Antibody-NKG2D Ligand Domain Fusion Protein

Abstract

The present disclosure provides an antibody fusion protein comprising: (i) a heavy chain comprising a variable region sequence comprising the amino acid sequence of SEQ ID NO: 1; and (ii) a light chain comprising a variable region sequence comprising the amino acid sequence of SEQ ID NO: 8, wherein the light chain is fused at its C-terminus to an A1-A2 domain comprising the amino acid sequence of SEQ ID NO: 11. Nucleic acids encoding all or a portion of the antibody fusion protein are provided, as well as methods of using the antibody fusion protein, e.g., in treating CD20-positive cancers. The present disclosure further provides mutant A1-A2 domain peptides.

Description

[Technical field]

[0001] The present invention relates to the A1-A2 domain of a non-natural NKG2D ligand that binds to a non-natural NKG2D receptor, and to an antibody fusion protein comprising this domain.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 208,407, filed June 8, 2021, the entire contents of which are hereby fully incorporated by reference.

[0003] Incorporation by Reference of Electronically Submitted Materials The computer readable nucleotide / amino acid sequence listing, submitted contemporaneously herewith and identified as follows, is incorporated herein by reference in its entirety: 63,727 byte ASCII (text) file entitled "56867_Seqlisting.txt", created on June 7, 2022. [Background technology]

[0004] Engineering patient-derived T cells to express chimeric antigen receptors (CARs) has changed the landscape of adoptive cell therapy, providing scientists and clinicians with the ability to harness the potent cytolytic capacity of T cells to direct them to specific antigen-expressing targets in an MHC-independent manner. Initial application to treat hematological tumors has yielded astounding responses, leading to a surge in research efforts to facilitate their effective use in non-hematological indications. However, CAR-T cell therapy is limited by the utilization of single-purpose targeting domains, lack of dose control that may contribute to cytokine release syndrome, inability to accommodate tumor antigen loss leading to disease recurrence, and immunogenicity of non-human targeting domains leading to lack of persistence. There is a need in the art for improved CAR-based cell therapies to address these limitations of current treatment options. Summary of the Invention

[0005] The present disclosure provides an antibody fusion protein comprising: (i) a heavy chain comprising a variable region sequence comprising the amino acid sequence of SEQ ID NO:1; and (ii) a light chain comprising a variable region sequence comprising the amino acid sequence of SEQ ID NO:8, wherein the light chain is fused at its C-terminus to an A1-A2 domain comprising the amino acid sequence of SEQ ID NO:11. In various embodiments, the heavy chain comprises a constant domain comprising the amino acid sequence of SEQ ID NO:3. Optionally, the A1-A2 domain is fused to the light chain via a linker comprising the amino acid sequence of SEQ ID NO:10. In this regard, the light chain, in various embodiments, comprises the amino acid sequence of SEQ ID NO:13. In various embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:7.

[0006] The present disclosure further provides a nucleic acid molecule comprising a nucleotide sequence encoding a light chain of an antibody fusion protein (e.g., a light chain comprising a variable region sequence comprising the amino acid sequence of SEQ ID NO:8, wherein the light chain is fused at its C-terminus to an A1-A2 domain comprising the amino acid sequence of SEQ ID NO:11). The present disclosure further provides a composition comprising a nucleic acid molecule encoding a light chain of an antibody fusion protein and a nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain of an antibody fusion protein described herein (e.g., a heavy chain comprising a variable region sequence comprising the amino acid sequence of SEQ ID NO:1). Also provided is an expression vector comprising a nucleic acid molecule encoding a light chain of an antibody fusion protein described herein, and optionally further comprising a nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain of an antibody fusion protein described herein. Further provided is a host cell comprising an expression vector described herein. The present disclosure provides a host cell comprising a nucleic acid molecule comprising a nucleotide sequence encoding a light chain of an antibody fusion protein and a nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain of an antibody fusion protein. Also provided is a method of producing an antibody fusion protein comprising culturing a host cell comprising a nucleic acid molecule comprising a nucleotide sequence encoding a light chain of the antibody fusion protein and a nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain of the antibody fusion protein, and recovering the antibody fusion protein.

[0007] Also provided are kits comprising one or more containers comprising an antibody fusion protein described herein. Optionally, the kit further comprises one or more containers comprising a mammalian cell (e.g., a human lymphocyte or a human macrophage) comprising a chimeric antigen receptor comprising SEQ ID NO: 15. In various embodiments, the chimeric antigen receptor further comprises SEQ ID NOs: 16-18.

[0008] The disclosure further provides a method of treating a subject suffering from a CD20 positive cancer, comprising administering to the subject an antibody fusion protein as described herein and a mammalian cell (e.g., a human lymphocyte or a human macrophage) comprising a chimeric antigen receptor comprising SEQ ID NO: 15. Optionally, the chimeric antigen receptor further comprises SEQ ID NOs: 16-18. Uses of the antibody fusion proteins and mammalian cells for treating CD20 positive cancer, as well as use of the antibody fusion proteins and mammalian cells in the preparation of a medicament for treating CD20 positive cancer, are provided.

[0009] The disclosure also provides an A1-A2 domain peptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:30, wherein the peptide comprises an alanine or glutamine at one or more of positions 40, 54, and / or 84 of SEQ ID NO:30. In various embodiments, the peptide comprises glutamine residues at positions 40 and 54 of SEQ ID NO:30. Optionally, the peptide comprises a glutamine at position 84 of SEQ ID NO:30, or an alanine at position 84 of SEQ ID NO:30.

[0010] Although various embodiments herein are presented using the term "comprising," it should also be understood that under various circumstances, relevant embodiments may be described using the terms "consisting of" or "consisting essentially of." The present disclosure contemplates embodiments described as "comprising" a feature, including embodiments "consisting of" or "consisting essentially of" the feature. The terms "a" or "an" refer to one or more. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein. The term "or" should be understood to encompass items alternatively or together, unless the context clearly dictates otherwise.

[0011] The recitation of ranges of values ​​herein is intended to serve as a shorthand method of referring individually to each individual value within the range and each endpoint, unless otherwise indicated herein, and each individual value and endpoint is incorporated herein as if it were recited individually herein. However, the description also contemplates the same range, but excluding lower and / or higher endpoints. When the term "about" is used, it means plus or minus 5%, 10%, or more of the recited number. The actual variation intended will be determined from the context.

[0012] All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any examples provided herein, or the use of exemplary language (e.g., "etc.") are intended merely to better illustrate the disclosure, and do not limit the scope of the disclosure unless otherwise claimed. Only those limitations described herein as being important to the invention should be considered as such, and variations of the invention lacking limitations not described herein as being important are contemplated as aspects of the invention.

[0013] Additional features and variations of the present invention will be apparent to those skilled in the art from the entirety of this application, including the drawings and detailed description, and all such features are intended as aspects of the present invention. Similarly, the features of the present invention described herein, regardless of whether a combination of features is designated as an aspect or embodiment of the present invention, can be recombined into additional embodiments, which are also intended as aspects of the present invention. It is intended that the entire document be related as a unified disclosure, and it is understood that all combinations of features described herein (even if described in separate sections) are contemplated, even if the combinations of features are not found together in the same sentence, paragraph, or section of this document. [Brief description of the drawings]

[0014] [Figure 1] 1 is a chart providing various sequences described herein. [Figure 2A] Octet BLI kinetic binding data for the interaction of His-tagged monomeric wild-type MIC ligands with either wild-type NKG2D or iNKG2D.YA are shown. Fc-wtNKG2D or Fc-iNKG2D.YA were captured on an anti-human IgG Fc capture (AHC) biosensor tip in association with a dilution series of each ligand after baseline establishment (values ​​in brackets indicate the highest concentration examined). ULBP4 was not included in this assay as it could not be expressed and purified as a monomer. Note that all axes are on the same scale (binding - 0 nm, 0.4 nm, 0.8 nm, 1.2 nm (y-axis); time - 0 s, 50 s, 100 s, 150 s, 200 s, 250 s, 300 s, 350 s (x-axis)). Data are from a single experiment. [Figure 2B]ELISA results confirming that iNKG2D.YA cannot associate with natural ligand. Ligand-Fc fusions (R&D Biosystems) were coated onto microtiter plates and titrations of biotinylated Fc-wtNKG2D (dashed line) or Fc-iNKG2D.YA (solid line) were applied and detected by streptavidin-HRP. (ELISA signal (OD450) (y-axis); nM Ligand Fc (x-axis).) [Figure 3A-3D]Selective binding of orthogonal U2S3 ligand (A1-A2 domain) to NKG2D Y152A / 199F (iNKG2D.AF) (NKG2D ectodomain of the present disclosure). Library design and phage panning performed as described for iNKG2D.YA, except biotinylated double mutant Fc-iNKG2D.AF was used during selection rounds against increasing concentrations of Fc-wtNKG2D competitor. Data represent Octet BLI binding data of a single experiment (FIG. 3A) for the interaction of monomeric ligand to either Fc-wtNKG2D or Fc-iNKG2D.AF. Data are from two experiments. (FIG. 3B) Lead variants selected from the phage display library were cloned as fusions to the C-terminus of the rituximab light chain and differentially bound to Fc-wtNKG2D, Fc-iNKG2D.YA, and Fc-iNKG2D.AF and quantified by ELISA. Shown are four variants that selectively engage Fc-iNKG2D.YA but not the other two receptors. In the line graph of FIG. 3B, wtNKG2D is represented by diamonds, iNKG2D is represented by squares, and iNKG2D.AF is represented by triangles. (FIG. 3C) ELISA showing exclusivity of U2S3 and U2R ligand binding to selected receptor variants (Fc-iNKG2D.YA and Fc-iNKG2D.AF, respectively). U2S3 ligands are further described, for example, in U.S. Patent Publication No. 2019 / 0300594, which is incorporated herein by reference. (FIG. 3D) Calcein release assay using CD8+ T cells expressing either iNKG2D.YA-CAR or iNKG2D.AF-CAR and Ramos target cells at an effector:target ratio (E:T) of 20:1, and titration of rituximab LC-U2S3 or rituximab LC-U2R. Error bars represent ±SD of technical replicates. [Figure 4A] Relative binding of selected phage to Fc-iNKG2D.YA and Fc-wtNKG2D after the third and fourth rounds of panning in the presence of increasing concentrations of wtNKG2D competitor. Phage clones in the part of the graph enclosed in triangles were selected for further characterization. [Figure 4B] Three phage variants - S1, S2, S3 - were expressed as MicAbodies as fusions to the C-terminus of the anti-FGFR3 antibody clone R3Mab heavy chain and tested for the ability of selected variants to retain Fc-iNKG2D.YA binding (solid lines) preferentially over Fc-wtNKG2D (dashed lines) along with wild-type ULBP2 and the R81W version. All purified MicAbodies retained binding to human FGFR3 (data not shown). [Figure 4C] Binding analysis of orthogonal U2S3 ligand binding to His-tagged monomeric wild-type ULBP2, ULBP2 R81W, and Fc-NKG2D and Fc-iNKG2D.YA. Fc-wtNKG2D or Fc-iNKG2D.YA were captured on an anti-human IgG Fc capture (AHC) biosensor tip and then correlated with a dilution series of ligand. Data are from a single experiment. [Diagram 5] Validation of U2S3 orthogonality when fused to the C-terminus of either the heavy or light chain of Rituximab by Octet BLI. Fc-wtNKG2D or Fc-iNKG2D.YA were captured on an anti-human IgG Fc capture (AHC) biosensor tip and then associated with a two-fold dilution series of MicAbody starting at 50 nM. The y-axis, corresponding to the binding response, was set to the same scale for all sensorgrams. Kd values ​​could only be calculated for the two positive binding interactions shown. [Figure 6] Schematic diagram of the iNKG2D.YACAR receptor starting from the N-terminus of the polypeptide on the left, including the signal sequence (SS) not present in the mature type I transmembrane protein. Underlined sequences correspond to the signal sequence, italicized sequences correspond to the iNKG2D domain (SEQ ID NO: 15), sequences without special indication correspond to the CD8a hinge / transmembrane domain (SEQ ID NO: 16), underlined italicized sequences correspond to the 4-1BB domain (SEQ ID NO: 17), bold sequences correspond to the CD3 zeta domain (SEQ ID NO: 18), double underlined sequences correspond to the linker, and dotted underlined sequences correspond to the eGFP (green fluorescent protein) sequence. [Figure 7A-7C]Elements of convertibleCAR. (Figure 7A) Overview of the operations to convert the components of the NKG2D-MIC axis into a convertibleCAR system. iNKG2D.YA and U2S3 became components of the second generation CAR receptor and bispecific adapter molecule (MicAbody), respectively. "TAA" is a tumor-associated antigen. (Figure 7B) Representative example of highly efficient lentiviral transduction of iNKG2D.YA-CAR into either CD4 or CD8 cells. Although transduction efficiency differed between donors, >70% GFP+ yield was consistently achieved. For comparison, the RITscFv-CAR is shown, which has the same architecture as the iNKG2D-CAR, except that an scFv based on the VH / VL domains of rituximab was used instead of iNKG2D.YA. (FIG. 7C) Surface expression of iNKG2D.YA-CAR was determined in CD8+ T cells by incubating cells with Rituximab.LC-U2S3 MicAbody followed by PE-conjugated mouse-anti-human kappa chain antibody staining. For comparison, non-transduced T cells are shown. [Figure 8A-8C]Ligand-dependent activation and MicAbody-dependent receptor internalization of CD8+ T cells expressing iNKG2D-CAR. (Figure 8A) CD8+ T cells were transduced with CAR constructs consisting of either wild-type NKG2D or iNKG2D.YA as the receptor domain. Wild-type His-tagged monomeric ligand or His-tagged monomeric U2S3 were coated onto wells of microtiter plates in a 1:3 dilution series starting at 10ug / mL. 1x105 CAR-expressing cells were introduced into wells in a volume of 150uL without exogenous IL2, and after 24 hours, supernatants were collected and the amount of cytokines produced and released was quantified by cytokine-specific ELISA. ULBP4 was not included in the assay because a His-tagged version could not be expressed and purified. (Figure 8B) CD8+ cells expressing either iNKG2D-CAR or RITscFv-CAR were co-cultured with Ramos cells at an E:T of 4:1 with increasing concentrations of Rit-S3 MicAbody (nM) in the case of iNKG2D-CAR cells. After 24 h, culture supernatants were harvested and released cytokines were quantified by ELISA. Cell lysis was measured by calcein release after 2 h of co-incubation. All error bars are ±SD of technical triplicate measurements. All data are from a single experiment. (Figure 8C) iNKG2D-CD8+ cells were pre-incubated with 5 nM of trastuzumab.LC-U2S3 MicAbody and then exposed to wells pre-coated with a titration of Her2. After 2 h, cells were incubated with anti-kappa-PE antibody to detect surface accessible MicAbody and GFP was examined to determine the total level of iNKG2D-CAR expressed. [Figure 9A-9D]In vitro characterization of convertibleCAR activity. (Figure 9A) Ramos (CD20+) target cells were exposed to convertibleCAR-CD8 cells at an E:T of 5:1 and co-cultured with increasing concentrations of Rituximab antibody (ADCC deficient), Rituximab.LC-U2S3 MicAbody, or Trastuzumab.LC-U2S3 MicAbody. After 24 hours, supernatants were harvested and IL-2 (solid bars) or IFNγ (hatched bars) were quantified by ELISA. Rit-U2S3 was the only sample that showed cytokine release at ≥5000 pg / mL. (Figure 9B) ConvertibleCAR-CD8 cells were incubated with increasing concentrations of Alexa Fluor 647-conjugated Rituximab.LC-U2S3 for 30 minutes, the excess was washed away, and MFI was quantified by flow cytometry. 5 nM marks the inflection point where the receptor is maximally occupied. (Figure 9C) ConvertibleCAR-CD8 cells were armed with increasing concentrations of Rituximab.LC-U2S3 as described in (B) and then co-incubated with Ramos cells supplemented with calcein at 20:1 E:T for 2 hours, after which the amount of calcein released was quantified. (Figure 9D) iNKG2D.YA-CAR CD8+ cells were pre-armed with 5 nM Rituximab.LC-U2S3, 5 nM Trastuzumab.LC-U2S3, or an equimolar mixture of 2.5 nM of each as described in (B) and then exposed to calcein-supplemented Ramos cells or CT26-Her2 cells at the two E:T ratios indicated. The amount of calcein released was quantified after 2 hours. Except for Figure 9B, data are from at least two independent experiments and are plotted as the average of technical triplicate measurements. [Figure 10A-10C]Comparison of heavy vs. light chain U2S3 fusions with Rituximab (ADCC-) antibody. (FIG. 10A) Pharmacokinetics of serum Rituximab-U2S3 MicAbody levels after 100ug intravenous administration in NSG mice in the absence of human T cells or tumor. All MicAbody and antibody controls used were ADCC deficient. The graph on the left is a comparison of light chain U2S3 fusions with parental antibody, and the graph on the right is a comparison of heavy chain U2S3 fusions with parental antibody. All error bars are ±SD of technical triplicate measurements. (FIG. 10B) In vitro calcein release assay after co-culture of iNKG2D-CAR CD8+ T cells with Ramos target cells at 20:1 E:T for 2 hours and titration of Rituximab-MicAbody. Error bars represent ±SD of experiments and data are from multiple experiments. The top line in the graph corresponds to Rituxumab.LC-U2S3, the middle line corresponds to Rituxumab.HC-U2S3, and the bottom line corresponds to Rituximab. (FIG. 10C) ELISA showing binding of Rituximab LC-U2S3 to mouse NKG2D. A480 absorbance values ​​are shown. Trastuzumab.LC-Rae1b, which has mouse wild-type Rae1b ligand that naturally binds to mouse NKG2D, was included as a positive control. [Figures 11A-11E] Control of disseminated Raji B cell lymphoma in NSG mice. Includes (FIG. 11A) mean luminescence output ± SD for each cohort and traces of individual animals from groups that received (FIG. 11B) 5×106 or (FIG. 11C) 15×106 total T cells. (FIG. 11D) T cell dynamics over the course of the study examining human CD3+ cells in blood and (FIG. 11E) bound MicAbody detected by anti-F(ab')2. Shown are cohort means ± SD, n=5. [Figures 12A-12C]Control of Raji tumors implanted subcutaneously in NSG mice by convertible CAR-T cells. (FIG. 12A) Mean tumor volume for each cohort, n=5. Error bars are not shown on the graph because tumors vary widely in size within each group. The two cohorts, inverted triangles (7M cCAR-T+60ug Ritux-S3) and squares (35M pre-armed cCAR-T), overlap and cannot be distinguished on the graph beyond day 26. (FIG. 12B) Bar graph showing serum Rit-S3 levels 14, 21, and 45 days after implantation. Three bars are provided for each time point: 35M pre-armed cCAR-T (left bar), 7M cCAR-T+60ug Ritux-S3 (middle bar), and 35M cCAR-T+60ug Ritux-S3 (right bar). Error bars indicate ±SD samples from mice in a given cohort. (FIG. 12C) CD3+ T cell kinetics in blood and quantification of the percentage of T cells by surface-associated MicAbodyF(ab')2 staining are shown with ±SD error bars. [Fig. 13A-13F]Targeted recruitment of complement factor C1q to iNKG2D.AF-CAR cells induces their complement-mediated depletion. (Figure 13A) Structure of an orthogonal ligand fusion to the Fc portion of human IgG expressed as either an N-terminal or C-terminal fusion. In addition to wild-type Fc, two sets of mutations in the CH2 domain that enhance C1q binding, S267E / H268F / S324T / G236A / I332E (“EFTAE”) and K326A / E333A (“AA”), were explored independently. (Figure 13B) ELISA examining the binding of human C1q to each purified fusion protein. The rank order of Kd was EFTAE < AA < wt (0.12, 0.35, and 0.67 nM, respectively), regardless of the direction of fusion. (Figures 13C and 13D) Complement-dependent cytotoxicity (CDC) assays for C1q binding enhance Fc fusion. iNKG2D.AF-CAR or non-transduced CD8+ T cells were incubated for 3 hours while titrating each fusion molecule and 10% normal human serum complement, and then dead T cells were enumerated using SYTOX Red. (Figures 13E and 13F) CDC assay using a U2S3 orthogonal ligand fusion to induce complement in iNKG2D.YA-CAR cells as described above (Figure 13C). All error bars are ±SD of triplicate technical measurements using iNKG2D-AF and iNKG2D-YA performed as separate experiments. [Figures 14A-14E]Targeted delivery of mutant IL2 cytokines to iNKG2D-CAR CD8+ T cells. (Figure 14A) In vitro expansion after 3 days of treatment of wtNKG2D-CAR (left bar) or iNKG2D.YA-CAR (right bar) with 30 IUe / mL of cytokine or cytokine-U2S2 fusion. Dark shading is to highlight selectivity. (Figure 14B) Low efficiency (45% GFP+) iNKG2D.YA-CAR transductants were cultured with 30 IUe / mL of non-selective (U2R81W) or iNKG2D.YA-selective (U2S2) mutIL2 fusion and maintained for 7 days. Cells were periodically examined by flow cytometry to quantitate the percentage of GFP+ cells in each population. The upper line corresponds to U2S2-hFc-mutlL2 (squares) and U2S2-mutlL2 (circles), the lower line corresponds to U2R80W-mutlL2 (circles) and U2R80W-hFc-mutlL2 (squares). (Figure 14C) iNKG2D-CAR CD8+ T cells were cultured with either wild type IL-2 or U2S3-hFc-mutIL2 at 30 IUe / mL and then co-cultured with Ramos cells at 20:1 E:T with increasing concentrations of Rituximab LC-U2S3. Free calcein was quantified and non-transduced CD8+ cells were maintained in rhIL-2, which served as a negative control. (Figure 14D) Non-transduced (right bars) or iNKG2D-CAR CAR CD8+ T cells (left bars) were incubated with various cytokine molecules for 3 days and proliferation was quantified. Control molecules included monomeric U2S3-hFc, and Rit-S3 MicAbody. Values ​​in brackets are IUe / mL concentrations tested. Data shown are the average of technical triplicate determinations. (FIG. 14E) Serum PK of U2S3-hFc-mutIL2 after IP injection of 60 ug in NSG mice (N=3). All error bars are ±SD of biological triplicate determinations. Data are from at least two experiments. [Figure 15A-15B]In vivo response of convertibleCAR-T cells to U2S3-hFc-mutIL2. (Figure 15A) NSG mice were injected with a total of 7x106 iNKG2D-transduced cells (CD4:CD8 1:1). After T cell contraction on day 14, mice were injected once a week with 30ug U2S3-hFc-mutIL2 or PBS (indicated by triangles) and T cell dynamics were monitored by flow cytometry. Percentage of human CD3+ T cells in peripheral blood is shown, each trace corresponds to an individual mouse, n=5. (Figure 15B) Plot of CD8+ cell proliferation and increase in percentage of GFP+ (CAR-expressing) cells upon U2S3-mutIL2 treatment. The upper cluster of lines corresponds to %GFP+ of CD8+ cells and the lower cluster of lines corresponds to %CD8+ in blood. [Figure 16] Responsiveness of human PBMC to U2S3-hFc-mutIL2. Human PBMC from three donors were incubated with increasing concentrations of U2S3-hFc-mutIL2 or U2S3-hFc-wtIL2 along with controls for four days. To quantify the proliferative response under each condition, each of the labeled cell types was examined for the marker Ki-67. Eleven bars are shown for each of donors 1, 2, and 3, and the bars represent, from left to right in each panel, untreated anti-CD3 [2ug / ml], IL-2 [300IUe / ml], mutIL2 [30IUe / ml], mutIL2 [300IUe / ml], mutIL2 [3000IUe / ml], mutIL2 [30000IUe / ml], wtIL2 [30IUe / ml], wtIL2 [300IUe / ml], wtIL2 [3000IUe / ml], and wtIL2 [30000IUe / ml]. Error bars are ±SD of triplicate determinations, and data represent a single experiment. [Figure 17A-17B]Tests evaluating MicAbodies with A1-A2 domains attached at different positions and using different linkers are shown. Figure 17A shows the constructs tested. Rit.HCd.S3 corresponds to a rituximab antibody comprising the U2S3 A1-A2 domain as described in the Examples fused to the heavy chain via a GGGS (SEQ ID NO: 14) linker. Rit.HCd.apts.S3 corresponds to a rituximab antibody comprising the U2S3 A1-A2 domain fused to the heavy chain via an APTSSGGGGS (SEQ ID NO: 10) linker. Rit.HCd.LC.S3 corresponds to a rituximab antibody comprising the U2S3 A1-A2 domain fused to the light chain via an APTSSGGGGS (SEQ ID NO: 10) linker. Rit.HCd.LC.gggs.S3 corresponds to a rituximab antibody comprising the U2S3 A1-A2 domain fused to the light chain via a GGGS (SEQ ID NO: 14) linker. FIG. 17B is a bar graph showing cell lysis (% max; y-axis) achieved using various concentrations of MicAbody in an in vitro calcein release assay after co-culture of iNKG2D-CAR CD8+ T cells with Ramos target cells for 2 hours at E:T of 20:1 and titration of Rituximab-MicAbody. For each construct, the maximum % of cell lysis is shown for 0 nM (1st bar), 0.008 nM (2nd bar), 0.04 nM (3rd bar), 0.2 nM (4th bar), 1 nM (5th bar), and 5 nM (6th bar) of MicAbody. Rit.HCd.LC.S3 (comprising A1-A2 domains on the light chain linked by an APTSSSGGGGS (SEQ ID NO: 10) linker) was superior to the version with the GGGS (SEQ ID NO: 14) linker and was superior to constructs with A1-A2 domains fused to the heavy chain, regardless of linker. [Figure 18] 1 is a line graph showing iNKG2D.YA capture using rituximab fusion proteins containing the A1-A2 domains of SEQ ID NO:30 (U2S3) or SEQ ID NO:11 (U2S3(NQ)) fused to the light chain of the antibody. The A1-A2 domains with substitutions at positions 40 and 54 relative to SEQ ID NO:30 performed similarly to the A1-A2 domain of SEQ ID NO:30. [Figure 19] 1 is a line graph showing the reduced ability of rituximab ("Rit") fusion proteins containing the A1-A2 domain of SEQ ID NO:30 (U2S3) or SEQ ID NO:11 (U2S3(NQ)) fused to the light chain of the antibody to bind wild-type NKG2D. "Rit.P" refers to the rituximab parent antibody that was not fused to the A1-A2 domain. "Rit.U2wt" refers to rituximab fused to a wild-type ULBP2 domain that is predicted to bind to wild-type NKG2D. "Rit.S3" and "Trast.S3" refer to rituximab or trastuzumab, respectively, fused to the U2S3 domain (SEQ ID NO:30). "Rit.NQ" refers to rituximab (SEQ ID NO:11) fused to U2S3(NQ). Introduction of the NQ mutation into the U2S3 domain did not affect the orthogonality of the construct and there was no reversion in terms of binding to wild-type NKG2D (ie, the construct did not bind to wild-type NKG2D). [Figure 20] 1 is a bar graph showing cell lysis (% max; y-axis) achieved using various concentrations of MicAbody in an in vitro calcein release assay after co-culture of iNKG2D-CAR CD8+ T cells with Ramos target cells for 2 hours with an E:T of 20:1 and titration of Rituximab-MicAbody. For each construct, the maximum % cell lysis is shown for 0 nM (1st bar), 0.008 nM (2nd bar), 0.04 nM (3rd bar), 0.2 nM (4th bar), 1 nM (5th bar), and 5 nM (6th bar) of MicAbody. Rit.S3 (containing the A1-A2 domain of SEQ ID NO:30), Rit.S3.NQ (containing the A1-A2 domain of SEQ ID NO:30 with glutamines at positions 40 and 54), Rit.S3.NQ.AYT (containing the A1-A2 domain of SEQ ID NO:30 with glutamines at positions 40 and 54 and alanine at position 82), and Rit.S3.NQ.QYT (containing the A1-A2 domain of SEQ ID NO:30 with glutamines at positions 40 and 54 and glutamic acid at position 82) were tested. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present disclosure provides fusion proteins comprising an antibody (or other antigen binding protein) and the A1-A2 domain of a non-natural NKG2D ligand. The non-natural NKG2D ligand selectively binds to a non-natural NKG2D receptor. In various embodiments of the present disclosure, the fusion proteins are used in conjunction with CAR-T cells that exhibit a non-natural NKG2D receptor to which the A1-A2 domain binds, thereby providing a powerful system for delivering tailored CAR-T cell therapy that overcomes many of the shortcomings of current CAR-T cell-based therapeutics. Unlike currently available CAR-T cell-based therapies, the fusion proteins and systems of the present disclosure allow for flexible targeting to direct T cell activity to selected antigens, multiplexing capabilities to reduce the likelihood of relapse associated with antigen loss, dose control for differential engagement of CAR-T cells, and selective delivery of modulating agents to CAR-expressing cells.

[0016] The present disclosure provides the A1-A2 domain of non-natural NKG2D ligands with particularly advantageous properties. NKG2D is an activating receptor expressed as a type II homodimeric integral membrane protein on natural killer (NK) cells, some myeloid cells, and certain T cells. Human NKG2D has eight different natural MIC ligands (MICA, MICB, ULBP1-ULBP6) that are upregulated on the surface of cells in response to various stresses, and their differential regulation provides the immune system with a means to respond to a wide range of urgent cues while minimizing collateral damage. Groh et al., Proc. Natl. Acad. Sci. USA 93, 12445-12450 (1996); Zwirner et al., Hum. Immunol. 60, 323-330 (1999); and Spies et al., Nat. Immunol. 9, 1013-1015 (2008). The structures of the NKG2D ectodomain, several soluble ligands, and the bound complex of the ligand to the ectodomain have been solved, revealing a saddle-shaped groove at the homodimer interface involving the structurally conserved A1-A2 domains of ligands with otherwise distinct amino acid identities (Li et al., Nat. Immunol. 2, 443-451 (2001), Radaev et al., Immunity 15, 1039-1049 (2001), Zuo et al., Sci Signal 10, (2017), and McFarland et al., Immunity 19, 803-812 (2003). The "A1-A2 domain" of the present disclosure is not a naturally occurring A1-A2 domain, but comprises an amino acid sequence that binds to a mutated version of the NKG2D ectodomain and does not bind to wild-type NKG2D (wtNKG2D) (or at least does not bind to wtNKG2D in a biologically relevant manner in vivo). This orthogonal A1-A2 domain, based on the U2S3 domain (SEQ ID NO: 30) described in the Examples, allows for a unique glycosylation pattern with advantageous properties.In various aspects, the disclosure provides A1-A2 domain peptides comprising an amino acid sequence having at least 95% identity to SEQ ID NO:30 (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:30), wherein the peptide comprises an alanine or glutamine at one or more of positions 40, 54, and / or 84 of SEQ ID NO:30. In this regard, the A1-A2 domain may comprise a glutamine at position 40, an alanine at position 40, a glutamine at position 54, an alanine at position 54, an alanine at position 40 and a glutamine at position 54 (optionally, glutamine or alanine at position 84), an alanine at position 40 and alanine at position 54 (optionally, glutamine or alanine at position 84), a glutamine at position 40 and alanine at position 54 (optionally, glutamine or alanine at position 84), a glutamine at position 40 and alanine at position 54 (optionally, glutamine or alanine at position 84), a glutamine at position 40 and alanine at position 54 (optionally, Alternatively, the A1-A2 domain may comprise a glutamine residue at positions 40 and 84, an alanine at position 40 and an alanine at position 84, an alanine at position 40 and an alanine at position 84, an alanine at position 40 and an alanine at position 84, an alanine at position 40 and an alanine at position 84, an alanine at position 54 and an alanine at position 84, an alanine at position 54 and an alanine at position 84, an alanine at position 54 and an alanine at position 84, or an alanine at position 54 and an alanine at position 84, where the positions refer to amino acid positions in SEQ ID NO:30. For example, the disclosure provides an A1-A2 domain peptide having at least 95% identity to the sequence of SEQ ID NO:30, wherein the peptide comprises glutamine residues at positions 40 and 54 with respect to the sequence of SEQ ID NO:30. Optionally, the A1-A2 domain comprises a glutamine at position 84 of SEQ ID NO:30. Alternatively, the A1-A2 domain may, in various embodiments, comprise an alanine at position 84 of SEQ ID NO: 30. In various embodiments, the A1-A2 domain peptide comprises (or consists of) SEQ ID NO: 11, SEQ ID NO: 31, or SEQ ID NO: 32.As further described herein, any of the A1-A2 domains of the present disclosure can be fused to the heavy or light chain of an antibody (or other antigen binding protein) to create, for example, a bispecific fusion protein that binds both the target antigen and a mutant NKG2D ectodomain. This form (antibody fused to the A1-A2 domains) is also referred to as a "MicAbody."

[0017] The present disclosure provides an antibody fusion protein comprising (i) a heavy chain comprising a variable region sequence of SEQ ID NO:1, and (ii) a light chain comprising a variable region sequence of SEQ ID NO:8. The light chain is fused at its C-terminus to an A1-A2 domain comprising the amino acid sequence of SEQ ID NO:11. The heavy and light chain variable regions of the antibody fusion protein of the invention are those of rituximab, a chimeric monoclonal antibody (IgG1 kappa immunoglobulin) that binds to the surface antigen CD20 displayed on B cells. Rituximab is further described, for example, in U.S. Patent Nos. 5,736,137, 5,776,456, and 5,843,439. B cells play a role in the pathogenesis of certain autoimmune diseases and cancer, and rituximab is effective in targeting and killing B cells to achieve beneficial effects in a variety of disorders. For example, rituximab has shown efficacy in the treatment of cancers such as leukemias (e.g., hairy cell leukemia (HCL) and chronic lymphocytic leukemia (CLL)) and lymphomas (e.g., non-Hodgkin's lymphoma (NHL, e.g., diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma (BL), Mantel cell lymphoma (MCL), and follicular lymphoma). Rituximab has also demonstrated efficacy in the treatment of autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), chronic inflammatory demyelinating polyneuropathy, and autoimmune-related anemia. Rituximab is also approved for the treatment of granulomatosis with polyangiitis (GPA) (Wegener's granulomatosis) and granulomatosis with microscopic polyangiitis (MPA).

[0018] The term "antibody" as used herein refers to an immunoglobulin having a full-length heavy chain and a light chain. The antibodies of the present disclosure are IgG antibodies, including four highly conserved subclasses (IgG1, IgG2, IgG3, and IgG4), which generally differ in their constant regions (e.g., hinge and / or CH2 domains). Optionally, the antibody fusion protein of the present disclosure comprises an IgG1 antibody, whose constant region may be modified to reduce or inactivate the ability of the antibody to induce antibody-dependent cell lysis (ADCC) (e.g., by introducing D265A / D297A substitutions into the Fc domain). In various embodiments, the heavy chain of the antibody fusion protein comprises a constant domain comprising the amino acid sequence of SEQ ID NO:3. The present disclosure also contemplates an antibody fusion protein, in which the heavy chain comprises a constant region comprising the amino acid sequence of SEQ ID NO:2. The present disclosure also contemplates an antibody fusion protein, wherein the heavy chain comprises an amino acid sequence at least 90% identical or at least 95% identical to SEQ ID NO:3, except that amino acids 234, 235, and 329 in SEQ ID NO:3 are alanine. In some embodiments, the antibody fusion protein comprises a heavy chain of SEQ ID NO:7. In this regard, the present disclosure provides an antibody fusion protein comprising a light chain of SEQ ID NO:21 and a heavy chain of SEQ ID NO:7. In other embodiments, the antibody fusion protein comprises a heavy chain of SEQ ID NO:6. In this regard, the present disclosure contemplates an antibody fusion protein comprising a light chain of SEQ ID NO:21 and a heavy chain of SEQ ID NO:6.

[0019] In various embodiments of the disclosure, the light chain of the antibody comprises the variable region sequence of SEQ ID NO: 8. Optionally, the light chain comprises a constant region comprising the amino acid sequence of SEQ ID NO: 9 (or a sequence at least about 90% identical or 95% identical to SEQ ID NO: 9). Thus, in various embodiments, the light chain of the antibody fusion protein of the disclosure comprises SEQ ID NO: 8 and SEQ ID NO: 9 (SEQ ID NO: 21).

[0020] The light chain is optionally fused at its C-terminus to an NKG2D ligand A1-A2 domain comprising the amino acid sequence of SEQ ID NO: 11. As explained in more detail below, fusion of the A1-A2 domain to the C-terminus of the light chain amino acid sequence resulted in superior activity compared to fusion of the A1-A2 domain to the heavy chain of an antibody of the disclosure. The superior properties of the domain arrangements on the antibody fusion proteins described herein could not have been predicted prior to the studies described in the Examples.

[0021] In various embodiments, the A1-A2 domains are fused to the C-terminus of the light chain via a linker, optionally comprising (or consisting of) SEQ ID NO: 10. As explained in more detail below, the linker of SEQ ID NO: 10 generated MicAbodies that were unexpectedly superior to other antibody fusion constructs in terms of B cell cytotoxicity. In an exemplary embodiment of the disclosure, an antibody fusion protein of the disclosure comprises a variable region sequence of SEQ ID NO: 8 and an A1-A2 domain of SEQ ID NO: 11 fused to the C-terminus of the light chain via a linker sequence of SEQ ID NO: 10, and optionally a light chain constant region of SEQ ID NO: 9. In this regard, in various embodiments of the disclosure, the light chain of the antibody fusion protein comprises the amino acid sequence of SEQ ID NO: 13.

[0022] The present disclosure provides an antibody fusion protein comprising a light chain of SEQ ID NO: 13 and a heavy chain of SEQ ID NO: 7. The present disclosure also provides an antibody fusion protein comprising a light chain of SEQ ID NO: 13 and a heavy chain of SEQ ID NO: 6. Methods for making antibodies and antibody fusion proteins are known in the art and are described, for example, in the Examples below.

[0023] The present disclosure also provides a kit comprising one or more containers comprising an antibody fusion protein as described herein. The kit may further comprise instructions and written information regarding the indication and use of the antibody fusion protein. Syringes, e.g., single-use or pre-filled syringes, sterile sealed containers, e.g., vials, bottles, vessels, and / or kits or packages comprising the antibody fusion protein are also contemplated, optionally with suitable instructions for use. In a further aspect, the present disclosure provides an article of manufacture or unit dose form comprising (a) a composition comprising an antibody fusion protein as described herein, (b) a container comprising said composition, and (c) a label affixed to said container or a package insert contained within said container that refers to the use of said antibody fusion protein in the treatment of a disease or disorder (e.g., cancer). Also provided herein is a composition comprising an antibody fusion protein (and, in various aspects, a mammalian cell expressing a CAR as described herein) and a pharma- ceutically acceptable carrier, excipient, or diluent. In an exemplary embodiment, the composition is a sterile composition.

[0024] The present disclosure further provides a system or kit comprising components of a cell therapy regimen targeting CD20-presenting cells. In various embodiments, the first component is an antibody fusion protein as described herein, i.e., a bispecific antibody-based fusion protein that binds both CD20 and a CAR comprising an NKG2D ectodomain. The second component is a mammalian cell (e.g., a human cell) genetically modified to express a chimeric antigen receptor (CAR) that is itself inactive (i.e., an unarmed CAR-T). In various embodiments, the mammalian cell is a lymphocyte or macrophage, e.g., a human lymphocyte (such as a human T cell) or a human macrophage. In various embodiments, the second component is a human NK (natural killer) cell (e.g., an autologous human NK cell), and the disclosure herein regarding T cells also applies to NK cells. The kit comprises one or more containers comprising a mammalian cell expressing a CAR and one or more containers comprising an antibody fusion protein. The kit may further comprise instructions and written information regarding the indication and use of the components described herein.

[0025] "Chimeric antigen receptor" or "CAR" refers to an artificial immune cell receptor engineered to recognize and bind to an antigen expressed by a target cell, such as a tumor cell. Generally, CARs are designed for T cells and are chimeras of the signaling domain of the T cell receptor (TCR) complex and the antigen recognition domain (e.g., single chain fragment (scFv) of an antibody or other antibody fragment). See, e.g., Enblad et al., Human Gene Therapy. 2015; 26(8): 498-505. T cells and NK cells can be modified using gene transfer techniques to directly and stably express transmembrane signaling receptors on their surface that confer novel antigen specificity. See, e.g., Gill & June, Immunological Reviews 2015. Vol. 263: 68-89; Glienke et al., Front. Pharmacol. doi: 10.3389 / fphar.2015.00021. CARs come in a variety of forms, each containing different components. "First generation" CARs link the antigen-binding domain to the CD3ζ intracellular signaling domain of the T cell receptor via a hinge and transmembrane domain. "Second generation" CARs incorporate additional domains, such as CD28, 4-1BB (41BB), or ICOS, to provide a costimulatory signal. "Third generation" CARs contain two costimulatory domains fused to the TCR CD3ζ chain. Third generation costimulatory domains can include, for example, a combination of CD3ζ, CD27, CD28, 4-1BB, ICOS, or OX40. CARs constructed in this way can, for example, trigger T cell activation when bound to a target antigen, similar to the endogenous T cell receptor, but independent of the major histocompatibility complex (MHC).

[0026] The chimeric antigen receptor of the present disclosure comprises a mutated NKG2D ectodomain that is unable to engage a natural ligand as the "antigen binding domain" of the CAR. Mutations of the NKG2D ectodomain are further described, for example, in Culpepper et al., Mol. Immunol. 48, 516-523 (2011) and in the Examples. The mutated NKG2D is referred to herein as "iNKG2D". In various embodiments, the iNKG2D domain comprises the amino acid sequence of SEQ ID NO: 15. The ectodomain is preferably associated with a transmembrane domain, an intracellular domain of a costimulatory molecule (e.g., 4-1BB or CD28), and / or a T cell receptor intracellular signaling domain. For example, in exemplary embodiments of the present disclosure, the iNKG2D ectodomain is fused to a CD8a hinge / transmembrane domain (e.g., comprising or consisting of the sequence of SEQ ID NO: 16), a 4-1BB domain (e.g., comprising or consisting of the sequence of SEQ ID NO: 17), and / or a CD3 zeta domain (e.g., comprising or consisting of the sequence of SEQ ID NO: 18). In various embodiments, the CAR comprises all of these components (e.g., SEQ ID NOs: 15-18 or SEQ ID NO: 19).

[0027] Since CARs are inactive, they can only form a productive immunological synapse with a target cell presenting an antigen and activate cytolysis when "armed" with its cognate antibody fusion protein non-covalently bound to its receptor. CAR-expressing cells are referred to herein as "convertibleCARs." An example of the system is shown in FIG. 7A. The antibody fusion proteins described herein can activate iNKG2D-CAR-expressing cells (e.g., T cells) only in the presence of cells expressing CD20. When used with additional MicAbodies (i.e., antibody fusion proteins with different variable regions that bind to different cell surface antigens) that target other antigens, convertibleCAR-T cells can target different antigens simultaneously or sequentially to mediate cytolysis. This approach can be useful, for example, to address tumor resistance and tumor escape as a result of target antigen loss without the need to generate, expand, and inject multiple different autologous CAR cells. This highly modular convertibleCAR system expands the possibilities of adoptive cell therapy and overcomes many of the shortcomings of existing cell therapies, including severe systemic toxicity, antigen escape, and limited and uncontrollable persistence of current CAR-T and CAR-NK cell therapies. Furthermore, cell manufacturing is simplified and less expensive, since a single CAR can be used in a variety of settings (as target specificity is determined by the antibody fusion protein administered, not the CAR).

[0028] CAR cell therapy can be an immunotherapy that utilizes a subject's or patient's own immune cells that have been engineered to produce a specific CAR on their surface. In some situations, cells (e.g., T cells) are harvested from the subject's or patient's body by apheresis. The cells (e.g., T cells) harvested from the body are then genetically engineered to produce a specific chimeric antigen receptor on their surface. The CAR-expressing cells are expanded by growing in the laboratory and then administered to the subject or patient, or to another subject or patient. The CAR-expressing cells recognize and kill cells (e.g., cancer cells) that express a target antigen on their surface. The cells may be isolated from the subject who will be the recipient of the treatment, or from a donor subject who is not the ultimate recipient of the treatment. In various embodiments, the cells are autologous CD4+ and CD8+ T cells.

[0029] The present disclosure further provides a method of treating a subject for a disease or disorder associated with cells expressing CD20, such as cancer (CD20-positive cancer). The method includes administering to the subject a CAR-expressing cell described herein (e.g., a T cell or NK cell expressing an iNKG2D-based CAR described herein) and administering to the subject an antibody fusion protein described herein. Examples of cancers include, but are not limited to, leukemias and lymphomas, such as hairy cell leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma (e.g., diffuse large B-cell lymphoma, Burkitt's lymphoma, Mantel cell lymphoma, and follicular lymphoma).

[0030] As used herein, the term "treat" and related words do not necessarily mean 100% or complete cure or remission. Rather, there are various degrees of treatment that one skilled in the art would recognize as having potential benefit or therapeutic effect. In this regard, the method of treating a disease or disorder can provide any amount or level of treatment. Furthermore, the treatment provided by the method can include treatment of one or more conditions or symptoms or signs of the disease being treated. For example, the treatment method of the present disclosure can suppress one or more symptoms of the disease. Also, the treatment provided by the method of the present disclosure can include slowing the progression of the disease.

[0031] Treatment of cancer can be determined by any of a number of methods. Any improvement in the subject's well-being is contemplated (e.g., at least or about 10% reduction, at least or about 20% reduction, at least or about 30% reduction, at least or about 40% reduction, at least or about 50% reduction, at least or about 60% reduction, at least or about 70% reduction, at least or about 80% reduction, at least or about 90% reduction, or at least or about 95% reduction in any of the parameters described herein). For example, a therapeutic response refers to one or more of the following improvements in disease: (1) a reduction in the number of neoplastic cells; (2) an increase in neoplastic cell death; (3) an inhibition of neoplastic cell survival; (5) an inhibition (i.e., a slowing to some degree, preferably a halt) of tumor growth or the appearance of new lesions; (6) a reduction in tumor size or burden; (7) the absence of clinically detectable disease; (8) a reduction in the level of a cancer marker; (9) an increase in patient survival; and / or (10) some relief from one or more symptoms associated with the disease or condition (e.g., pain). Additionally, therapeutic efficacy can be characterized in terms of responsiveness to other immunotherapeutic treatments or chemotherapy. In various embodiments, the methods of the present disclosure further comprise monitoring the treatment in the subject.

[0032] The subject may be, but is not limited to, a mammal of the order Rodentia, such as mice and hamsters, and a mammal of the order Lagomorpha, such as rabbits, a mammal of the order Carnivora, including cats (Felines) and dogs (Canines), a mammal of the order Artiodactyla, including cats (Bovines) and pigs (Pigs), or a mammal of the order Perissodactyla, including horses (Equus). In some embodiments, the mammal is of the order Primates, Ceboids, or Simoids (Monkeys), or Anthropoids (Humans and Apes). In some embodiments, the mammal is a human. The therapeutic composition may be delivered to the subject using any of a variety of routes, including parenteral, topical, oral, intrathecal, or local administration. Indeed, the composition may be administered subcutaneously, intradermally, intradermally, intravenously, intraarterially, intratumorally, parenterally, intraperitoneally, intramuscularly, intraocularly, intraosseously, epidurally, intradurally, intratumorally, and the like.

[0033] The present disclosure also provides (i) a nucleic acid molecule (i.e., an isolated nucleic acid) encoding a light chain of an antibody fusion protein described herein, and (ii) a nucleic acid molecule (i.e., an isolated nucleic acid) encoding a heavy chain of an antibody fusion protein described herein, and compositions comprising (i) and / or (ii). The present disclosure further provides a nucleic acid molecule encoding any of the A1-A2 domain peptides disclosed herein. The nucleic acids of the present disclosure include nucleic acids encoding any of the amino acid sequences disclosed herein, as well as nucleic acids comprising a nucleotide sequence having at least 80%, more preferably at least about 90%, more preferably at least about 95%, and most preferably at least about 98% identity to the nucleic acids of the present disclosure (i.e., the nucleic acid sequences set forth in the sequence listing). The nucleic acids of the present disclosure also include complementary nucleic acids. In some cases, the sequences are perfectly complementary (no mismatches) when aligned. In other cases, there may be up to about 20% mismatches in the sequences. The present disclosure provides nucleic acid molecules that include nucleic acid sequences encoding both the heavy and light chains of the antibody fusion proteins of the present disclosure.

[0034] The nucleic acids of the present disclosure can be cloned into an expression vector, such as a plasmid, cosmid, bacmid, phage, artificial chromosome (BAC, YAC) or virus, into which another genetic sequence or element (either DNA or RNA) can be inserted to effect replication of the linked sequence or element. In some embodiments, the expression vector can contain a constitutively active promoter segment (such as, but not limited to, CMV, SV40, elongation factor or LTR sequences) or an inducible promoter sequence, such as the steroid-inducible pIND vector (Invitrogen), to control expression of the nucleic acid. The expression vector of the present disclosure can further include a control sequence, for example, an internal ribosome entry site. A secretory signal peptide sequence can also be optionally encoded by the expression vector operably linked to the coding sequence of interest such that the expressed polypeptide can be secreted by the recombinant host cell to more easily isolate the polypeptide of interest from the cell. The expression vector can be introduced into the cell, for example, by transfection.

[0035] Recombinant host cells comprising the nucleic acid molecules (optionally contained in an expression vector) are also provided. The recombinant host cells can be prokaryotic cells, e.g., E. coli cells, or eukaryotic cells, e.g., mammalian cells or yeast cells. Yeast cells include, e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris cells. Mammalian cells include, e.g., Vero, HeLa, Chinese hamster ovary (CHO), W138, baby hamster kidney (BHK), COS-7, MDCK, human embryonic kidney cell line 293, African green monkey kidney cells, and COS cells. Recombinant protein production cells of the present disclosure also include any known insect expression cell line, e.g., Spodoptera frugiperda cells. In one embodiment, the cells are mammalian cells, e.g., CHO cells.

[0036] A method of producing the antibody fusion protein is further provided by the present disclosure. The method includes culturing a host cell (isolated host cell) that includes a nucleic acid molecule that includes a nucleotide sequence that encodes a light chain of the antibody fusion protein and a nucleic acid molecule that includes a nucleotide sequence that encodes a heavy chain of the antibody fusion protein. The method further includes recovering the antibody fusion protein. The present disclosure also provides a method of producing the A1-A2 domain peptide described herein. The method includes culturing a host cell (isolated host cell) that includes a nucleic acid molecule that includes a nucleotide sequence that encodes the A1-A2 domain peptide. The method further includes recovering the domain peptide (optionally fused to another peptide). Culture conditions and methods for making recombinant proteins, such as antibody proteins, are known in the art. Similarly, protein purification methods are known in the art and are utilized herein for recovery of recombinant proteins from cell culture media. In some embodiments, purification methods for proteins and antibodies include filtration, affinity column chromatography, cation exchange chromatography, anion exchange chromatography, and concentration. Optionally, the method includes formulating the antibody fusion protein or the A1-A2 domain peptide.

[0037] While the foregoing disclosure focuses on anti-CD20 antibodies as fusion partners for A1-A2 domain peptides in various embodiments, it should be understood that the A1-A2 domain peptides of the present disclosure may be fused to other peptides, including other antigen-binding peptides, such as other antibodies. The foregoing disclosure regarding the structure of anti-CD20 antibodies also applies to other antigen-binding proteins and antibodies (i.e., antibodies that bind to other targets). The antibodies may be monoclonal or multispecific antibodies (e.g., bispecific antibodies). The A1-A2 domain peptides may be fused to an antigen-binding fragment of an antibody. Examples of antibody fragments include Fab, Fab', F(ab') and / or F(ab') fragments. 2, and Fv fragments. Other antigen binding proteins include diabodies, linear antibodies, single chain antibody molecules, and the like. The antigen binding protein may target any suitable antigen, such as an antigen expressed on the surface of a cancer cell. Examples of antigens include, but are not limited to, CD19, BMCA, HER2, EGFR, EpCAM, CEA, BCMA, PSMA, CD19, CD20, CD22, CD33, CD37, CD38, CD123, CD276 (B7-H3), GPC2, GPC3, GPRC5D, WT-1, NY-ESO-1, CLDN4, CLDN6, CLDN18.2, PSCA, and TSPAN8. The present disclosure further provides a method of treating a subject for a disease or disorder (e.g., cancer). The method includes administering to the subject a CAR-expressing cell described herein (e.g., a T cell or an NK cell expressing an iNKG2D-based CAR described herein) and administering to the subject an antigen-binding fusion protein comprising an A1-A2 domain peptide described herein. Examples of cancers are discussed above and apply to this aspect of the disclosure.

[0038] The following examples are provided merely to illustrate the invention and are not intended to limit its scope. EXAMPLES

[0039] This example describes an exemplary method of generating CAR-T cells comprising an antibody fusion protein of the present disclosure and an NKG2D ectodomain. The example further demonstrates the ability of an antibody fusion protein comprising a variable region sequence from rituximab and an A1-A2 domain fused to the C-terminus of the light chain to selectively bind to a CAR-T cell comprising an amino acid sequence of SEQ ID NO: 15-18, and the ability of the combination of the antibody fusion protein and the CAR-T cell to kill CD20-bearing cancer cells in vivo.

[0040] Materials and Methods Cloning, Expression, and Purification: The wild-type ectodomain of NKG2D (UniProtKB P26718, residues 78-216; https: / / www.uniprot.org) was expressed as a fusion to the C-terminus of human IgG1 Fc via a short factor Xa-recognizing Ile-Glu-Gly-Arg linker (Fc-wtNKG2D). Inactive NKG2D variants containing either a single Y152A (iNKG2D.YA) or double Y152A / Y199A substitutions (iNKG2D.AF) were generated by PCR-mediated mutagenesis or synthesis (gBlocks®, IDT). DNA constructs of Fc-NKG2D molecules were expressed in Expi293™ cells (Thermo Fisher Scientific) and dimeric secreted proteins were purified by Protein A affinity chromatography (Pierce™ #20334, Thermo Fisher). The eluted material was characterized and further purified by size-exclusion chromatography (SEC) on an AKTA Pure system using a Superdex 200 column (GE Life Sciences). Correctly assembled, appropriately sized monomeric material was fractionated into phosphate-buffered saline (PBS).

[0041] The A1-A2 domains of human MICA*001 (UniProtKB Q29983, residues 24–205), MICB (UniProtKB Q29980.1, 24–205), ULBP1 (UniProtKB Q9BZM6, 29–212), ULBP2 (UniProtKB Q9BZM5, 29–212), ULBP3 (UniProtKB Q9BZM4, 30–212), ULBP5 (NCBI accession no. NP_001001788.2, 29–212), and ULBP6 (UniProtKB, 29–212) were cloned with a C-terminal 6x-His tag. Monomeric protein was purified from Expi293™ supernatant Ni-NTA resin (HisPur™, Thermo Fisher) and the eluted material was exchanged into PBS using Sephadex G-25 in a PD-10 desalting column (GE Life Sciences).

[0042] MIC ligands and orthogonal variants were cloned by ligation-independent assembly (HiFi DNA Assembly Master Mix, NEB #E2621) as fusions to the C-terminus of either the kappa light or heavy chain of a human IgG1 antibody via either the APTSSGGGGS or GGGS linker, respectively. In addition, D265A / N297A (Kabat numbering) mutations were introduced in the CH2 domain of the heavy chain of all antibody and MicAbody clones to eliminate antibody-dependent cellular cytotoxicity (ADCC) function. Heavy and light chain plasmid DNA (in mammalian expression vector pD2610-V12(ATUM)) of a given antibody clone was co-transfected into Expi293™ cells and purified by Protein A. For any monoclonal antibody fusions made, the appropriate VL or VH domain was replaced with either the kappa light chain or the ADCC-deficient IgG1 heavy chain.

[0043] Engineering inactive NKG2D and orthogonal ligands: Biolayer interferometry (BLI) using a ForteBio Octet system (Pall ForteBio LLC) was performed to confirm loss of wild-type MIC ligand binding by iNKG2D. Fc-wtNKG2D, Fc-iNKG2D.YA, or Fc-iNKG2D.AF were captured onto an anti-human IgG Fc capture (AHC) biosensor tip and association / dissociation kinetics were monitored in a titration series of monomeric MIC-His ligands. Additionally, ELISA (enzyme-linked immunosorbent assay) binding assays were performed using MICA-Fc, MICB-Fc, ULBP1-Fc, ULBP2-Fc, ULBP3-Fc, or ULBP4-Fc (R&D Systems) coated onto microtiter plates, titrated with biotinylated Fc-wtNKG2D or Fc-iNKG2D.YA, detected with streptavidin-HRP (R&D Systems #DY998), and developed with 1-Step Ultra TMB ELISA (Thermo Fisher #34208).

[0044] Using phage display, orthogonal ULBP2 A1-A2 variants were identified that showed exclusive binding to either iNKG2D.YA or iNKG2D.AF. A synthetic NNK (N=A / C / G / T and K=G / T, displaying all 20 amino acids with no stop codons) DNA library was generated targeting codons in the bound state of helix 2 (residues 74-78, numbering based on the mature protein) or helix 4 (residues 156-160), located proximal to position Y152 on the native NKG2D receptor. Muller et al., PLoS Pathog. 6, e1000723 (2010). Libraries probing helix 2 alone, helix 4 alone, or combinations thereof were cloned as fusions to the minor coat protein pill of M13 phage, and phage particles displaying mutagenized A1-A2 domain variants were produced in SS320 E. coli cells according to standard methods. These A1-A2 phage libraries were captured with either biotinylated Fc-iNKG2D.YA or Fc-iNKG2D.AF proteins (EZ-Link™ NHS-Biotin Kit, Thermo Fisher no. 20217) and enriched by repeated four rounds of selection with increasing concentrations of non-biotinylated Fc-wtNKG2D competitor. Positive phage clones were verified for selective binding to plate-bound Fc-iNKG2D.YA or Fc-iNKG2D.AF compared to Fc-wtNKG2D by spot ELISA, and bound phage were detected with biotinylated M13 phage coat protein monoclonal antibody E1 (Thermo Fisher no. MA1-34468) followed by incubation with streptavidin-HRP.

[0045] Phage variants were sequenced and then cloned as human IgG1 monoclonal antibody fusions for further validation. To confirm that the selectivity of the orthogonal variants was maintained in the bivalent MicAbody format, ELISA wells were coated with 1 μg / mL of Fc-wtNKG2D, Fc-iNKG2D.YA, or Fc-iNKG2D.AF, and bound MicAbody was detected with an HRP-conjugated mouse anti-human kappa chain antibody (Abcam no. ab79115). The affinity of both monomeric and antibody-fused ULBP2 variants was also determined by Octet analysis as previously described.

[0046] Generation of convertible CAR-T cells: Human codon-optimized DNA (GeneArt, Thermo Fisher) containing the CD8α chain signal sequence, NKG2D variant, CD8α hinge and transmembrane domain, 4-1BB, CD3ζ, and eGFP was cloned into the pHR-PGK transfer plasmid for second-generation Pantropic VSV-G pseudotyped lentivirus production along with the packaging plasmids pCMVdR8.91 and pMD2.G48. (GGGGS) 3 The VH and VL domains of rituximab, separated by a linker, were replaced with the NKG2D module to generate rituximab scFv-based CARs (RITscFv-CARs). For each batch of lentivirus generated, 6 × 10 6Lenti-X 293T (Takara Bio #632180) cells were seeded in a 10 cm dish the day before transfection. Then, 12.9 μg pCMVdR8.91, 2.5 μg pMD2.G and 7.2 μg pHR-PGK-CAR construct were combined in 720 μl Opti-MEM™ (Thermo Fisher #31985062) and then mixed with 67.5 μl Fugene HD (Promega Corp. E2311), vortexed briefly, incubated at room temperature for 10 minutes, and then added to the dish of cells. After 2 days, the supernatant was collected by centrifugation and passed through a 0.22 μm filter. Five-fold concentrated PEG-6000 and NaCl were added to give a final concentration of 8.5% PEG-6000 (Hampton Research No. HR2-533) and 0.3 M NaCl, incubated on ice for 2 hours, and then centrifuged for 20 minutes at 4° C. The concentrated viral particles were resuspended in 0.01 volume of PBS and stored frozen at −80° C.

[0047] For isolation of primary human T cells, human peripheral blood Leuko Pak (Stemcell Technologies #70500.1) from an anonymous donor was diluted with an equal volume of PBS+2% FBS and then centrifuged at 500×g for 10 min at room temperature. Cells were diluted to 5×10 in PBS+2% FBS. 7CD4+ or CD8+ cells were enriched by negative selection (Stemcell EasySep™ Human CD4 T Cell Isolation Kit #17952 or EasySep Human CD8 T Cell Isolation Kit #17953) by resuspending at 100 cells / ml, adding 50 μl of isolation cocktail per ml of cells and incubating at room temperature for 5 minutes. Subsequently, 50 μl of RapidSpheres™ per ml of cells was added to fill the sample (21 ml cells, 14 ml PBS each). Cells were isolated with the EasySEP™ magnet for 10 minutes, followed by removal of the buffer while maintaining the magnetic field. The enriched cells were transferred to a new tube containing fresh buffer and the magnet was reapplied for a second round of enrichment, after which the cells were resuspended, counted and counted at 10–15 × 10 6 Cells were stored frozen in cryovials (RPMI-1640, Corning #15-040-CV; 20% human AB serum, Valley Biomedical #HP1022; 10% DMSO, Alfa Aesar #42780).

[0048] To generate CAR-T cells, one vial of cryopreserved cells was thawed and added to 10 mL of T cell medium "TCM" (TexMACS medium, Miltenyi 130-097-196; 5% human AB serum, Valley Biomedical number HP1022; 10 mM neutralized N-acetyl-L-cysteine; 1X 2-mercaptoethanol, Thermo Fisher number 21985023, 1000X; 45 IUe / ml human IL-2IS "rhIL-2", Miltenyi number 130-097-746) (added at the time of addition to cells). Cells were centrifuged at 400 x g for 5 minutes and then resuspended in 10 ml of TCM and diluted to 1 x 10 6The cells were adjusted to 1 ml / ml and seeded at 1 ml / well in a 24-well plate. After overnight settling, 20 μL of Dynabeads™ Human T-Activator CD3 / CD28 (Thermo Fisher #1131D) were added per well and incubated for 24 hours. Concentrated lentiviral particles (50 μL) were added per well and the cells were incubated overnight and then transferred to a T25 flask with 6 ml of TCM. After 3 days of growth, the Dynabeads were removed (MagCellect magnet, R&D Systems MAG997) and transduction efficiency was assessed by flow cytometry for GFP, with 5×10 5 Dilute back to 4 x 10 cells / mL 6 Cell density was monitored daily to ensure that it did not exceed 100 cells / mL. Surface expression of iNKG2D was correlated with GFP expression using MicAbody and detection with PE-anti-human kappa chain (Abcam#ab79113) or by direct conjugation of Rituximab-MicAbody to Alexa Fluor 647 (Alexa Fluor Protein Labeling Kit#A20173, Thermo Fisher) when necessary. The amount of iNKG2D expression on the surface of convertibleCAR-CD8 cells was quantified using Rituximab-MicAbody conjugated to Alexa Fluor 647 and median fluorescence intensity was correlated with Quantum™ MESF 647 beads (Bangs Laboratories#647). All flow cytometry was performed on either a Bio-Rad S3e Cell Sorter or a Miltenyi MACSQuant Analyzer 10 instrument.

[0049] Cell lines and in vitro assays: Ramos human B-cell lymphoma cells (ATCC number CRL-1596) were cultured in RPMI supplemented with 20 mM HEPES and 10% FBS. The murine colon carcinoma line CT26 transfected to express human Her2 was also used. No additional mycoplasma testing or authentication was performed except for verifying by flow cytometry that the target antigen was expressed.

[0050] For calcein release assays, tumor cells were centrifuged and eluted at 1–2 × 10 in 4 mM probenecid (MP Biomedicals no. 156370) + 25 μM calcein-AM (Thermo Fisher no. C1430) in T cell medium. 6 Resuspend and wash once at 8 × 10 cells / ml for 1 h at 37 °C. 5 CD8+ CAR-T cells were pelleted and diluted to 4×10 in 60 IUe / ml IL-2 and 4 mM probenecid in TCM. 6 After resuspension at 1000 cells / mL, the effector:target ratio was adjusted according to the desired ratio (not adjusted for transduction efficiency). 25 μL of target cells were seeded, followed by 25 μL of medium or diluted MicAbody. Then, 100 μL medium (minimal lysis), medium + 3% Triton-X100 (maximal lysis), or CAR-T cells were added and the plate was incubated at 37 °C for 2 h. Cells were pelleted and 75 μL of supernatant was transferred to a black clear-bottom plate and fluorescence was acquired (excitation 485 nm, emission cutoff 495 nm, emission 530 nm, 6 flashes per read) on a SpectraMax M2e plate reader (Molecular Devices). For experiments with armed convertibleCAR-CD8+, T cells were preincubated at 37 °C for 30 min with either saturating (5 nM) or titrated MicAbody, then washed to remove unbound MicAbody and co-cultured with target cells supplemented with calcein.

[0051] To quantify target-dependent activation of T cells, experiments were set up as described above, except that calcein preloading was omitted and assays were set up in T cell medium not supplemented with IL-2. After 24 hours of co-culture, supernatants were harvested and stored at -80°C until the amount of free cytokine could be quantified using ELISA MAX™ Human IL-2 or Human IFN-g detection kits (BioLegend #431801 and #430101).

[0052] MicAbody binding curve data was generated by ProMab Biotechnologies, Inc. (Richmond, CA). 5 convertibleCAR-CD8+ cells were seeded into 96-well V-bottom plates and incubated with Alexa Fluor 647-labeled Rituximab.LC-U2S3 MicAbody for 30 min at room temperature in a final volume of 100 μL RPMI+1% FBS using a titration curve starting at 200 nM. Cells were then rinsed and the median fluorescence intensity was determined at each titration point by flow cytometry.

[0053] Animal Study: For PK analysis of serum levels of MicAbody, 6-week-old female NSG mice (NOD.Cg-Prkdcscid IL2rgtm1Wjl / SzJ, The Jackson Laboratory #005557) were injected intravenously (IV) with 100 μg of either parental rituximab antibody (ADCC deficient), rituximab heavy chain U2S3 fusion (Rituximab.HC-U2S3), or light chain fusion (Rituximab.LC-U2S3). Collected serum was subjected to ELISA by capture with human anti-rituximab idiotypic antibody (HCA186, Bio-Rad Laboratories), detected with rat anti-rituximab-HRP antibody (MCA2260P, Bio-Rad), and serum levels were interpolated using standard curves of either rituximab or rituximab-U2S3. PK analysis of U2S3-hFc-mutIL2 was performed in NSG mice by IP injection of 60 μg followed by periodic serum collection. Samples were examined by ELISA capture with Fc-iNKG2D and detection with biotinylated rabbit-anti-human IL-2 polyclonal antibody (Peprotech no. 500-P22BT) followed by incubation with streptavidin-HRP. Half-lives were calculated in GraphPad Prism based on the beta phase of the curve using nonlinear regression analysis, exponential monophasic decay analysis with the plateau limited to zero.

[0054] For disseminated Raji B-cell lymphoma studies, Raji cells (ATCC number CCL-86) stably transfected to constitutively express luciferase from Luciola italica (Perkin Elmer RediFect Red-FLuc-GFP number CLS960003) were implanted intravenously into 6-week-old female NSG mice. The start of treatment administration is detailed in each in vivo study figure. In all experiments, CD4 and CD8 primary human T cells were independently transduced and combined after expansion in a 1:1 mixture of CD4:CD8 cells without normalizing for transfection efficiency between cell types or CAR constructs, and the mixture was verified by flow cytometry before intravenous injection. Administration of MicAbody or control antibody was by intraperitoneal (IP) route unless otherwise stated, and in vivo imaging of bioluminescence was performed using the Xenogen IVIS system (Perkin Elmer). Animals were bled periodically to monitor human T cell dynamics by flow cytometry and staining with APC anti-human CD3 (clone OKT3, no. 20-0037-T100, Tonbo Biosciences), GFP was monitored, and cell-associated MicAbody levels were examined with biotinylated anti-human F(ab')2 (no. 109-066-097, Jackson ImmunoResearch Laboratories Inc.) followed by streptavidin-PE detection (BD no. 554061). Serum ELISA to monitor MicAbody levels was performed as previously described.

[0055] For subcutaneous tumor studies, 1 x 10 cells in Matrigel 6 Raji cells were implanted into the right flank of 6-week-old female NSG mice until tumors grew to 70–100 mm 3 Treatment was initiated when the cohort received armed convertibleCAR-T cells. For the cohorts that received armed convertibleCAR-T cells, cells were incubated ex vivo with 5 nM Rituximab.LC-U2S3 MicAbody for 30 min at room temperature, then washed and finally mixed to achieve the desired 1:1 CD4:CD8 ratio and cell concentration. Biotinylated anti-human F(ab') 2Confirm the arming by flow cytometry using antibodies to GFP and F(ab') 2 A strong correlation between MFI and MFI was evident. These mice did not receive a separate MicAbody treatment. Caliper measurements were taken periodically to estimate tumor volume (L x W x W x 0.5 = mm 3 ), and the final tumor volume was measured.

[0056] Complement-Mediated Ablation of iNKG2D.AF-CAR Cells: To generate Fc reagents with enhanced complement binding and targeted delivery to iNKG2D.AF-expressing T cells, orthogonal ligands were cloned as fusions to either the N-terminus (U2R-Fc) or C-terminus (Fc-U2R) of human IgG1 Fc via a GGGS linker, where the Fc included the hinge, CH2, and CH3 domains. In addition to the wild-type Fc, a set of mutants with enhanced C1q binding were explored, including K326A / E333A21 (Kabat numbering, "AA") and S267E / H268F / S324T / G236A / I332E20 ("EFTAE"). All were expressed in Expi293T cells and purified and fractionated as described above. Confirmatory ELISA was performed by capturing with Fc-NKG2D.AF followed by binding U2R / Fc variant fusions at a concentration of 1 μg / mL, titrating with human C1q protein (Abcam no. ab96363), and then detecting with polyclonal sheep anti-C1q-HRP antibody (Abcam no. ab46191). Complement-dependent cytotoxicity (CDC) assays were performed by iQ Biosciences (Berkeley, CA). Briefly, 5×10 NKG2D.AF-CAR-transduced CARs were collected from 100 mice. 4CD8+ cells were seeded in 96-well plates and incubated with serial dilutions of each U2R / Fc variant fusion in triplicate for 3 h in the presence of normal human serum complement (Quidel Corporation) at a final concentration of 10% (v / v). Cells were then harvested, resuspended with SYTOX™ Red dead cell staining reagent (Thermo Fisher) at a final concentration of 5 μg / mL, and analyzed by flow cytometry. EC50 values ​​for cytotoxicity were calculated with GraphPad Prism by fitting nonlinear regression curves.

[0057] Delivery of mutant IL2 to T cells expressing iNKG2D-CAR: A heterodimeric Fc strategy was employed to generate a reagent that is a monomer of the U2S3 ligand and a monomer of mutant IL-2, lacking the ability to bind IL-2Rα (mutIL2, R38A / F42K) (Heaton et al., Cancer Res. 53, 2597-2602 (1993); Sauve et al., Proc. Natl. Acad. Sci. USA 88, 4636-4640 (1991)) but retaining serum stability. Gunasekaran et al., J. Biol. Chem. 285, 19637-19646 (2010). U2S3 was fused to the N-terminus of the Fc hinge of one chain with K392D / K409D (Kabat numbering) mutations, and mutIL2 was fused to the C-terminus of the second Fc chain with E356K / D399K mutations. Additionally, D265A / N297A mutations were introduced into both Fc chains to render the Fc ADCC-deficient. Expression and purification in Expi293T cells was as previously described. Properly assembled U2S3-hFc-mutIL2 material was fractionated by SEC and the presence of individual appropriately sized polypeptides was confirmed by denaturing SDS-PAGE. Direct fusions between the orthogonal ligand and mutIL2 expressed as a single polypeptide, using linkers containing a glycine-serine bond, a flag tag, and a 6xHis tag, were also formed and purified by Ni-NTA exchange chromatography. Ghasemi et al., Nat Commun 7, 12878 (2016). Determination of IUe activity equivalents was based on the calculation that a 4.4 μM solution of wild-type IL-2 has the equivalent of 1000 IU / μL. IL-15 with the V49D mutation, which reduced binding to IL-15Rα but retained bioactivity, was similarly formatted in U2S3. Bernard et al., J. Biol. Chem. 279, 24313-24322 (2004).

[0058] CAR-T cell proliferation in response to various cytokines or U2S3-cytokine fusions was quantified with WST-1 cell proliferation reagent (Millipore Sigma no. 5015944001). Briefly, CAR-T cells were pelleted, resuspended in T cell medium without IL-2, and plated in 96-well plates at 4 × 10 4 Cells / well were dispensed and an appropriate amount of diluted U2S3-cytokine fusion was added to achieve a concentration of 30 IUe / mL or higher as needed in a final assay volume of 100 μL per well. Recombinant human IL2 and IL15 (Peprotech no. 200-02 and no. 200-15) were included as controls. After 3 days of incubation at 37°C, 10 μL of WST-1 was added to each well and allowed to incubate for 30-60 minutes before quantitating the intensity of color development on a plate reader. Changes in the percentage of GFP+CAR expressing cells in response to U2S3-cytokine fusions were monitored by flow cytometry. To monitor activation of STAT3 or STAT5 upon engagement of cytokine fusions, cells were left overnight in TCM medium without IL-2 supplementation and then treated with 150 IUe / mL of IL-2, IL-15, U2S3-hFc-mutIL2, or U2S3-hFc-mutIL15 for 2 hours before fixing and staining for intracellular phospo-STAT3 (Biolegend PE anti-STAT3 Tyr705 clone 13A3-1) and -STAT5 (BD Alexa Fluor 647 anti-STAT5 pY694 clone 47). To monitor temporal responses, treated convertibleCAR-CD8 T cells were fixed and then stained at 0, 30, 60, and 120 minutes after exposure to cytokines or U2S3-hFc-cytokine fusions.

[0059] Human PBMC stimulation and immunophenotyping studies were performed. Briefly, normal PBMCs from three donors were cultured at 1×10 5 Cells / well were seeded into a 96-well plate and incubated in a 10-fold dilution series of either U2S3-hFc-mutIL2 or U2S3-hFc-wtIL2 (wild-type IL2) at 4°C for 30 min in 5% CO. 2The wells were exposed to 100 μg / mL of anti-human CD3 (OKT2) and 300 IUe / mL of rhIL-2 for 4 days at 37°C. Positive controls included wells coated with 2 μg / mL anti-human CD3 (OKT2) and 300 IUe / mL of rhIL-2. After incubation, cells were treated with TruStain FcX Block (BioLegend #422301) and subsequently stained with BioLegend antibody panels for proliferating T cells (CD8 clone RPA-T8 #301050, CD4 clone OKT4 #317410, CD3 clone OKT3 #300430, Ki-67 #350514) and Treg cells (Fox3 clone 206D #320106, CD4 clone OKT4, CD3 clone OKT3, Ki-67).

[0060] result Engineering orthogonal NKG2D-ligand interactions: The two central tyrosine residues in each NKG2D monomer have a key role in driving receptor-ligand interactions. Culpepper et al., Mol. Immunol. 48, 516-523 (2011). Mutations at these residues were explored in depth and the Y152A mutant ("iNKG2D.YA") and the Y152A / Y199F double mutant ("iNKG2D.AF") were selected for further testing and confirmed by biolayer interferometry (BLI) (Figures 2A and 3A) and ELISA (Figure 2B) that binding to all naturally occurring human ligands was abolished. The ULBP2 A1A2 domain, which is not polymorphic, was therefore selected for phage display-based selection of mutants that exhibited high affinity binding to each of the iNKG2D variants. In NNK libraries interrogating helices 2 and 4, only helix 4 variants were found, and even then only in association with the spontaneous R81W mutation, which likely has a stabilizing role for the ULBP2 A1A2 domain. Competitive selection in rounds with increasing concentrations of wtNKG2D (Figure 4A) yielded three variants, U2S1, U2S2, and U2S3, that reproducibly bound exclusively to iNKG2D.YA, even when reformatted as a fusion to the C-terminus of the IgG1 heavy chain of the anti-FGFR3 antibody clone R3Mab (Figure 4B). Although the R81W mutation alone enhanced affinity for both wtNKG2D and iNKG2D.YA (Figures 4B and 4C), its presence in the iNKG2D-selected variants was deemed essential, since reversion to its wild-type residues resulted in loss of binding to iNKG2D.YA. U2S3 consistently showed greater binding differentials and was therefore more thoroughly characterized and shown to be a monomer with 10-fold higher affinity for iNKG2D.YA than wild-type ULBP2 for wtNKG2D (Figure 4C). The A1-A2 domain of SEQ ID NO: 11 is based on the U2S3 ligand (SEQ ID NO: 30). Picomolar binding to iNKG2D.YA was measured with bivalent rituximab antibody fusions, and orthogonality was maintained by both light chain (LC) and heavy chain (HC) fusion configurations (Figure 5).

[0061] Candidate orthogonal variants were identified as for iNKG2D.AF, and ELISAs comparing rituximab-LC fusions to Fc-wtNKG2D, Fc-iNKG2D.YA, and Fc-iNKG2D.AF identified four variants that selectively bound only to iNKG2D.AF (Figure 3B), with the U2R variant being the most selective. ELISAs comparing binding of rituximab.LC-U2S3 and rituximab.LC-U2R to both iNKG2D.YA and iNKG2D.AF confirmed that these two independently selected orthogonal ligands were exclusively associated with the inactive NKG2D variants from which they evolved (Figure 3C).

[0062] Expression of iNKG2D.YA as a chimeric antigen receptor: Lentiviral transduction of iNKG2D.YA fused to 4-1BB, CD3ζ, and eGFP into primary human T cells efficiently generated convertibleCAR-T cells that displayed robust transgene expression comparable to that of a rituximab-scFv-based CAR construct (RITscFv-CAR) with the same hinge, transmembrane, and intracellular architecture (Figure 2b and Figure 6). Surface staining of iNKG2D.YA with rituximab LC-U2S3 MicAbody strongly correlated with GFP expression, suggesting a direct relationship between the efficiency of CAR expression and the presentation of iNKG2D on the T cell surface (Figure 7C). Using flow cytometry of rituximab.LC-U2S3 MicAbody conjugated with Alexa Fluor 647 and standard quantification beads, the median amount of iNKG2D.YA expressed on the surface was estimated to be 21,000 molecules. Direct engagement of the iNKG2D.YA-CAR receptor by incubation of convertibleCAR-CD8+ cells on microtiter plates coated with wild-type or U2S3 ligands resulted in activation and release of IL-2 and IFNγ only with U2S3, whereas wtNKG2D-CAR-bearing cells responded only to the wild-type ligand, supporting the selectivity of orthogonal interactions in the context of T cells (Figure 8A). Furthermore, activation of convertibleCAR-T cell function was dependent on the presence of the appropriate cognate ULBP2 variant. Only iNKG2D.YA-expressing or iNKG2D.AF-expressing T cells lysed Ramos (CD20+) target cells when armed with MicAbodies bearing the respective orthogonal ligands, i.e., U2S3 or U2R (Figure 3D). Co-culture of Ramos cells alone was not sufficient to drive activation of convertibleCAR-CD8+ cells. Neither rituximab antibody nor trastuzumab LC-U2S3 activated CAR cells, whereas rituximab LC-U2S3 elicited maximal cytokine release in the range of 32–160 pM, thus requiring an appropriate antigen-targeted MicAbody instead.Furthermore, cytokine release by convertibleCAR-T cells bearing the rituximab.LC-U2S3 MicAbody exceeded that of RITscFv-CAR cells (Figure 8B). These data demonstrated that appropriate antigen-targeted MicAbodies are required to drive robust activation of T cell function, likely forming junctions between targets and convertibleCAR-T cells similar to those characterized for scFv-CAR19 (Figure 9A).

[0063] Staining of convertibleCAR-CD8+ cells with fluorescently labeled Rituximab LC-U2S3 MicAbody revealed saturation of all iNKG2D.YA-CAR receptors at 5 nM (Figure 9B). However, in co-culture killing experiments of convertibleCAR-CD8+ cells armed with decreasing amounts of Rituximab LC-U2S3, the lytic activity of Ramos target cells reached a saturation response at 30 pM, two orders of magnitude less than required for full receptor occupancy (Figure 9C). This result suggests that excess unoccupied iNKG2D.YA-CAR receptors can be armed with heterologous MicAbodies to induce activity against multiple targets simultaneously. To test this directly, convertibleCAR-CD8+ cells were armed with Rituximab.LC-U2S3, Trastuzumab.LC-U2S3 (targeting Her2), or an equimolar mixture of the two MicAbodies and exposed to either Ramos cells or CT26-Her2. Whereas CAR cells armed with a single MicAbody induced lysis only in tumor cells expressing the cognate antigen, dual-armed CARs targeted both tumor cell lines without compromising lytic potential (Fig. 9D).

[0064] Convertible CAR-T cells suppress disseminated B-cell lymphoma: Pharmacokinetics of both HC and LC rituximab-U2S3 MicAbodies in NSG mice (Figure 10A) revealed a beta phase comparable to the parental antibody with a steeper alpha phase of the MicAbody due to retention of U2S3 binding to endogenous mouse wild-type NKG2D (Figure 10C). LC-U2S3 fusions (i.e., antibody fusion proteins in which the A1-A2 domains are fused to the light chain of the antibody) had longer terminal half-lives than HC-fusion MicAbodies (i.e., antibody fusion proteins in which the A1-A2 domains are fused to the heavy chain of the antibody). LC-U2S3 fusions also outperformed HC fusions in in vitro killing assays with Ramos target cells (Figure 10B) and appeared to be more effective at earlier time points in suppressing growth of Raji B-cell lymphoma in NSG mice. In summary, antibody fusion proteins containing the A1-A2 domains fused to the N-terminus of the antibody light chain surprisingly outperformed antibody constructs in which the A1-A2 domains were fused to the heavy chain.

[0065] Rituximab LC-U2S3 (Rit-S3; an antibody fusion protein in which the A1-A2 domain of U2S3 is fused to the light chain of an antibody) was used in further experiments to explore dosing parameters for controlling lymphoma. A medium Rit-S3 dose of 20 μg was shown to be most effective, as higher concentrations may result in oversaturation of the receptors on CAR cells and antigens on tumor cells, thereby preventing productive engagement. Furthermore, more frequent Rit-S3 dosing, every 2 days versus every 4 days, was used in combination with a higher dose (10×10 6 Combining Rit-S3 with convertible CAR-T cells from 1000- and 1000-fold increased tumor growth in mice treated with Rit-S3 alone. Rit-S3 alone had no effect on tumor control, but graft-versus-tumor effects were consistently observed in both the untransduced and convertibleCAR-only cohorts. Rit-S3 was detectable in the serum of mice throughout the study, with peak levels occurring sooner with more frequent dosing.

[0066] A Raji disseminated lymphoma model using optimized convertibleCAR-T administration was performed, with 20 μg of Rit-S3 administered every 2 days, to treat 5 × 10 6 (5M) and 15×10 6 (15M) of convertibleCAR-T cells were compared. As a positive control, RITscFv-CAR cells, which have comparable in vitro Ramos killing power to convertibleCAR-T cells, were also included (Figure 8B). At a total of 5M T cells, both RITscFv-CAR and convertibleCAR+Rit-S3 were effective in controlling tumors. The mean tumor bioluminescence signal was lower in the RITscFv-CAR cohort (Figure 11A), and tumor tissue appeared to disappear in 4 of 5 mice in that cohort, but in 3 of 5 mice in the convertibleCAR+Rit-S3 cohort (Figure 11B). When the total injected dose of CAR-T cells was increased to 15M cells, both RITscFv-CAR and convertibleCAR+Rit-S3 were able to completely block tumor growth (Figures 11A and 11B). In all studies, peak levels of peripheral human CD3+ T cells consistently appeared around 7 days after infusion, and by 14 days, contraction was observed in most mice with both scFv-CAR and convertibleCAR-T cells (Figure 11D). There was delayed expansion of CD3+ cells in the non-transduced and convertibleCAR-only cohorts, which coincided with the onset of the graft-versus-tumor response and was likely the result of the expansion of specific reactive clones. In the convertibleCAR+Rit-S3 cohort, MicAbody-associated convertibleCAR-T cells were observed in the blood of mice (Figure 11E).

[0067] Convertible CAR-T cells suppress subcutaneous lymphoma: Raji B cells were implanted subcutaneously to evaluate the ability of the convertible CAR system to suppress the growth of solid tumor masses. After tumors were established for 10 days, 7 × 10 6 (7M) or 35×106 In addition, one cohort received 35M cells pre-armed with a saturating concentration of Rit-S3 prior to administration, but no additional MicAbody-delivering injections. Administration of 7M convertibleCAR-T cells with Rit-S3 (7M+Rit-S3) reduced tumor size compared to convertibleCAR-T cells alone (Figure 12A). Furthermore, tumor growth was completely suppressed in the 35M+Rit-S3 cohort. Tumor growth in the cohort that received 35M pre-armed cells was also inhibited. By 2 days post-infusion, pre-armed convertibleCAR-T cells had no detectable surface-associated Rit-S3 MicAbody (Figure 12C), likely a result of disarming due to activation-induced cell proliferation in combination with armed receptor turnover. Serum levels of Rit-S3 were comparable for both CAR-T cell doses throughout the study, persisting through day 21 (Figure 12B), and were detected at approximately 600 ng / mL (3.2 nM), corresponding to high levels of armed peripheral CAR cells (Figure 12C). By day 45 of the study, the 35M+Rit-S3-fed cohort maintained relatively high CD3+ T cell counts but were not fully armed with MicAbody, while the 7M+Rit-S3 cohort had cells that maintained surface-associated MicAbody. This suggested that high CAR-T cell levels were unable to maintain CAR arming because MicAbody levels were below the detectable limit in plasma. An alternative possibility is that the higher CD3+ cell counts in the 35M+Rit-S3 cohort reflect the expansion of a graft-versus-tumor subset of cells that do not express the CAR construct. However, the lack of elevated CD3+ cell counts in the 35M pre-armed cohort suggests that this is not the case. In summary, pre-armed convertibleCAR-T could exert potent antitumor responses that inhibited tumor growth. Moreover, convertibleCAR-T could effectively control solid lymphoma when adequate in vivo levels of convertibleCAR-T cell arming were maintained.

[0068] Selective delivery of biomolecules to convertible CAR-T cells: The privileged interaction between iNKG2D variants and their orthogonal ligands enables the selective delivery of drugs to iNKG2D-CAR-expressing cells simply by fusing them as payloads to the orthogonal ligands themselves. To demonstrate the utility of this feature, two different applications were explored: targeted ablation using the complement system and selective delivery of activating cytokines. In the first application, the U2R variant was fused to either the N-terminus or C-terminus of the wild-type human IgG1 Fc domain, or to the mutant Fc domains S267E / H268F / S324T / G236A / I332E (「EFTAE」) and K326A / E333A (「AA」) that enhance C1q binding (Figure 13A). In contrast to using a complete therapeutic antibody that targets epitope-tagged CAR cells, using only the Fc portion avoids the concomitant effects of opsonization of non-iNKG2D-expressing cells. Enhanced C1q binding was confirmed by ELISA with a Kd in the relative order of EFTAE < AA < wt (Figure 13B). iNKG2D.AF-CAR cells were sensitive to killing by human complement in a manner that depended on both concentration and C1q affinity (Figures 13C and 13D), while non-transduced cells were not affected. Interestingly, the orientation of the U2R fusion was important for function, and the N-terminal fusion that oriented the Fc in a manner consistent with the antibody was far more effective. Similar results were obtained with the pairing of U2S3 and iNKG2D.YA (Figures 13E and 13F).

[0069] The potential ability of orthogonal ligands to selectively deliver cytokines to iNKG2D-CAR expressing cells has the advantage of not only promoting their proliferation but also potentially exploiting differential cytokine signaling to control T cell phenotype and function. As a general design principle, mutant cytokines with reduced binding to their native receptor complexes were used to reduce engagement with immune cells not expressing CAR and minimize toxicity associated with wild-type cytokines. Furthermore, cytokine fusions were kept monovalent to preclude binding and signaling with enhanced avidity. To this end, the R38A / F42K mutation in IL-2 (mutIL2)25 and the V49D mutation in IL-15 (mutIL15) dramatically reduce binding of each cytokine to its respective Rα subunit while maintaining engagement of the IL-2Rβ / γ complex. Initial experiments with the iNKG2D.YA orthogonal variant U2S2 fused to either mutIL2 or mutIL15 promoted proliferation of iNKG2D.YA-CAR expressing cells but not cells expressing wtNKG2D-CAR (Figure 14A). Both cell populations expanded in the presence of the ULBP2.R81W variant, which does not distinguish between wtNKG2D and iNKG2D.YA. Fusion to the ligand directly or via heterodimeric Fc linkage (e.g., U2S2-hFc-mutIL2) promoted proliferation of GFP+convertibleCAR-T cells to densities exceeding those of existing untransduced cells (Figure 14B), and these expanded convertibleCAR-T cells maintained their cytolytic capacity (Figure 14C). Engagement of iNKG2D.YA with MicAbody, monovalent U2S3-hFc (without cytokine payload), or mutIL2 alone was insufficient to promote proliferation of convertibleCAR-CD8 cells (Figures 14A and 14D). Flow cytometric characterization of STAT3 and STAT5 phosphorylation (pSTAT3 and pSTAT5) revealed that exposure to wild-type IL-2 or IL-15 led to an increase in pSTAT3 and pSTAT5 in both non-transduced and convertibleCAR-CD8 cells.Treatment of non-transduced cells with U2S3-hFc-mutIL2 only resulted in a minimal shift in pSTAT5 compared to non-cytokine controls, consistent with retention of mutIL2 in IL-2Rβ / γc binding. ConvertibleCAR-CD8 cells responded to both U2S3-hFc-mutIL2 and U2S3-hFc-mutIL15 with increased pSTAT5 levels via γ-chain activation of JAK3. Unlike wild-type cytokines, no increase in pSTAT3 signaling was observed, indicating reduced JAK1 activation by IL-2Rβ in both scenarios as a result of disruption of Rα binding, supporting the hypothesis of a role for IL-15Rα in increasing the affinity of IL-15 for IL-2Rβ. The kinetics of the U2S3-hFc-mutIL2 and U2S3-hFc-mutIL15 responses were nearly identical, suggesting functional redundancy in their mutant forms.

[0070] U2S3-hFc-mutIL2 was shown to have an in vivo PK half-life of several days (Figure 14E). ConvertibleCAR-T cells administered to NSG mice in the absence of tumors underwent homeostatic proliferation, peaking at 3 days, followed by contraction. Three injections of U2S3-hFc-mutIL2, given stepwise at 1-week intervals, led to a dramatic expansion of human T cells in peripheral blood (Figure 15A), with a reduction in T cell numbers after cessation of U2S3-hFc-mutIL2, corroborated by CD8+ T cells driving the majority of the proliferation. In parallel with the proliferation, the percentage of GFP+CD8+ T cells increased to 100%, demonstrating selective expansion of iNKG2D-CAR-expressing cells, but not non-transduced cells (Figure 15B).

[0071] The effect of U2S3-hFc-mutIL2 on normal human PBMCs from three donors was explored in vitro by flow-based quantification of cells positive for the proliferation marker Ki-67 after 4 days of exposure to increasing concentrations of the drug (Figure 16). In addition to the -mutIL2 fusion, a wild-type IL2 fusion (U2S3-hFc-wtIL2) was included to directly demonstrate that the reduced mutIL2 bioactivity was a result of the mutations used and not the fusion format itself. CD4+ and CD8+ T cells responded robustly to both anti-CD3 and wild-type IL-2 positive controls, as well as to the lowest dose of U2S3-hFc-wtIL2. The proliferative response to U2S3-hFc-mutIL2 occurred in a dose-dependent manner, with proliferation observed across donors at levels above 300 IUe / mL, but not reaching levels equivalent to those of the IL-2 positive control until 30,000 IUe / mL. Treg responses were comparable to those of CD4+ and CD8+ cells, except for cells from one donor that responded to U2S3-hFc-mutIL2 at lower concentrations than the other donors (which additionally showed a weak response to anti-CD3 stimulation). Taken together, these data support the hypothesis that normal human PBMCs do not respond to U2S3-hFc-mutIL2 except at supraphysiological levels, potentially providing a broad administration window for selectively delivering ligand-fused mutIL2 to convertibleCAR cells while minimizing toxicity and Treg activation.

[0072] Comparison of A1-A2 domain location and linkers: In addition to the above tests, constructs containing different linkers connecting the antibody heavy or light chain to the A1-A2 domain were examined. See FIG. 17A. Rituximab antibodies were generated that contain U2S3-based A1-A2 domains linked to the heavy chain (Ritux.HCd) via a GGGS (SEQ ID NO: 14) linker (Ritux.HCd.S3) or an APTSSSGGGS (SEQ ID NO: 10) linker (Ritux.HCd.apts.S3). Similar constructs were generated in which the A1-A2 domains were fused to the light chain of the Rituximab antibody via the same linkers (Ritux.HCd.LC.S3 (APTSSSGGGGS linker (SEQ ID NO: 10)) and Ritux.HCd.LOC.gggs.S3 (GGGS linker (SEQ ID NO: 14))). The constructs were examined using the methods described herein in connection with FIG. 10B. The results are shown in FIG. 17B. An antibody fusion construct comprising a heavy chain comprising the variable region sequence of SEQ ID NO: 1 and a light chain comprising the variable region sequence of SEQ ID NO: 8, the light chain fused C-terminally to the A1-A2 domain, was superior in killing tumor cells to a construct in which the A1-A2 domain was linked to the heavy chain. Furthermore, a construct in which the A1-A2 domain was fused to the light chain via an APTSSGGGGS linker (SEQ ID NO: 10) was surprisingly superior to all constructs tested at nearly all concentrations (0.04 nM, 0.2 nM, 1 nM, and 5 nM).

[0073] Mutations affecting glycosylation: Glycosylation introduced during the production of protein therapeutics can lead to undesired heterogeneity in the final product. The ones present at positions 40 and 54 in SEQ ID NO: 30 were mutated to introduce alanine or glutamine via substitution. These substitutions reduced N-glycosylation when the peptide was expressed in HEK293 cells. Surprisingly, N-glycosylation was observed in the mutant A1-A2 domain peptide when the peptide was produced in CHO cells. An additional mutation was introduced into the sequence at position 84 to introduce alanine or glutamine via substitution.

[0074] The activity of mutant A1-A2 domains containing substitutions at positions 40, 54, and / or 84 was confirmed in the context of U2R ligands fused to rituximab. Rituximab fusion proteins containing U2R ligands (1) containing alanines at positions 40 and 54 of the A1-A2 domain, or (2) containing glutamines at positions 40 and 54 of the A1-A2 domain, were tested in a killing assay using iNKG2D.AF-CAR cells against CD20+ve Ramos cells. Fusions containing mutant A1-A2 domains performed similarly to fusions with the parent A1-A2 domain (no substitutions at positions 40 and 54).

[0075] Substitutions were also made at positions 40 and 54 of SEQ ID NO:30 (U2S3(NQ)) to generate a fusion protein containing the mutant A1-A2 domain fused to the light chain of rituximab. SDS-PAGE confirmed that the mutant A1-A2 domain fusion showed reduced N-glycosylation when expressed in Expi-293 cells. Similar results were observed in CHO cells. ELISA assays were performed using methods similar to those described above to confirm that antibodies containing the U2S3(NQ) domain bound to iNKG2D.YA similarly to antibodies containing the unmodified U2S3 domain of SEQ ID NO:30. See FIG. 18. Binding to wild-type NKG2D was also tested. See FIG. 19. MicAbodies containing U2S3 and U2S3(NQ) showed substantially reduced binding to wild-type NKG2D, and it was observed that introduction of glutamines at positions 40 and 54 resulted in a surprisingly more inactive fusion with respect to wild-type NKG2D binding (i.e., the A1-A2 domain containing the substitutions bound iNKG2D.YA to a similar extent as the parent domain without the substitutions, but showed further reduced binding to wild-type NKG2D).

[0076] When the U2S3(NQ) domain was expressed in CHO cells, N-glycosylation was observed, despite the fact that N-glycosylation appeared to be virtually absent when expression was performed in HEK293 cells. Further substitutions of the U2S3(NQ)A1-A2 domain were made to introduce an alanine (U2S3(AYT)) or a glutamine (U2S3(QYT)) at position 84. Octet binding experiments were performed with this additional mutation in the context of the Rituximab-MicAbody. Substitution with alanine or glutamine at position 84 did not alter binding to iNKG2D.YA. Cytotoxicity was also confirmed. See FIG. 20. Ramos target cells were loaded with calcein and co-cultured with iNKG2D-CAR CD8+ T cells in the presence of increasing nM concentrations of each MicAbody tested (rituximab fused to U2S3, U2S3(NQ), U2S3(AYT), and U2S3(QYT)) at an E:T ratio of 20:1 for 2 hours. The amount of calcein released was quantified. All MicAbodies mediated comparable levels of cytotoxicity, indicating that (a) CD20 engagement was not compromised and (b) iNKG2D engagement was not compromised by the substitutions described herein. Thus, the A1-A2 domains described herein demonstrated reduced glycosylation and mediated comparable levels of target cell binding and cytotoxicity using iNKG2D-CAR, and further demonstrated reduced binding to wild-type NKG2D.

[0077] Consideration The present disclosure describes the engineering of a privileged receptor-ligand (iNKG2D.YA and U2S3) pairing composed of human components of a highly adaptable CAR resulting in a versatile and extensively controllable platform. The iNKG2D.YA-CAR receptor itself is immutably maintained on T cells with CAR function that is easily directed to potentially any antigen of interest by conjugating the orthogonal ligand to an appropriate antigen-recognizing antibody. In this way, the same convertibleCAR-T cells can be retargeted as needed, for example, if the original tumor antigen is downregulated during the course of treatment. This targeting flexibility is not limited to sequential engagement of antigens, but can also be multiplexed to simultaneously direct T cells to multiple antigens to reduce the chance of tumor escape due to antigen loss, to address the problem of intratumoral antigen expression heterogeneity, or to simultaneously target suppressive cellular components of the tumor and tumor microenvironment. Conventional scFv-CAR cells are generally heavily committed to fixed expression levels of receptors that reduce the ability to distinguish between antigen levels present on healthy cells and those present on abnormal cells. The use of a switch / adaptor strategy, such as MicAbody with convertible CAR-T cells, may offer the opportunity to differentially engage CAR-T to achieve a therapeutic index that reduces the risk of serious adverse events.

[0078] The use of privileged receptor-ligand interactions to deliver payloads specifically to iNKG2D-bearing cells without additional cell engineering is another advantage. The ability to harness interleukin function to drive proliferation and activation, prevent exhaustion, or even promote suppression in a controlled and targeted manner can have beneficial consequences for efficacy and safety. The introduction of cytokine-ligand fusions during CAR manufacturing can address the qualitative and quantitative limitations of patient T cells, and their administration after CAR infusion can expand the number of CAR-T cells, and their persistence with CD19-CAR therapy positively correlates with response rates. While most CAR therapies require lymphodepletion preconditioning methods to promote CAR cell engraftment and expansion, one rationale is to provide a more immature immunological setting for CARs to grow. Robust and controllable expansion of convertibleCAR-T in patients can replace the need for lymphodepletion, allowing the retention of endogenous immune functions with sufficient capacity to support initial convertibleCAR-mediated antitumor activity. Another clinical strategy could be to deliver cytokine-ligand fusions to enhance convertibleCAR-T function, possibly with a cycling regimen to reduce T cell exhaustion and promote maintenance of memory T cells. And finally, since CARs have been demonstrated to persist in humans for years after infusion, the ability to recall resident convertibleCAR-Ts to attack primary or secondary malignancies (either with the original targeted MicAbody or a different one) without the need to redesign or create new batches of CAR cells would be highly advantageous. Unlike the scenario in which CARs are engineered to constitutively express cytokines, the delivery of exclusive cytokines to convertibleCAR-T cells could be tuned according to manufacturing or clinical needs.

[0079] By design, each component of the convertibleCAR system, the iNKG2D-based CAR receptor and the MicAbody (ADCC-deficient), are themselves functionally inactive. This has advantages during manufacturing, particularly in relation to indications such as T-cell malignancies where traditional scFv-based CARs encounter proliferation difficulties due to friendly fire. Furthermore, this enhances the control of CAR function during treatment. This disclosure demonstrates that convertibleCAR-T cells can be armed with MicAbody prior to administration to provide an initial burst of antitumor activity comparable to traditional scFv-CARs. In addition to activation-induced replication, these cells also internalize the engaged CAR receptor in a manner consistent with that observed with other 4-1BB / CD3ζ scFv-CARs. As a result of these two processes, convertibleCAR-T cells rapidly disarm after initial proliferation and target engagement, providing an opportunity to then re-arm and re-engage in a manner controlled by MicAbody administration.

[0080] In addition to the ULBP2-based iNKG2D-U2S3 pairing, the present disclosure identifies high affinity orthogonal MicA and ULBP3 variants to iNKG2D.YA that are not redundant in amino acid composition through the helix 4 domain. Furthermore, a completely independent iNKG2D.AF and U2R pairing is described. Having mutually exclusive receptor-ligand pairs allows, for example, to introduce them into separate cell populations (e.g., CD4 and CD8 T cells) and differentially engage them as needed. Furthermore, within the same cell, two iNKG2D variants can be expressed with split intracellular signaling domains to provide dual antigen-dependent activation and enhance tumor selectivity. Alternatively, two iNKG2D variants can be differentially linked to either the activation domain or the immunosuppressive domain to enhance the discrimination of T cells between tumors or healthy tissues, respectively.

[0081] In summary, the system described herein demonstrated the ability to be easily targeted to distinct cell surface antigens as well as selectively exogenously engaged to drive cell proliferation. The privileged receptor-ligand interactions developed are cell type independent and can be engineered into any cell of interest as long as the cell is provided with the appropriate signaling domain. Furthermore, the adoptive cell therapy field is actively pursuing the development of allogeneic cells to reduce the time, complexity, and cost of manufacturing to provide a more consistent and readily available product. Highly adaptable CAR systems are highly synergistic with allogeneic efforts, and once a truly universal allogeneic CAR system is demonstrated, the therapeutic field will be characterized by the relative ease of development and implementation of libraries of adaptor molecules that can perform personalized selection. This strategy also expands the potential area of ​​application to any pathogenic cell bearing a targetable surface antigen.

[0082] All references cited in this specification, including publications, patent applications, and patents, are hereby incorporated by reference as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

Claims

1. An A1-A2 domain peptide comprising an amino acid sequence having at least 95% identity with SEQ ID NO: 30, wherein the peptide contains alanine or glutamine at one or more of the 40th, 54th, and / or 84th positions of SEQ ID NO:

30.

2. The A1-A2 domain peptide according to claim 1, wherein the peptide comprises glutamine residues at positions 40 and 54 of SEQ ID NO:

30.

3. The A1-A2 domain according to claim 1, wherein the peptide contains glutamine at position 84 of SEQ ID NO:

30.

4. The A1-A2 domain according to claim 2, wherein the peptide contains glutamine at position 84 of SEQ ID NO:

30.

5. The A1-A2 domain according to claim 1, wherein the peptide contains alanine at position 84 of SEQ ID NO:

30.

6. The A1-A2 domain according to claim 2, wherein the peptide contains alanine at position 84 of SEQ ID NO:

30.

7. The A1-A2 domain peptide according to claim 1, wherein the peptide comprises SEQ ID NO: 11, SEQ ID NO: 31, or SEQ ID NO:

32.

8. An A1-A2 domain peptide according to any one of claims 1 to 7, fused to an antibody light chain.

9. The A1-A2 domain peptide according to claim 8, wherein the antibody light chain comprises a variable region sequence containing the amino acid sequence of SEQ ID NO:

8.

10. An A1-A2 domain peptide according to any one of claims 1 to 7, fused to an antibody heavy chain.

11. The A1-A2 domain peptide according to claim 10, wherein the antibody heavy chain comprises a variable region sequence including the amino acid sequence of SEQ ID NO:

1.

12. The A1-A2 domain peptide according to any one of claims 1 to 7, for use in the treatment of cancer in subjects requiring it.

13. The A1-A2 domain peptide according to claim 12, wherein the A1-A2 domain peptide is present in the antibody fusion protein.

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