPDCs to which a therapeutic moiety is conjugated can be used to treat cancer.

[0233] In embodiments, membrane proteins on cancer cells can be targeted to degrade and / or modulate the proteins (e.g., epidermal growth factor receptor or EGFR, intercellular adhesion molecule-1 or ICAM1, poliovirus receptor or PVR, monocarboxylate transporter 1 or MCT1, L-type amino acid transporter 1 or LAT1, and bone marrow stromal antigen 2 or BST2). In some examples, FPDCs can be used to improve anti-tumor responses in this way.

[0234] In embodiments, the reagents and methods disclosed herein can be used with cells that are not cancer cells.

[0235] In embodiments, the FPDCs to which a therapeutic moiety is conjugated can be used to treat neurodegenerative disease (e.g., ALS). In embodiments, membrane proteins on neurons can be targeted to degrade and / or modulate the proteins (e.g., EPHA4, Ephrin Type-A Receptor 4 or CD90, Thy-1 Cell Surface Antigen).DOCKET NO: 91016-434826

[0236] Herein, “subject” or “patient” can be used interchangeably, and can include both human patients and veterinary subjects, including human and non-human mammals.

[0237] Table 5 lists possible targets, therapeutic moieties and indications for administrations of the FPDC disclosed herein.Therapeutic Preparations

[0238] Aspects of the invention are drawn towards therapeutic preparations. As used herein, the term “therapeutic preparation” can refer to any compound or composition that can be used or administered for therapeutic effects (e.g., targeting compositions). As used herein, the term “therapeutic effects” can refer to effects sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.

[0239] Embodiments as described herein can be administered to a subject in the form of a pharmaceutical composition or therapeutic preparation prepared for the intended route of administration. Such compositions and preparations comprises, for example, the active ingredient(s) (e.g., targeting composition) and a pharmaceutically acceptable carrier, diluent, or excipient. Such compositions and preparations can be in a form adapted to parenteral (such as, intravenous, intraperitoneal). Other routes of administration are subcutaneous, intraperitoneal and intravenous, and such compositions can be prepared in a manner well-known to the person skilled in the art, e.g., as described in “Remington's Pharmaceutical Sciences”, 17. Ed. Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, Pa., U. S. A., 1985 and more recent editions and in the monographs in the “Drugs and the Pharmaceutical Sciences” series, Marcel Dekker. The compositions and preparations can appear in conventional forms, for example, solutions and suspensions for injection in the form of enteric formulations, e.g., as disclosed in U. S. Pat. No. 5,350,741.

[0240] Sterile injectable solutions can be prepared by incorporating the compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, examples of useful preparation methods are vacuumDOCKET NO: 91016-434826drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0241] For example, the pharmaceutical composition can be administered by bolus injection or by infusion. A bolus injection can refer to a route of administration in which a syringe is connected to the IV access device and the medication is injected directly into the subject. The term “infusion” can refer to an intravascular injection.

[0242] Embodiments as described herein can be administered to a subject one time (e.g., as a single injection, bolus, or deposition). Alternatively, administration can be once or twice daily to a subject for a period of time, such as from about 2 weeks to about 28 days. Administration can continue for up to one year. In embodiments, administration can continue for the life of the subject. It can also be administered once or twice daily to a subject for period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or a combination thereof.

[0243] In embodiments, compositions as described herein can be administered to a subject chronically. “Chronic administration” can refer to administration in a continuous manner, such as to maintain the therapeutic effect (activity) over a prolonged period of time.

[0244] A specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the targeting compositions, variant or derivative thereof used, the patient's age, body weight, general health, sex, and diet, and the time of administration, rate of excretion, drug combination, and the severity of the particular disease being treated. Judgment of such factors by medical caregivers is within the ordinary skill in the art. The amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.

[0245] A therapeutically effective amount of a reagent or therapeutic composition of the invention can be the amount needed to achieve a therapeutic objective. As noted herein, this can be a binding interaction between a composition provided herein and POI that, in certain cases, interferes with the functioning of the POI. The compositions disclosed herein limit toxicity and side effects, while demonstrating efficacy for delivering the conjugated drug to cells expressing a protein of interest. The compositions herein exhibit reduced toxicity and increased efficacy though, for example, effectively internalizing and clearing the FPDCs from target cells;DOCKET NO: 91016-434826exhibiting affinity and selectivity for a target cell or molecule; and including a drug conjugate linkage with sufficiently tight binding to avoid early, unintended, and off-target release as well as efficiently releasing the payload in the target cell. A lower dose of the compositions disclosed herein can be used relative to typical antibodies given the enhanced selectivity and potency. The amount required to be administered will furthermore depend on the binding affinity of the reagent or therapeutic composition for its specific target and will also depend on the rate at which an administered reagent or therapeutic composition is depleted from the free volume other subject to which it is administered. The dosage administered to a subject (e.g., a patient) of the binding polypeptides described herein is about 0.1 mg / kg to 100 mg / kg of the patient's body weight, between 0.1 mg / kg and 20 mg / kg of the patient's body weight, or 1 mg / kg to 10 mg / kg of the patient's body weight. 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 reagent or therapeutic composition of the disclosure may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation. Common ranges for therapeutically effective dosing of targeting compositions of the invention can be, by way of nonlimiting example, from about 0.1 mg / kg body weight to about 50 mg / kg body weight. Common dosing frequencies can range, for example, from twice daily to once a week.

[0246] Where fragments (e.g., antibody fragments) are used, the smallest inhibitory fragment that specifically binds to the binding domain of the POI is contemplated. For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and / or produced by recombinant DNA technology. (See, e g., Marasco et al, Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). The formulation can also contain more than one active compound as necessary for the specific indication being treated, for example, those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition comprises an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine (e.g., IL-15), chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.DOCKET NO: 91016-434826

[0247] The compositions and / or preparations can be sterilized by conventional sterilization techniques which are well known in the art. The resulting aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with the sterile aqueous solution prior to administration. The compositions and / or preparations can contain pharmaceutically and / or therapeutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents, for instance sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.DOCKET NO: 91016-434826EXAMPLES

[0248] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.Example 1

[0249] Described in the studies herein are compositions and methods for binding to an internalizing receptor (i.e., Transferrin receptor 1 (TfRl)) on the surface of cells and delivering a therapeutic agent.

[0250] Cloning and Expression for Example 1 FPDC. Plasmids were constructed using standard cloning methods. Gene fragments encoding target protein binders or TfRl binders were synthesized and subcloned into a pFuse-hlgGl vector (InvivoGen®) with an optimized IL2 signal peptide for mammalian expression. Proteins were expressed in Expi293 cells (Thermo Fisher Scientific®) using a polyethylenimine (Polysciences®) transfection protocol or other Expi293 expression methods.

[0251] On-Bead Antibody-Payload Conjugation for Example 1 FPDC. Cell media containing the produced antibody was collected by centrifugation at 4,000 rpm for 15 minutes at 4 °C to remove cell pellets and then combined with protein A beads. This mixture was allowed to bind for 60 minutes at 4 °C, after which the beads were washed three times with 5 mL phosphate-buffered saline (PBS). 2 mM dithiothreitol (DTT) and 2 mM ethylenedi aminetetraacetic acid (EDTA) in PBS (pH 7.4) were added to the beads to reduce the antibodies, and the mixture was shaken overnight (9-12 hours) at 4 °C. The beads were then washed three times with 5 mL PBS containing 2 mM EDTA. For oxidation, 1 mM dehydroascorbic acid (DHAA) in PBS with 2 mM EDTA was added to the beads and shaken at 4 °C for 3 hours. Finally, the beads were incubated with 100 pM payloads in PBS containing 2 mM EDTA while shaking overnight. The beads were subsequently washed three times with 5 mL PBS containing 2 mM EDTA. Elution was performed by adding 0.5 mL of 0.1 M glycine (pH 2.0) at 4 °C. Immediately after elution, 40 pL of 1 M Tris buffer (pH 11) and 30 pL of 5 M NaCl were added to each elution tube. The pH of the protein solution was then checked and adjusted to approximately 7.4.DOCKET NO: 91016-434826

[0252] Referring to FIG. 1, a schematic diagram of exemplary fusion protein-drug conjugates disclosed herein is shown. R1 is a protein of interest (POI) binder (POIB). R2 and R2’ are IgG Fc regions. R3 is a transferrin receptor (TR) binder (TRB). D is a therapeutic moiety.

[0253] Referring to FIG 2, a schematic diagram of an example FPDC disclosed herein is shown interacting with the cell surface of a normal, noncancerous cell (left) and a cancerous cell (right). The cancerous cell expresses a POI to which the POIB of the FPDC can bind. FPDC binding to the cell is dependent on POI on the cell surface.

[0254] Referring to FIG. 3, binding to Jurkat cells with fusion protein-drug conjugate is shown. Figure 3 A shows example data for an FPDC having a TRB with a binding affinity of 1.073 x 10-1[VHHA, SEQ ID NO: 1], Figure 3B and 3C show example data for FPDCs having TRBs with binding affinities of 1.242 x 102nM [VHHA-12; SEQ ID NO: 2] and 2.588 x 10 nM (VHHA-7), respectively. The relative affinities of the TRBs for transferrin receptor in these studies is A > C > B. The FPDCs used in this binding study did not include a therapeutic moiety. The protein of interest binder is an antibody specific for CCR6. The dimer is a knob and hole configuration. In the graphs, cells having a POI to which the POIB of the FPDC can bind are shown with a red line. Cells not having a POI to which the POIB of the FPDC can bind are shown with a black line.

[0255] Referring to FIG. 4A-C, binding to Jurkat cells with fusion protein-drug conjugate is shown. The FPDCs used in Figure 4A and 4B have transferrin receptor binders having a binding affinity of X [VHHA, SEQ ID NO: 1] compared to transferrin receptor binder having a binding affinity of Y1 [VHHA-12; SEQ ID NO: 2], Y2 [VHHA-12+5; SEQ ID NO: 3] and Y3 [VHHA-12+7; SEQ ID NO: 4], The relative binding affinities of the TRBs for transferrin in these studies is VHHA > VHHA-12 > (VHHA 12+7 ~ VHHA 12+5). This binding study did not include a therapeutic moiety. The protein of interest binder is a CCR6 antibody. The FPDCs are homodimers of the configuration in Figure 5C. The cells either have Protein of Interest on the surface (POI+) or do not have the Protein of Interest on the surface (POI-). (A) shows binding of 1 nM of each embodiment to cells. (B) shows binding of 10 nM of each embodiment to cells.

[0256] Referring to FIG. 5A-B a cell viability assay comparing the anti-tumor activity of fusion protein drug conjugates (red, EGFRFPDC2(MMAF*6); SEQ ID NO:82; TRB heavy chain, SEQ ID NO: 85; POIB light chain, TRB heavy chain, SEQ ID NO: 82; POIB light chain, SEQ ID NO: 83, traditional antibody drug conjugates (EGFR-IgG Fc-MC-MMAF in green), andDOCKET NO: 91016-434826TfRl-only binder-MC-MMAF in blue. Panel A is triple-negative breast cancer cell line MDA-MB-231, and panel B is Hs578T cells is provided. The protein of interest is EGFR. The therapeutic moiety (D) is monomethyl auristatin F (MMAF) with a non-cleavable linker for conjugation. Fusion protein drug conjugates exhibit significantly enhanced activity compared to traditional antibody drug conjugates. In contrast, the TfRl-only binder (which is the same as FPDC except R1 is deleted) did not demonstrate activity, indicating that the efficacy of the fusion protein drug conjugates depends on binding to the specific cell surface POI.

[0257] Referring to FIG. 6A- B, cell viability assay results are provided. Figure 6A is a western blot assay showing different cancer cell lines expressing heterogenous levels of EGFR. Figure 6B shows cell viability assays comparing the anti-tumor activity of fusion protein drug conjugates (EGFR FPDC2(MMAF*6); SEQ ID NO: 82; R1 is an EGFR antibody (SEQ ID NO: 84); R2 is IgG Fc (SEQ ID NO: 81); R3 is SEQ ID NO: 85; D is MC MMAF; n- Y in blue), traditional antibody-drug conjugates (EGFR-IgG Fc-MC-MMAF in green), TfRl-only binder-MC-MMAF in red, and unconjugated controls in triple-negative breast cancer cell lines (Figure 6B) MDA-MB-231, KRAS G12S lung cancer A549 cell line, EGFR-driven lung cancer PC9 Dell9 cells, and EGFR / cMET driven lung cancer HCC827GR6 cells. These molecules target EGFR as the cell surface antigen (POI) and utilize MMAF as the payload with a non-cleavable linker for conjugation. Fusion protein drug conjugates exhibit significantly enhanced activity compared to traditional antibody drug conjugates across all four cell lines despite varying EGFR expression levels. The FPDC is R1 is SEQ ID NO: 84; R2 is IgG Fc of SEQ ID NO: 81; R3 is SEQ ID NO: 85; D is MC MMAF; n is 6).

[0258] Referring to FIG. 7A-B, a cell viability assay when the POI is ICAM1 is shown. The data compare the anti-tumor activity of fusion protein drug conjugate in red (ICAM FPDC2(MMAF*6), FPDC wherein R1 is SEQ ID NO: 90; R2 is IgG Fc of SEQ ID NO: 81; R3 is SEQ ID NO: 85; D is MC MMAF; n is 6; TRB heavy chain, SEQ ID NO: 88; POIB light chain, SEQ ID NO: 89); traditional antibody drug conjugates (IgG Fc-MC-MMAF in green), and TfRl-only binder-MC-MMAF in blue in triple-negative breast cancer cell lines MDA-MB-231 (A)) and Hs578T (B)). These molecules target ICAM1 as the cell surface antigen (POI) and utilize MMAF as the payload with a non-cleavable linker for conjugation. Fusion protein drug conjugates of the present invention exhibit significantly enhanced activity compared to traditional antibody drug conjugates. In contrast, TfRl-only binder-MC-MMAF (which is theDOCKET NO: 91016-434826same as FPDC except R1 is deleted) did not demonstrate activity, indicating that the efficacy of the fusion protein conjugates depends on binding to the specific cell surface antigen.

[0259] Referring to FIG. 8A-B, a cell viability assay when the POI is PVR is shown. The data compare the anti-tumor activity of fusion protein drug conjugate in red (PVR FPDC2(MMAF*6) FPDC wherein R1 is SEQ ID NO: 95; R2 is IgG Fc of SEQ ID NO: 81; R3 is SEQ ID NO: 85; D is MC MMAF; n is 6; TRB heavy chain, SEQ ID NO: 93; POIB light chain, SEQ ID NO: 94); traditional antibody drug conjugates (IgG Fc-MC-MMAF in green), and TfRl-only binder-MC-MMAF in blue in triple-negative breast cancer cell lines MDA-MB-231 (A) and Hs578T (B). These molecules target PVR as the cell surface antigen (POI) and utilize MMAF as the payload with a non-cleavable linker for conjugation. Fusion protein drug conjugates of the present invention exhibit significantly enhanced activity compared to traditional antibody drug conjugates. In contrast, TfRl-only binder-MC-MMAF (which is the same as FPDC except R1 is deleted) did not demonstrate activity, indicating that the efficacy of the fusion protein conjugates depends on binding to the specific cell surface antigen.

[0260] Referring to FIG. 9A-B, a cell viability assay when the POI is MCT1 is shown. The data compare the anti-tumor activity of fusion protein drug conjugate in red (MCT1 FPDC2(MMAF*6) FPDC wherein R1 is SEQ ID NO: 100; R2 is IgG Fc of SEQ ID NO: 81; R3 is SEQ ID NO: 85; D is MC MMAF; n is 6; TRB heavy chain, SEQ ID NO: 98; POIB light chain, SEQ ID NO: 99); traditional antibody drug conjugates (IgG Fc-MC-MMAF in green), and TfRl-only binder-MC-MMAF in blue in triple-negative breast cancer cell lines MDA-MB-231 (A) and Hs578T (B). These molecules target MCT1 as the cell surface antigen (POI) and utilize MMAF as the payload with a non-cleavable linker for conjugation. Fusion protein drug conjugates of the present invention exhibit significantly enhanced activity compared to traditional antibody drug conjugates. In contrast, TfRl-only binder-MC-MMAF (which is the same as FPDC except R1 is deleted) did not demonstrate activity, indicating that the efficacy

[0261] Referring to FIG. 10A-B, a cell viability assay when the POI is LAT1 is shown. The data compare the anti-tumor activity of fusion protein drug conjugate in red (LAT1 FPDC2(MMAF*6) FPDC wherein R1 is SEQ ID NO: 105; R2 is IgG Fc of SEQ ID NO: 81; R3 is SEQ ID NO: 85; D is MC MMAF; n is 6; TRB heavy chain, SEQ ID NO: 103; POIB light chain, SEQ ID NO: 104); traditional antibody drug conjugates (IgG Fc-MC-MMAF in green), and TfRl-only binder-MC-MMAF in blue in triple-negative breast cancer cell lines MDA-MB-DOCKET NO: 91016-434826231 (A) and Hs578T (B). These molecules target LAT1 as the cell surface antigen (POI) and utilize MMAF as the payload with a non-cleavable linker for conjugation. Fusion protein drug conjugates of the present invention exhibit significantly enhanced activity compared to traditional antibody drug conjugates. In contrast, TfRl-only binder-MC-MMAF (which is the same as FPDC except R1 is deleted) did not demonstrate activity, indicating that the efficacy of the fusion protein conjugates depends on binding to the specific cell surface antigen.

[0262] Referring to FIG. 11A-B, a cell viability assay when the POI is BST2 is shown. The data compare the anti-tumor activity of fusion protein drug conjugate in red (BST2 FPDC2(MMAF*6) FPDC wherein R1 is SEQ ID NO: 110; R2 is IgGFc of SEQ ID NO: 81; R3 is SEQ ID NO: 85; D is MC MMAF; n is 6; TRB heavy chain, SEQ ID NO: 108; POIB light chain, SEQ ID NO: 109); traditional antibody drug conjugates (IgG Fc-MC-MMAF in green), and TfRl-only binder-MC-MMAF in blue in triple-negative breast cancer cell lines MDA-MB-231 (upper panel) and Hs578T (lower panel). These molecules target BST2 as the cell surface antigen (POI) and utilize MMAF as the payload with a non-cleavable linker for conjugation. Fusion protein drug conjugates of the present invention exhibit significantly enhanced activity compared to traditional antibody drug conjugates. In contrast, TfRl-only binder-MC-MMAF (which is the same as FPDC except R1 is deleted) did not demonstrate activity, indicating that the efficacy of the fusion protein conjugates depends on binding to the specific cell surface antigen.

[0263] Referring to FIG. 12, a cell viability assay showing efficacy with alternative therapeutic moieties (D) is shown. The data compare the anti-tumor activity of fusion protein drug conjugate in red (EGFR FPDC2(DOX*6) in left panel or EGFR FPDC2(RSL3*6) in right panel; FPDC wherein R1 is SEQ ID NO: 84; R2 is IgG Fc of SEQ ID NO: 81; R3 is SEQ ID NO: 85; D is MC-caproyl-hydrazone-doxorubicin or MC-RSL3; n is 6, in red); traditional antibody drug conjugates (IgG Fc- MC-caproyl-hydrazone-doxorubicin (left panel) or MC-RSL3 (right panel) in green), and TfRl-only binder- MC-caproyl-hydrazone-doxorubicin or MC-RSL3 in black in triple-negative breast cancer cell lines MDA-MB-231. These molecules target EGFR as the cell surface antigen (POI) and vary the payload (D) with a non-cleavable linker for conjugation. Fusion protein drug conjugates of the present invention exhibit significantly enhanced activity compared to traditional antibody drug conjugates and the TfRl-only binderDOCKET NO: 91016-434826indicating that the efficacy of the fusion protein conjugates is achieved with multiple therapeutic moieties.

[0264] Referring to FIG. 13, a comparison of non-cleavable and cleavable linkers showing little difference in anti -turn or activity is shown. Cell viability assay comparing the anti -tumor activity of fusion protein drug conjugates (FPDC wherein R1 is SEQ ID NO: 84; R2 is IgG Fc of SEQ ID NO: 81; R3 is SEQ ID NO: 85; D is MMAE that is non-cleavable MC or cleavable MC-VC; n is 6; in red), in lung cancer A549 cells (A) and PC9 Del 19 cells (B). These molecules target EGFR as the cell surface antigen and utilize Monomethyl auri statin E (MMAE) as the payload with either a non-cleavable linker or a cleavable linker (VC) for conjugation. Fusion protein drug conjugates exhibit significantly enhanced activity compared to traditional antibodydrug conjugates. TfR1-only binder did not demonstrate activity, indicating that the efficacy of the fusion protein conjugates depends on binding to the specific cell surface antigen. No significant differences in anti-tumor activity were observed between the two linker types, indicating that the fusion protein drug conjugates can use different linker types.

[0265] Referring to FIG. 14, a pHrodo internalization assay in Raji wildtype (WT) and CD20 knockout (KO) Cells is shown. A pHrodo internalization assay was conducted using Raji wildtype (WT) cells (CD20 positive) and Raji CD20 knockout (KO) cells to demonstrate antigen-dependent internalization of fusion protein conjugates of the present invention. An anti-CD20 antibody (binding affinity about 10 nm) (black), fusion protein conjugates containing a TfR1 binder VHHA, in red, and fusion protein conjugates (CD20 FP4; R1 is POIB of SEQ ID NO: 124; R2 is TRB0comprising SEQ ID NO: 1 (binding affinity about 1.2-1.3 x 102nM) without payload, D; green) were labeled with pHrodo, a pH-sensitive fluorescent dye. Raji WT cells and CD20 KO Raji cells were treated with 5 nM of each test protein for 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, or 10 h. Fluorescence was measured using a flow cytometer to assess the internalization of the molecules. Only the low binding affinity CD20 Fc-TfR1 protein conjugates (CD20 FP2; R1 is POIB of SEQ ID NO: 124; R2 is TRB’ comprising SEQ ID NO: 4; green line) exhibited antigen (CD20)-dependent internalization. Fusion protein conjugates containing VHHA (red) showed internalization in both CD20 positive and CD20 negative cells. The anti-CD20 antibody-mediated internalization (black line) was significantly lower than that of the fusion protein conjugates (green).DOCKET NO: 91016-434826

[0266] Referring to FIG. 15, fusion protein conjugates having low binding affinity are shown. TfR1 binder -125 (binding affinity for 12+5; CD20 FP3) and TfR1 binder-127 (CD20 FP2) show minimal on-cell binding on antigen-negative cells, including both wildtype Jurkat cells (left) and Raji CD20 knockout cells (right). These molecules target CD20 as the cell surface antigen (POI).

[0267] Referring to FIG. 16, demonstrated is that CD20-targeted fusion proteins exhibit reduced on-cell binding in CD20-negative cells. CD20-targeted fusion proteins, CD20 FP1 and CD20 FP2 demonstrate significantly lower on-cell binding compared to CD20 FP4 across various concentrations on Jurkat cells (CD20-negative).

[0268] Referring to FIG. 17, schematics and quantification of the percentage of cells in coculture of Raji WT cells (green) / Raji CD20 knockout cells (red) treated with traditional drug conjugates or fusion protein drug conjugates (FPDC wherein R1 is SEQ ID NO; X; R2 is IgG Fc of SEQ ID NO X; R3 is SEQ ID NO X (CD20 is POI / antigen); D is MMAF; n is 2) is provided. The results show that the fusion protein drug conjugates selectively kill the antigen positive, Raji WT cells, and do not kill the Raji CD20 knockout cells, demonstrating the antigen selectivity of these molecules. The traditional ADC shows no observable killing effects of either cell type. These molecules target CD20 as the cell surface antigen and utilize MMAF as the payload with a non-cleavable linker for conjugation.

[0269] Referring to FIG. 18A-C, a cell viability assay comparing the anti-tumor activity of fusion protein drug conjugates (FPDC) containing variable number of payload (D) is shown. Data are provided for n=2 (CD20 FPDC2(MMAF*2)) and n=6 (CD20 FPDC2(MMAF*6) by conjugating to cysteines. These molecules target CD20 as the cell surface antigen (POI) and utilize MMAF as the payload with a non-cleavable linker for conjugation. In Raji cells (A)., fusion protein drug conjugates exhibit significantly enhanced activity compared to traditional antibody drug conjugates. Fusion protein drug conjugates containing the additional drug conjugate in the TfR1 binding domains show higher activity than Fusion protein drug conjugates not containing the additional cysteines. The TRB’ control did not demonstrate activity, indicating that the efficacy of the fusion protein conjugates depends on binding to the specific cell surface antigen. In Raji CD20 knock-out cells (B), these molecules target CD20 as the cell surface antigen and utilize MMAF as the payload with a non-cleavable linker for conjugation. No molecules show cell killing effects, indicating their activities depend on presence of the specific cell surface antigen (POI). Figure 18C illustrates the conjugation of the therapeutic moiety (D).DOCKET NO: 91016-434826

[0270] Referring to FIG. 19, a cell viability study with fusion protein drug conjugates with a TfR1 H7 TRB (CD20 FPDC4(MMAF*2)) is shown. The cell viability assays compare the antitumor activity of fusion protein drug conjugates containing the TfRl H7 binder (binding affinity X) on CD20-positive and CD20-negative cells. These molecules target CD20 as the cell surface antigen (POI) and utilize MMAF as the payload with a non-cleavable linker for conjugation. Unlike fusion protein drug conjugates of the present invention, those with the TfR1 H7 binder kill both CD20-positive cells (Raji and CD20-overexpressing Jurkat cells) and CD20-negative cells (Raji CD20 KO and wildtype Jurkat cells), lacking the necessary antigen specificity for cancer-specific killing.

[0271] Referring to FIG. 20, a cell viability assays in primary human T cells is shown, demonstrating that a CD20-targeted fusion protein drug conjugate (FPDC) does not induce cytotoxicity in T cells, highlighting antigen selectivity in a primary cell setting. The T cells were either inactivated (left panel) or activated (right panel).Example 2

[0272] The TransTAC-ADC design was applied to Trop2, a clinically validated target in TNBC, using a Trop2-DXd ADC backbone (datopotamab deruxtecan; GGFG-Dxd; DAR = 4). In addition to the TfRlb-low variant (vl.l), we tested a medium-affinity TfRlb variant (F51I) with a monovalent KD of 129 nM, which remains over 150-fold weaker in affinity than the TAA binders (datopotamab, KD= 0.74nM) (FIG. 22). This TfRlb-med variant was evaluated in both monovalent (vl.2) and bivalent (vl.3) formats (FIG. 23). A standard Trop2 ADC matching the clinically validated datopotamab deruxtecan design was also generated to benchmark the TransTAC-ADC molecules. Across three Trop2-positive TNBC lines, all Trop2 TransTAC-ADC designs enhanced activity compared to the parental Trop2 ADC, with vl.3 showing the greatest potency, while maintaining no or over 500-fold reduced activity in the non-malignant MCF10A control (FIG.24). Across the TfRl affinity series, IC50improvements ranged from approximately 10- to 340-fold (FIG. 24).

[0273] The molecules were also tested in non-malignant cell models to further evaluate tumor selectivity. Trop2 TrasnTAC-ADCs showed no or negligible toxicity in BEAS-2B (human bronchial epithelial), NK. TerT (telomerase-immortalized normal epithelial), HS27 (human boneDOCKET NO: 91016-434826marrow-derived stromal fibroblast), and TMR-90 (primary human fetal lung fibroblast) cells, with undeterminable IC50 values at protein concentrations up to 100 nM (FIG. 25A, FIG. 25B).

[0274] Trop2 TransTAC-ADC vl.1 was further tested in an MDA-231 TNBC xenograft model at single doses of 1 mg kg-1and 5 mg kg-1(FIG. 26A). Both doses led to strong tumor growth inhibition and outperformed the control ADC. These results show that Trop2 TransTAC-ADCs have stronger antitumor activity both in vitro and in vivo compared with the clinically validated datopotamab deruxtecan design, even with the lowest-affmity TfRl variant (FIG. 26B). The molecules also showed a good safety profile, with no weight loss and no signs of toxicity in complete blood count (CBC), liver, or kidney assays (FIG. 26A, FIG. 26B).

[0275] TransTAC-ADCs are designed to not depend on the natural internalization properties of TAAs for drug delivery. Therefore, they may enable targeting of membrane proteins that were previously not suitable for ADC therapies. To explore this possibility in TNBC, published TNBC cell-surface proteomics datasets were analyzed and established a bioinformatics and antibody-screening workflow to identify additional candidate targets (FIG.28). Based on antibody binding performance (FIG. 29) and expression (FIG. 30) across TNBC models, this approach narrowed a large list of surface proteins to three candidates: EGFR, PVR, and ICAM1. Consistently, across TNBC cohorts, EGFR, PVR (CD 155) and ICAM1 (CD54) are frequently overexpressed, with reported immunohistochemistry positivity or high-expression rates of ~52–76% for EGFR, ~41–72% for PVR, and ~63% for ICAM1, supporting their prioritization as therapeutically tractable targets in TNBC.

[0276] TransTAC-ADC vl.l molecules for EGFR and PVR with an MC-MMAF payload (DAR = 2) were generated and evaluated them across nonmalignant MCF10A cells and multiple TNBC lines. The EGFR antibody alone showed some inhibitory activity in MCF10A, possibly because of the role of EGFR signaling in supporting proliferation in certain epithelial cell lines (FIG. 31). EGFR TransTAC-ADC vl.l exhibited enhanced cytotoxicity in EGFR-positive TNBC models compared with the matched ADC, while showing similar activity to the ADC in MCF10A cells (FIG. 31). This is consistent with the hypothesis that TransTAC-ADC-mediated enhancement is dependent on TfRl expression. Similarly, improved activity of PVR TransTAC-ADC vl.l compared with the matched ADC was observed only in TNBC cells and not in MCF10A (FIG. 32). These results highlight that elevated TfRl expression in tumor cells relative to most healthy tissues can preferentially enhance ADC uptake in tumors.DOCKET NO: 91016-434826

[0277] ICAM1 is highly expressed across many TNBC models and an exploratory TNBC drug target. ICAM1 bispecific Abs iBsAbs were expressed and observed all three proteins bind ICAM1 -positive cells whereas the paired Fc-TfRlb controls showed little binding, validating target-dependent activity across all three TfRlb variants (FIG.33A). ICAM1 TransTAC-ADC conjugates were further constructed vl.l-vl.3 with an MC-MMAF payload (DAR = 2) and observed an approximately 10- to 330-fold improvement in activity compared with matched ADC controls (FIG.33A, FIG. 33B). Minimal activity was detected in MCF10A cells with vl.l and vl.2. With vl.3 the potency was > 500-fold lower in MCF10A than in TNBC lines, indicating a favorable therapeutic window (FIG. 33A, FIG.33B).

[0278] The molecules were further tested in non-malignant cell models. Similar to Trop2 TransTAC-ADCs, ICAM1 TransTAC-ADCs showed negligible toxicity in IC AMI -negative non-malignant cell models, including BEAS-2B, NK. TerT, HS27, and IMR-90 cells (FIG. 34A, FIG. 34B). In contrast, the non-affinity-optimized TranTAC-ADC vl. O exhibited broad activity against ICAM1 -negative non-malignant cells, underscoring the importance of TfR.1 affinity tuning to achieve antigen-dependent activity, consistent with our observations with CD20 (FIG.35).Table 7. Nucleic acid and amino acid sequences of Example 2SEQ Name SequenceID *the CDR regions are shown in underlineNO142 hVHHA Amino EVQLVESGGGVVQPGGSLRLSCAASGTDFSINFIRWYR based on acid QAPGKGLEFVAGFTATGNTNYADSVKGRFTISRDNSKN Lamanade TLYLQMNSLRAEDTAVYYCYMLDKWGOGTLVTVSS 143 hVHHA Amino QVQLVESGGGLVQPGGSLRLSCAASGTDFSINFLGWFR based on acid QAPGQGLEAVAAFTATGNTYYADSVKGRFTISRDNSKN h- TLYLQMNSLRAEDTAVYYCYMLDKWGQGTLVTVSS NbBclllOFGLAscaffold144 hVHHA Amino EVQLVESGGGLVQPGGSLRLSCAASGTDFSINFMYWVR based on acid QAPGKGLEWVSEFTATGNTKYPDSVKGRFTISRDNAKN Ozoralizu TL YLQMN SLRPEDT A V Y YC YMLDKRGQGTL VT V S S mab CDRGraftingDOCKET NO: 91016-434826hVHHA Amino EVQLVESGGGLVQPGGSLRLSCAASGTDFSINFMGWFR based on acid QAPGKGRELVAAFTATGNTYYCaplacizu PDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCY mab CDR MLDKWGQGTQVTVSSGraftinghVHHA Amino QVQLQESGGGLVQPGGSLRLSCAASGTDFSINFMAWFR based on acid QAPGKERERVAKFTATGNTYLADSVKGRFTISQNNAKS Envafolim T VYLQMNSLKPEDTAM YYC YMLDK WGQGTQ VT VS S ab CDRGraftinghVHHA Amino EVQLVESGGGVVQPGGSLRLSCAASGTDFSINFIRWYR based on acid QAPGKGLEFVAGFTATGNTNYADSVKGRFTISRDNSKN Lamanade TLYLQMNSLRAEDTAVYYCYRLDKWGQGTLVTVSS (CDR3changed)hVHHA Amino QVKLEESGGGLVQAGRSLRLSCAASGTDFSINFMGWFR based on acid QAPGKERESVAVFTATGNTSYADSVKGRFTISRDNAKK Ciltacabta TLYLQMNSLKPEDTAVYYCYMLDKWGQGTQVTVSS gene CDRGraftingDatopota Amino QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQW mab HC- acid VRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISAD VHHA12 TSTSTAYLQLSSLKSEDTAVYYCARSGFGSSYWYFDVW GQGTLVTVSSCSTKGPSVFPLAPSSKSTSGGTAALGCLV KD YFPEP VT VS WNSGALT SGVHTFP AVLQ S SGLYSL S S VVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSS GGSGGSGGSEVQLVESGGGVVQPGGSLKLSCVASGTDF SINFIRWYRQ APGKQREF VAGITATGNTNYAD SMKGRF TISRDNTKNAVYLQIDSLKPEDTAVYYCYMLDKWGQG TQVTVSSDatopota Amino DIQMTQ SP S SL S AS VGDRVTITCKASQD VST A VAWYQQ mab LC acid KPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSL QPEDFAVYYCQQHYITPLTFGQGTKLEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWCVDNAL Q S GNS QE S VTEQD SKD S T YSL S STLTL SK AD YEKHK VY ACEVTHQGLSSPVTKSFNRGECDatopota Amino QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQW mab HC- acid VRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISAD knob T ST STA YLQL S SLK SEDT A VYYC ARSGFGS S YW YFD VWGQGTLVTVSSCSTKGPSVFPLAPSSKSTSGGTAALGCLVDOCKET NO: 91016-434826KD YFPEP VT VS WNSGALT SGVHTFP AVLQ S SGLYSL S S VVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGST YRVVSVLTVLAQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPS DIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGSS HHHHHHDatopota Amino QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQW mab HC- acid VRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISAD VHHA12- T ST STAYLQL S SLKSEDT AVYYC ARSGFG S S YWYFD VW hole GQGTLVTVSSCSTKGPSVFPLAPSSKSTSGGTAALGCLV KD YFPEP VT VS WNSGALT SGVHTFP AVLQ S SGLYSL S S VVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQ VYTLPP SRDELTKNQ VSLSC AVKGF YP S DIAVE WESNGQPENNYKTTPPVLDSDGSFFLVSKLTVD KSRWQQGNVF SC S VMHE ALHNHYTQKSL SL SPGKGS S GGSGGSGGSEVQLVESGGGVVQPGGSLKLSCVASGTDF SINFIRWYRQAPGKQREFVAGITATGNTNYADSMKGRF TISRDNTKNAVYLQID SLKPEDT AVYYC YMLDKWGQG TQVTVSSDatopota Amino QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQW mab HC- acid VRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISAD VHHA12 T ST STAYLQL S SLK SEDT AVYYC ARSGFGS S YWYFD VW GQGTLVTVSSCSTKGPSVFPLAPSSKSTSGGTAALGCLV KD YFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQ VYTLPP SREEMTKNQ VSLTCLVKGFYP S DIAVEWE SNGQPENNYKTTPP VLD SDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSS GGSGGSGGSEVQLVESGGGVVQPGGSLKLSCVASGTDF SINFIRWYRQAPGKQREFVAGITATGNTNYADSMKGRF TISRDNTKNAVYLQID SLKPEDT A VYYCAMLDKWGQG TQVTVSSDatopota Amino QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQW mab HC acid VRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISAD T ST STAYLQL S SLK SEDT AVYYC ARSGFGS S YWYFD VW GQGTLVTVSSCSTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEP VT VS WNSGALT SGVHTFP AVLQ S SGLYSL S SDOCKET NO: 91016-434826VVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPP SREEMTKNQ VSLTCLVKGF YPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKControl - Amino DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT Fc- acid CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY VHHA12 GSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE +5 KTISKCKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YP SDIAVEWESNGQPENNYKTTPP VLD SDG SFFLYSKLT VDK SRWQQGNVF SC S VMHEALHNHYTQKSL SL SPGKG SSGGSGGSGGSEVQLVESGGGVVQPGGSLKLSCVASGT DF SINFIRWYRQ APGKQREF VAGITATGNTNYAD SMKG RFTISRDNTKNAVYLQIDCLKPEDTAVYYCAMLDKWG QGTQVTVSSControl - Amino DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT Fc- acid CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY VHHA12 GSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKCKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YP SDIAVEWE SNGQPENNYKTTPP VLD SDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKG SSGGSGGSGGSEVQLVESGGGVVQPGGSLKLSCVASGT DF SINFIRWYRQ APGKQREF VAGITATGNTNYAD SMKG RFTISRDNTKNAVYLQIDCLKPEDTAVYYCYMLDKWG QGTQVTVSSTrop2 Amino QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQW TransTAC acid VRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISAD vl.l HC TSTSTAYLQLSSLKSEDTAVYYCARSGFGSSYWYFDVW GQGTLVTVSSCSTKGPSVFPLAPSSKSTSGGTAALGCLV KD YFPEP VT VS WNSGALT SGVHTFP AVLQ S SGLYSL S S VVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS K AKGQPREPQ VYTLPP SREEMTKNQ VSLTCLVKGF YPS DIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSS GGSGGSGGSEVQLVESGGGVVQPGGSLKLSCVASGTDF SINFIRWYRQ APGKQREF VAGITATGNTNYAD SMKGRF TISRDNTKNAVYLQIDSLKPEDTAVYYCAMLDKWGQG TQVTVSSTrop2 Amino DIQMTQ SP S SL S AS VGDRVTITCKASQD VST A VAWYQQ TransTAC acid KPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLvl.l LC QPEDFAVYYCQQHYITPLTFGQGTKLEIKRTVAAPSVFIDOCKET NO: 91016-434826FPPSDEQLKSGTASVVCLLNNFYPREAKVQWCVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC TROP2 Amino QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQW (POI) acid VRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISAD Binder TSTSTAYLQLSSLKSEDTAVYYCARSGFGSSYWYFDVW HC GQGTLVTVSSCSTKGPSVFPLAPSSKSTSGGTAALGCLV KD YFPEP VT VS WNSGALT SGVHTFP AVLQ S SGLYSL S S VVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVI< FNWYVDGVEVHNAI< TI< PREEQYGST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD K SRWQQGNVF SC S VMHE ALHNHYTQKSL SL SPGK HC Knob Amino QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQW acid VRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISAD TSTSTAYLQLSSLKSEDTAVYYCARSGFGSSYWYFDVW GQGTLVTVSSCSTKGPSVFPLAPSSKSTSGGTAALGCLV KD YFPEP VT VS WNSGALT SGVHTFP AVLQ S SGLYSL S S VVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVI< FNWYVDGVEVHNAI< TI< PREEQYGST YRVVSVLTVLAQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPS DIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGSS HHHHHH TROP2 Amino QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQW (POI) acid VRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISAD Binder T ST STAYLQL S SLKSEDT AVYYC ARSGFGS S YWYFD VW HC GQGTLVTVSSCSTKGPSVFPLAPSSKSTSGGTAALGCLV KD YFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGST YRVVSVLTVLAQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQ VYTLPP SRDELTKNQ VSLWCL VKGF YP S DIAVEWE SNGQPENNYKTTPP VLD SDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGK TROP2 Amino QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQW (POI) acid VRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISAD Binder T ST STAYLQL S SLKSEDT AVYYC ARSGFGS S YWYFD VW HC to GQGTLVTVSSCSTKGPSVFPLAPSSKSTSGGTAALGCLV SEQ ID KD YFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSS163 VVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKDOCKET NO: 91016-434826THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS K AKGQPREPQ VYTLPP SRDELTKNQ VSLSC AVKGF YP S DIAVEWE SNGQPENNYKTTPP VLD SDGSFFLVSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKTrop2 Amino QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQW TransTAC acid VRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISAD V1.2HC T ST STA YLQL S SLK SEDT AVYYC ARSGFGS S YWYFD VW Hole GQGTLVTVSSCSTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS K AKGQPREPQ VYTLPP SRDELTKNQ VSLSC AVKGF YP S DIAVEWE SNGQPENNYKTTPP VLD SDGSFFLVSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSS GGSGGSGGSEVQLVESGGGVVQPGGSLKLSCVASGTDF SINFIRWYRQAPGKQREFVAGITATGNTNYADSMKGRF TISRDNTKNAVYLQIDSLKPEDTAVYYCYMLDKWGQG TQVTVSSTrop2 Amino QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQW TransTAC acid VRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISAD vl.3 HC T ST STA YLQL S SLK SEDT AVYYC ARSGFGS S YWYFD VW GQGTLVTVSSCSTKGPSVFPLAPSSKSTSGGTAALGCLV KD YFPEP VT VS WNSGALT SGVHTFP AVLQ S SGLYSL S S VVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVI< FNWYVDGVEVHNAI< TI< PREEQYGST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS K AKGQPREPQ VYTLPP SREEMTKNQVSLTCLVK GF YPS DIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSS GGSGGSGGSEVQLVESGGGVVQPGGSLKLSCVASGTDF SINFIRWYRQ APGKQREF VAGITATGNTNYAD SMKGRF TISRDNTKNAVYLQIDSLKPEDTAVYYCYMLDKWGQG TQVTVSS EGFR LC Amino DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRT acid NGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESED lADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWCVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC EGFR HC Amino QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRacid QSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSDOCKET NO: 91016-434826QVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGT LVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP S S SLGTQTYICN VNHKP SNTK VDKK VEPK SCGSGS VDG DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVI< FNWYVDGVEVHNAI< TI< PREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YP SDIAVEWESNGQPENNYKTTPP VLD SDGSFFLYSKLT VDKSRWQQGNVF SC S VMHEALHNHYTQKSLSLSPGK EGFR Amino QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVR TransTAC acid QSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKS vl.l HC QVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGT LVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP S S SLGTQTYICN VNHKP SNTK VDKK VEPK SCGSGS VDG DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVI< FNWYVDGVEVHNAI< TI< PREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YP SDIAVEWESNGQPENNYKTTPP VLD SDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKG SSGGSGGSGGSEVQLVESGGGVVQPGGSLKLSCVASGT DF SINFIRWYRQ APGKQREF VAGITATGNTNYAD SMKG RFTISRDNTKNAVYLQIDSLKPEDTAVYYCAMLDKWG QGTQVTVSS PVR LC Amino DIQMTQ SP S SL S AS VGDRVTITCRASQD VGT AVVWYQQ acid KPGKAPKLLIYW AS SRHEGVP SRF SGSGSGTDFTLTIS SL QPEDFATYFCQQYSRYPLTFGQGTKLEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWCVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC PVR HC Amino QVQLVQSGAEVKKPGASVKVSCKATGYTFSNYWIEWV acid RQAPGQGLEWIGEIFPGSGRINFNEKFKGRVTFTADTSIS TTYMELSRLRSDDTAVYYCARTKIYGNSFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKKAEPKSCGGSGSGGS GSGVDGDKTHTCPPCPAPELLRGPSVFLFPPKPKDTLMI SRTPEVTC V V V D V SHEDPEVKFNW YVDGVEVHNAKTK PREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA RPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDOCKET NO: 91016-434826PVR Amino QVQLVQSGAEVKKPGASVKVSCKATGYTFSNYWIEWV TransTAC acid RQAPGQGLEWIGEIFPGSGRINFNEKFKGRVTFTADTSIS vl.l HC TTYMELSRLRSDDTAVYYCARTKIYGNSFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKKAEPKSCGGSGSGGS GSGVDGDKTHTCPPCPAPELLRGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA RPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVF SC S VMHEALHNHYTQKSL SLSPGKGSSGGSGGSGGSEVQLVESGGGVVQPGGSLKL SCVASGTDFSINFIRWYRQAPGKQREFVAGITATGNTNY ADSMKGRFTISRDNTKNAVYLQIDSLKPEDTAVYYCA MLDKWGQGTQVTVS S ICAM1 Amino QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQR LC acid LPGAAPQLLIYNNDQRPSGIPDRFSGSKSGTSGSLVISGL QSEDEADYYCASWDDSLNGRVFGGGTKLTVLGQPKAN PTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWCAD GSP VKAGVETTKP SKQ SNNK YAAS S YL SLTPEQWKSHR S YSC Q VTHEGST VEKTVAPTEC S ICAM1 Amino QVQLQQSGPGLVKPSETLSLTCTVSGYSISSGYFWGWIR HC acid QPPGKGLEWIGSIYQSGSTYYNPSLKSRVTISLDTSKNQF SLKLSSVTAADTAVYYCARDGYCSGGSCYPFDYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCGGSGSG GSGSGVDGDKTHTCPPCPAPELLRGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSN KARPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCL VKGF YP SDIA VEWESNGQPENNYKTTPPVLD SDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK ICAM1 Amino QVQLQQSGPGLVKPSETLSLTCTVSGYSISSGYFWGWIR TransTAC acid QPPGKGLEWIGSIYQSGSTYYNPSLKSRVTISLDTSKNQF vl.l HC SLKLSSVTAADTAVYYCARDGYCSGGSCYPFDYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCGGSGSG GSGSGVDGDKTHTCPPCPAPELLRGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSN KARPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGF YP SDIA VEWESNGQPENNYKTTPPVLD SDGDOCKET NO: 91016-434826SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGKGSSGGSGGSGGSEVQLVESGGGVVQPGGSL KLSCVASGTDFSINFIRWYRQAPGKQREFVAGITATGNT NYADSMKGRFTISRDNTKNAVYLQIDSLKPEDTAVYYC AMLDKWGQGTQ VT VS S ICAM1 Amino QVQLQQSGPGLVKPSETLSLTCTVSGYSISSGYFWGWIR TransTAC acid QPPGKGLEWIGSIYQSGSTYYNPSLKSRVTISLDTSKNQF vl.2 HC SLKLSSVTAADTAVYYCARDGYCSGGSCYPFDYWGQG Knob TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCGGSGSG G SG SGVDGDKTHTCPPCP APELLGGP S VFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYGSTYRVVSVLTVLAQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LWCL VKGF YP SDIAVEWESNGQPENNYKTTPP VLD SDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQK SLSLSPGKGSSHHHHHH ICAM1 Amino QVQLQQSGPGLVKPSETLSLTCTVSGYSISSGYFWGWIR POIB to acid QPPGKGLEWIGSIYQSGSTYYNPSLKSRVTISLDTSKNQF SEQ ID SLKLSSVTAADTAVYYCARDGYCSGGSCYPFDYWGQG176 TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCGGSGSG GSGSGVDGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK ICAM1 Amino QVQLQQSGPGLVKPSETLSLTCTVSGYSISSGYFWGWIR TransTAC acid QPPGKGLEWIGSIYQSGSTYYNPSLKSRVTISLDTSKNQF vl.2 HC SLKLSSVTAADTAVYYCARDGYCSGGSCYPFDYWGQG Hole TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCGGSGSG GSGSGVDGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGKGSSGGSGGSGGSEVQLVESGGGVVQPGGSLKLSCVASGTDFSINFIRWYRQAPGKQREFVAGITATGNTDOCKET NO: 91016-434826NYADSMKGRFTISRDNTKNAVYLQIDSLKPEDTAVYYC YMLDKWGQGTQ VT VS S177 ICAM1 Amino QVQLQQSGPGLVKPSETLSLTCTVSGYSISSGYFWGWIR TransTAC acid QPPGKGLEWIGSIYQSGSTYYNPSLKSRVTISLDTSKNQF vl.3 HC SLKLSSVTAADTAVYYCARDGYCSGGSCYPFDYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCGGSGSG GSGSGVDGDKTHTCPPCPAPELLRGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSN KARPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCL VKGF YP SDIA VEWESNGQPENNYKTTPPVLD SDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGKGSSGGSGGSGGSEVQLVESGGGVVQPGGSL KLSCVASGTDFSINFIRWYRQAPGKQREFVAGITATGNT NYADSMKGRFTISRDNTKNAVYLQIDSLKPEDTAVYYCYMLDKWGQGTQ VTVS STarget degradation tracks with TransTAC-ADC potency

[0279] The data herein support a positive association between target protein degradation efficiency and ADC-mediated tumor cell killing suggesting that degraders represent strong “backbones” for TransTAC-ADC design. For (CD20 TransTAC vl. O vs vl.l). See, e.g. FIG. 39, which illustrates the ratio of binding affinity of the fusion protein-drug conjugates.

[0280] CD20 ADC does not effectively kill Raji cells, whereas CD20 TransTAC vl. O shows stronger tumor cell killing than vl.l. The CD20 antibody alone cannot drive CD20 degradation, while CD20 TransTAC vl. O induces more robust CD20 degradation than vl.l. Together, these results suggest that greater CD20 degradation efficiency correlates with improved functional efficacy (tumor killing) in the TransTAC-ADC framework.Generality across targets / generations

[0281] Multipe additional TransTAC geography also demonstrate target protein degradation, indicating these formats are ADC-compatible and can be expanded as a broader TransTAC-ADC design space (i.e., degraders as a screening criterion for selecting next ADC scaffolds).DOCKET NO: 91016-434826EQUIVALENTSThose skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention.

Claims

1. DOCKET NO: 91016-434826CLAIMSWhat is claimed is:

1. A fusion protein-drug conjugate of the formula:(Fl -Fl)- (D)nwherein:D is a therapeutic moiety;n is 1 to 8;Fl is a protein of the formula:R1 -R2 -R3:wherein:R1 is a protein of interest (POI) binder (POIB);R2 is a complementary IgG Fc region; andR3 is a transferrin receptor (TfR) binder (TRB), wherein the TRB to TfR binding affinity (KD) is about 10 nM to about 100 pM; andF1-F1 form a homodimer.

2. A fusion protein-drug conjugate of the formula:(Fl -Fl)- (D)nwherein:D is a therapeutic moiety;n is 1 to 8;Fl is a protein of the formula:R1– R2 – R3:wherein:R1 is a means for binding a protein of interest (POI);R2 is a complementary IgG Fc region; andR3 is a means for binding a transferrin receptor (TfR), wherein the means for binding the TB to TfR has a binding affinity (KD) of of about 10 nM to about 100 pM; andF1-F1 form a homodimer.DOCKET NO: 91016-4348263. A fusion protein-drug conjugate of the formula:(Fl -F2)-(D)nwherein:D is a therapeutic moiety;n is 1 to 8;Fl is a protein of the formula:R1 -R2 orRl -R2’:wherein:R1 is a protein of interest (POI) binder (POIB); andR2 is an IgG Fc region complementary to R2’;F2 is a protein of the formula:R3 -R2’ orR3 -R2:wherein:R2’ is an IgG Fc region complementary to R2; andR3 is a transferrin receptor (TfR) binder (TRB), wherein the TRB to TfR binding affinity (KD) is about 10 nM to about 100 pM; andF1-F2 form a heterodimer.

4. A fusion protein-drug conjugate of the formula:(Fl -F2)- (D)nwherein:D is a therapeutic moiety;n is 1 to 8;Fl is a protein of the formula:R1-R2 orRl -R2’:wherein:R1 is a means for binding a protein of interest (POI); andR2 is an IgG Fc region complementary to R2’;F2 is a protein of the formula:R3 - R2’ or R3 - R2:wherein:DOCKET NO: 91016-434826R2’ is an IgG Fc region complementary to R2; andR3 is a means for binding a transferrin receptor (TfR), wherein the means for binding the TfR has a binding affinity (KD) to TB of about 10 nM to about 100 pM; andF1-F2 form a heterodimer.

5. The fusion protein-drug conjugate of any one of claims 3-4 wherein the R2 and R2’ have complementary knob and hole structures.

6. The fusion protein-drug conjugate of claim 1, wherein D is conjugated to Fl at Rl.

7. The fusion protein-drug conjugate of claim 1, wherein D is conjugated to F 1 at R2.

8. The fusion protein-drug conjugate of claim 1, wherein D is conjugated to Fl at R3.

9. The fusion protein-drug conjugate of any one of claims 1-5, wherein D is conjugated to Fl atRl, and to Fl atR3.

10. The fusion-protein drug conjugate of any one of claims 1-9, wherein the TRB is an scFv or a heavy chain variable region (VHH).

11. The fusion protein-drug conjugate of any one of claims 1-10, wherein the TRB comprises:a. a VH CDR1 comprising the amino acid sequence of GTDFSINF (SEQ ID NO:70), a VH CDR2 comprising the amino acid sequence of 1TATGNT (SEQ ID NO:73), and a VH CDR3 comprising the amino acid sequence of YMLDK (SEQ ID NO: 72) [[VHHA-12]; orb. a VH CDR1 comprising the amino acid sequence of GTDFSINF (SEQ ID NO:70), a VH CDR2 comprising the amino acid sequence of ITATGNT (SEQ ID NO: 73), and a VH CDR3 comprising the amino acid sequence of AMLDK (SEQ ID NO: 74) [[VHHA-12+5]; orc. a VH CDR1 comprising the amino acid sequence of GTDFSINF (SEQ ID NO:70), a VH CDR2 comprising the amino acid sequence of ITATGNT (SEQ ID NO:DOCKET NO: 91016-43482673), and a VH CDR3 comprising the amino acid sequence of YMADK (SEQ ID NO: 75)-[[VHHA-12+7],12. The fusion protein-drug conjugate of any one of claims 1-11, wherein the POIB is an antibody or antibody fragment.

13. The fusion protein-drug conjugate of any one of claims 1-12, wherein the fusion proteindrug conjugate binds the POI with an affinity (KD) of between 0.1 nM - 10 nM.

14. The fusion protein-drug conjugate of any one of claims 1-13, wherein the fusion proteindrug conjugate binds the TfR and the POI with a ratio of affinities (TfR KD / POI KD) of between about 0.01-5000.

15. The fusion protein-drug conjugate of claim 1-14, wherein the fusion protein-drug conjugate binds TfR. with an affinity (KD) of about 10 nM to about 10 μM.

16. The fusion protein-drug conjugate of any one of claims 1-15, wherein the therapeutic moiety comprises a microtubule inhibitor, a DNA-damaging agent, a topoisomerase inhibitor, a cell death inducer, or a combination thereof.

17. The fusion protein-drug conjugate of any one of claims 1-16, wherein the POI is selected from the group consisting of EGFR, CD20, CCR6, MCT1, ICAM1, BST2, PVR, LAT1, and Trop-2.

18. The fusion protein-drug conjugate of any one of claims 1-17, wherein the POI is selected from the group consisting of HERZ, CD30, CD22, CD33, CD79b, Nectin-4, BCMA, CD19, HER3, and CD25.

19. A nucleic acid encoding the fusion- protein drug conjugate of any one of claims 1-18.

20. A pharmaceutical composition comprising the fusion protein-drug conjugate of any one of claims 1-18 and a pharmaceutically acceptable carrier, diluent, or excipient.DOCKET NO: 91016-43482621. A method of administering a therapeutic moiety to a subject, comprising administering an effective amount of the fusion protein-drug conjugate of any one of claims 1-18 or the pharmaceutical composition of claim 20 to the subject.

22. A method of treating a subject afflicted with a neurogenerative disease, an autoimmune disease or a cancer, comprising administering an effective amount of the fusion proteindrug conjugate of any one of claims 1-18, or the pharmaceutical composition of claim 20.

23. A method of treating a symptom of a neurogenerative disease, an autoimmune disease or a cancer, comprising administering to a subject in need thereof an effective amount of the fusion protein-drug conjugate of any one of claims 1-18, or the pharmaceutical composition of claim 20.

24. The fusion protein-drug conjugate of any one of claims 1-18, wherein the TRB comprises a serine at position 7 of SEQ ID NO: 2, 3, or 4 substituted for a cysteine, a serine at position 84 of SEQ ID NO: 2, 3, or 4 substituted for a cysteine, or a combination thereof.

25. The fusion protein-drug conjugate of claim 24, wherein the TRB comprising SEQ ID NO: 2, 3, or 4 is humanized.- 111 -

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