Humanized Anti-CD8 Antibody and Uses thereof

Abstract

The present disclosure provides a humanized antibody that binds to CD8α and its antigen-binding domain, and other antibody formats comprising such antigen-binding domain, and uses thereof as a targeting moiety on lipid nanoparticles (tLNP) for delivering a therapeutic payload (e.g., a nucleic acid molecule) or other types of payload. The present disclosure further relates to a pharmaceutical composition comprising a CD8-targeted tLNP and a humanized anti-CD8α antibody that encapsulates a payload.

Description

Technology Field

[0001] Reference to the electronic sequence list

[0002] The present application comprises a sequence list that was submitted electronically and is incorporated herein by reference in its entirety. The sequence list was created on December 15, 2024, has a filename of "23-1742-US _SequenceListing.xml", and has a size of 250,755 bytes.

[0003] Cross-reference of related applications

[0004] The present application claims priority to U.S. Provisional Application No. 63 / 610,917 filed December 15, 2023; U.S. Provisional Application No. 63 / 654,930 filed May 31, 2024; and U.S. Provisional Application No. 63 / 708,461 filed October 17, 2024; the disclosures thereof are expressly incorporated herein by reference. Background Technology

[0005] Differentiation cluster 8 (CD8) antigen is a cell surface glycoprotein found on most cytotoxic T lymphocytes, which helps mediate efficient cell-cell interactions. CD8 antigen binds to Class I major histocompatibility complex (MHC) molecules and acts as a co-receptor with the T cell receptor (TCR) on T lymphocytes to recognize antigens displayed by antigen-presenting cells (APCs) in the context of Class I MHC molecules. The co-receptor acts as a homodimer composed of two alpha chains (CD8 subunit alpha—CD8α) or as a heterodimer composed of one CD8α and one CD8 beta chain (CD8β).

[0006] CD8-positive T cells are mediators of adaptive immunity. These include cytotoxic T cells, which are important for killing cancer cells, virus-infected cells, or other pathogenic cells, and CD8-positive suppressor T cells, which suppress certain types of immune responses.

[0007] Several anti-CD8 antibodies have been developed. However, many were generated in mice, where these antibodies possess the ability to induce an immune response when introduced into humans. Therefore, there remains a need for humanized (or human) anti-CD8 antibodies that possess reduced immunogenic potential while maintaining biological activity, particularly in binding to CD8-expressing cells. Furthermore, such humanized anti-CD8 antibodies must also possess biophysical properties suitable for clinical development and manufacturing. Thus, there is a need to develop improved anti-CD8 antibodies for therapeutic applications. Brief explanation of the drawing

[0008] The present disclosure will be better understood by considering the following detailed description of the invention, and features, aspects, and advantages other than those presented above will become clear. Such detailed description refers to the following drawings. Figure 1a shows the humanized sequence of the variable heavy chain (VH) of CBD1017vh (mouse anti-CD8 antibody clone CT8) in the IGHV1-46*01 (VH1-46) germline. Text in bold indicates the CDR region based on the AbM definition (see bioinf.org.uk / abs / ). Text underlined indicates framework residues reverse-mutated to amino acids found in the maternal mouse antibody. Other potential changes are indicated below the sequence. Seq: Simple sequential amino acid numbering. AbM: Chothia amino acid numbering. Figure 1b shows the humanized sequence of the variable light chain (VL) of CBD1017vl (mouse anti-CD8 antibody clone CT8) in the IGKV1-39*01 (VK1-39) germline. Text in bold indicates the CDR region based on the AbM definition. Text underlined indicates framework residues reverse-mutated to amino acids found in the maternal mouse antibody. Other potential changes are indicated below the sequence. Seq: Simple sequential amino acid numbering. AbM: Chothia amino acid numbering. Figure 1c shows the humanized sequence of the variable heavy chain (VH) of CBD1017vh (mouse anti-CD8 antibody clone CT8) in the modified IGHV1-18*01 (VH1-18) germline. Dark bars represent the CDR region. Amino acid residues within CBD1017vh that differ from the VH1-18 sequence are shaded, similar to residues in the humanized sequence using mouse residues. Simple sequential amino acid numbering was used. Figure 1d shows the humanized sequence of the variable light chain (VL) of CBD1017vl (mouse anti-CD8 antibody clone CT8) in the modified IGVK3D-11*01 (VK3D-11) germline. Dark bars represent the CDR region. Amino acid residues within CBD1017vh that differ from the VH1-18 sequence are shaded, as are residues in the humanized sequence using mouse residues. Simple sequential amino acid numbering is used. Figure 2a is a summary of the binding constants of different humanized anti-CD8 antibody binding fragments (Fabs) based on VH1-46 and VK1-39 germlines as measured by biolayer interferometer (BLI) kinetic assays at 30°C and 37°C. Figure 2b shows the binding kinetics sensogram of CBD1033 Fab for CD8αα antigen at indicated concentrations in the biolayer interferometric kinetics assay at 30°C and 37°C. As described below, overall fitting was performed using a 1:1 fitting model with GatorOne software. Figure 2c is a summary of the steady-state binding constants of different humanized anti-CD8 antibodies with hIgG1-LALAPA (human IgG1(hIgG1) isoforms with Fc-silencing mutants L234A, L235A, and P329A(LALAPA)) (Sequence No. 43) humanized into the VH1-46 and VK1-39 germline framework regions as measured by biolayer interferometer kinetics assay. Figure 2d shows the binding kinetics sensogram of the interaction between CBD1033 IgG1 and human CD8αα antigen at the indicated concentrations in the biolayer interferometer kinetics assay. As described below, overall fitting was performed using a 1:1 fitting model with GatorOne software. Figure 2e shows a steady-state analysis of the humanized antibody CBD1033 (CBD1033-hIgG1-LALAPA) (Sequence No. 43) having a human IgG1 constant region with Fc-silencing mutants L234A, L235A, and P329A (LALAPA) interacting with human CD8αα based on the data in Figure 2d. Figure 2f is a summary of the steady-state binding constants of different humanized anti-CD8 antibodies with hIgG1-LALAPA (human IgG1 (hIgG1) isoforms (sequence number 43) with Fc-silencing mutants L234A, L235A, and P329A (LALAPA)) based on VH1-18 and VK3D-11 germlines interacting with human CD8αα as measured by biolayer interferometer kinetics assay. Figure 2g is K reported in Figure 2f DThis shows the biolayer interferometric binding kinetics sensogram plot for the humanized CBD1043 anti-CD8 antibody at the indicated concentrations of human CD8αα used for determination. As described below, overall fitting was performed using a 1:1 fitting model with GatorOne software. Figure 2h is based on the data in Figure 2g and K reported in Figure 2f D This represents the steady-state analysis of the humanized CBD1043 anti-CD8 antibody used in the determination. Figure 2i is a summary of the binding constants of CBD1033 and CBD1017ch Fab for human and cyanomolgus CD8αα homomers and CD8αβ heteromers as measured by biolayer interferometric kinetics assay at 37°C or 30°C. Figure 2j shows the binding kinetics sensogram of humanized CBD1033 and CBD1017ch Fab interacting with the cyanomolgus macaque CD8αα homomer antigen at the indicated concentrations in the biolayer interferometric kinetics assay at 30°C. As described below, overall fitting was performed using a 1:1 fitting model with GatorOne software. Figure 2k is a summary of the binding constants of the CBD1033 total antibody against human or cynomolgus macaque CD8αα homomers or CD8αβ heteromers as measured by surface plasmon resonance (SPR). Figure 2l shows the binding kinetics sensogram of the humanized CBD1033 whole antibody against human or cyanomorphic CD8αα homomer or CD8αβ heteromer in the SPR assay. Except for cyanomorphic CD8αβ heteromer, binding was analyzed at 2-fold serial dilutions of recombinant CD8αα homomer or CD8αβ heteromer proteins from 200 nM to 6.25 nM. For cyanomorphic CD8αβ heteromer, binding was analyzed at 2-fold serial dilutions from 400 nM to 12.5 nM. Overall fitting was performed using a 1:1 fitting model with GatorOne software as described below. Figure 3a shows the binding of an anti-CD8 total antibody with human IgG1 (hIgG1) homotype (SEQ No. 43) containing Fc-silencing mutants L234A, L235A, and P329A (LALAPA) at different concentrations to CD8-overexpressing HEK293T cells using a normalized geometric median fluorescence intensity (gMFI) assay. Binding was measured by fluorescence from an anti-human Fc secondary antibody conjugated to a BV421 fluorophore that recognizes the binder. gMFI was normalized to the gMFI of the secondary antibody in the absence of the anti-CD8α binder. Figure 3b shows the EC of the anti-CD8α antibody determined by normalized gMFI in the experiment shown in Figure 3a. 50 This is a summary of. Figures 4a to 4d show the transfection rates (Figures 4a and 4b) and expression levels (Figures 4c and 4d) of primary human T cells derived from different donors using a set of targeted lipid nanoparticles (tLNPs) conjugated with the indicated anti-CD8 antibody and encapsulating mCherry-encoding mRNA. The tLNPs added to the cells provided mRNA at concentrations of 0.6, 0.3, 0.15, 0.075, and 0 μg. The transfection rate was measured by the percentage of CD4-CD8+ T cells expressing mCherry. Figures 5a to 5c show a comparison of mCherry expression after transfection using CBD1017ch; humanized anti-CD8 binders derived from anti-CD8 antibody clones CT8 and OKT8; and mCherry-encoding mRNA encapsulated in CD8-targeted tLNPs using cetuximab (negative control) as a targeting moiety. Figure 6 shows the fold-in-fold increase in normalized gMFI of fluorescence measured by flow cytometry upon binding of the anti-CD8α binder CBD1033 at different concentrations to CD8-expressing lymphoma T cells SupT1 and HPB-ALL. Binding was measured by fluorescence from an anti-human Fc secondary antibody conjugated to a BV421 fluorophore that recognizes the binder. gMFI was normalized to the gMFI of the secondary antibody in the absence of the anti-CD8α binder. Figures 7a and 7b show the % mCherry positivity and transfection rate (% mCherry positivity) for an equivalent soluble fluorescent dye molecule (MESF) as measured by flow cytometry of cynomolgus macaque CD8+ T cells transfected with mCherry-encoding mRNA encapsulated in anti-CD8α binder-targeted lipid nanoparticles (tLNP). Figures 8a and 8b are normalized to show the background-contrast increase of gMFI measured by flow cytometry demonstrating the binding of the chimeric anti-CD8α binder and the humanized anti-CD8α binder at different concentrations to CD4-CD8+ resus, cynomolgus macaque, or human T cells, respectively. Binding was measured by fluorescence from a BV421 fluorescence-conjugated anti-human Fc secondary antibody that recognizes the binder. gMFI was normalized to the gMFI of the secondary antibody in the absence of the anti-CD8α binder. Figures 9a and 9b show EC in μg / mL and nM, respectively. 50Listing the values, this indicates similar CD8-specificity across T cells from different species (interspecies) of anti-CD8 binders, and the lack of binding to CD4+ T cells compared to humanized 5D7 antibodies (anti-CD5 antibodies). Figure 10 illustrates the efficient and cell-specific in vivo delivery of mCherry mRNA to CD8+ T cells using the anti-CD8 binder CBD1033 Fc silenced IgG1 whole antibody as a targeting moiety on tLNP. The antibody-to-mRNA (w / w) ratios on tLNP were 0.37, 0.72, and 1.09 as indicated. Figure 11a shows the affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS) test scores of six anti-CD8α hIgG1-LALAPA antibodies. All six binders showed a low propensity for self-binding. Vocosizumab was used as a positive control, while alirocumab and vococizumab NEI were used as negative controls. Figure 11b is a schematic summary of the results shown in Figure 11a. The results for the positive and negative controls are plotted as horizontal lines, and the results for the six anti-CD8α antibodies are plotted as circles. Fig. 12a shows the melting temperature (T) of the anti-CD8α hIgG1-LALAPA antibody. m It shows the dot plot distribution. The shaded area above 65°C indicates the range of good development potential. All results were within a sufficient range for chemical, manufacturing, and management (CMC) development potential. Figure 12b shows T as measured by differential scanning fluorescence (DSF). m and agglutination temperature (T) as measured by static light scattering (SLS) of anti-CD8 antibodies agg This is a summary of ). Figure 13a shows the dot plot distribution of the multiplex reactivity ELISA scores of six anti-CD8 binders for double-stranded DNA (dsDNA) and insulin. Figure 13b is a table of experimental values ​​plotted in Figure 13a. Figure 14 shows data from a baculovirus particle (BVP) multiplexer ELISA. PC: Positive control. NC: Negative control. The assay measured non-specific binding to an array of membrane proteins on baculovirus particles carrying multiple proteins from the host cell where the virus was produced. Figure 15 shows the binding interactions of anti-CD8α binders to human cell membrane proteins in a membrane proteome array assay. No additional significant specific interactions were observed other than the gene product of CD8A. Figure 16a shows the percentage of deamidation at the N55 position within the VH-CDR1 region for CBD1017ch at high pH (8.5) and high temperature (40°C) after 7 days under these conditions. Figure 16b shows the percentage of deamidation at the N55 position within the VH-CDR2 region at high pH (8.5) and high temperature (40°C) for anti-CD8α binders CBD1033, CBD1035, and CBD1039 after 7 days under these conditions. Figure 16c shows the percentage of deamidation at the N55 position within the VH-CDR2 region over time for CBD1033 at high pH (8.5) and high temperature (40°C). Figure 16d shows the minimum loss of binding affinity of the anti-CD8α binder due to high pH stress. Figure 16e shows the effect of the mutation on the binding affinity at the N55 position of the VH-CDR2 region. Figure 16f shows the effect of mutations at the N55 position of the VH-CDR2 region or the D30 position of VL-CDR1, or both, on the binding affinity of the Fab fragment. Figures 17a and 17b show the reduction of mCherry transfection by anti-CD8α tLNP in two separate donors by a mutation at N55 to aspartate (D) mimicking deamidation. A mutation at the N55 position of the VH-CDR2 region to encode glutamine (Q), serine (S), or alanine (A) does not result in a loss of these functions. Figure 17c is a summary of the mutations run and tested in Figures 17a and 17b. FIGS. 18a and 18b show the transfection efficiency and expression level of mCherry in T cells by anti-CD8α-targeted tLNPs, respectively. The anti-CD8α antigen-binding domain CBD1033 of various antibody formats (diabody synthetic hinge [Groups (GRP) 1 to 3], diabody IgG3 hinge [Groups 4 to 7], recombinant IgG1 F(ab')2 [Groups 8 to 10], recombinant IgG4 F(ab')2 [Groups 11 to 13], or enzymatically degraded IgG1 F(ab')2 [Groups 14 to 16]) was conjugated to tLNPs. Antibody density and reduction conditions were also varied (for details, refer to Example 8 and Table 15 therein). tLNPs containing CBD1033 Fc silenced IgG1 total antibodies were used as a positive control (group 17), and non-transfected cells (NTD) were used as a negative control. Figure 19a summarizes the design strategy for the disulfide-manipulated F(ab') construct. Figure 19b shows the absence of F(ab') dimers due to purification under reducing conditions without breaking the manipulated interchain disulfide bonds of F(ab'). Figure 20a shows the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of anti-CD8 F(ab') analogs CBD1033.37 or CBD1033.24 under fully reduced (R) or non-reduced (NR) conditions. Figure 20b shows the 280 nm absorbance peaks corresponding to the anti-CD8 Fab' binders CBD1033.37 and CBD1033.24 in the size exclusion high-performance liquid chromatography (SEC-HPLC) chromatogram. Figure 20c shows the results obtained from non-reducing liquid chromatography-mass spectrometry (LC-MS) analysis of the anti-CD8 F(ab') fragment of CBD1033.37 or CBD1033.24 having a high abundance peak corresponding to the F(ab') fraction. Figure 21a is a summary of the coupling constants of various engineered anti-CD8 coupled F(ab') fragments as measured by biolayer interferometer kinetics assay. Figure 21b shows the sensogram of the binding kinetics of CBD1033.37 F(ab') interacting with CD8αα homomers at the concentrations indicated in the biolayer interferometer kinetics assay. As described below, overall fitting was performed using a 1:1 fitting model with GatorOne software. Figure 22a demonstrates the conjugation reaction between humanized anti-CD8 F(ab') and maleimide-PEG-biotin. Figure 22b shows the immunoblotting of biotin-conjugated F(ab') analogs detected by streptavidin-horseradish peroxide (HRP). Figure 22c shows the 280 nm absorbance peak corresponding to the biotin-conjugated F(ab') fraction in the SEC-HPLC chromatogram. Figure 22d shows the results obtained from the LC-MS analysis of maleimide-PEG-biotin conjugated CBD1033.24 and CBD1033.37, each having a high abundance peak corresponding to the F(ab') fraction. Figure 22e shows the abundance ratios of detected modifications on conjugated CBD1033.24 and CBD1033.37 from peptide mapping analysis. The results demonstrate site-specific and quantitative conjugation of maleimide-PEG-biotin to CBD1033.24 and CBD1033.37. Amino acid positions follow sequential numbering. C227 and C230 correspond to C239 in the Kabat numbering of IgG4 and IgG1, respectively. Figure 23a is a summary of the binding constants for human CD8α mouse Fc fusion proteins of maleimide-PEG-biotin conjugated CBD1033.24 and CBD1033.37 F(ab') as measured by biolayer interferometric kinetic assay at 37°C. Figure 23b shows the binding kinetics sensogram of maleimide-PEG-biotin-conjugated CBD1033.37 against human CD8α mouse Fc fusion protein at the indicated concentrations in the biolayer interferometric kinetics assay at 37°C. Overall fitting was performed using a 1:1 fitting model with GatorOne software as described below. Figure 24a shows the level of mCherry expression as gMFI in primary human activated T cells transfected with anti-CD8α F(ab')-conjugated lipid nanoparticles (tLNP) encapsulating mCherry-encoding mRNA. The tLNP added to the cells was formulated at binder-to-mRNA ratios (w / w) of 0.1, 0.3, 0.5, 0.75, or 0.35. Transfection rates were measured by the percentage of CD4-CD8+ T cells expressing mCherry. tLNP conjugated to CBD1033.29, a total IgG1 containing a LALAPA Fc silencing mutant (CBD1033.3) and thiolized by the AJICAP process, was used as a positive control. Figure 24b illustrates the efficient and cell-specific in vivo delivery of mCherry mRNA to CD8+ T cells in the blood and spleen of NCG mice engrafted with human PBMCs using natural and engineered disulfide anti-CD8 F(ab') as targeting moiety on tLNP. The antibody-to-mRNA (w / w) ratio on tLNP was 0.35 for the total antibody (CBD1033.29) and 0.3 for F(ab'). CBD1033.29 is the CBD1033 antigen-binding domain linked to Sequence No. 43, which is an IgG1 constant region thiolized by the AJICAP process and possesses the Fc silencing LALAPA mutation (CBD1033.3). Figures 25a and 25b show the expression levels in expanded human CD8+ T cells from two donors transfected in vitro with modified tLNP-98219, in which the whole antibody as the targeting moiety was substituted with various engineered anti-CD8 F(ab') analogs. Transfection was performed in duplexes with a dose of 0.6 μg mRNA. CAR expression in duplexes transfecting CD8+ T cells from two donors with their respective tLNPs is shown as transfection efficiency (percentage of CAR+) (Figure 25a) and CAR expression levels (median fluorescence intensity) at 24 hours post-transfection (Figure 25b). tLNP-98219 is an anti-CD8 targeting composition F9 tLNP that encapsulates RM_61461 mRNA (sequence number 195) encoding anti-CD19 CAR2. The CBD1033.29 positive control was CBD1033.3 Fc-silenced whole antibody IgG1 (LALAPA) thiolized by the AJICAP process and conjugated to LNP. Figures 26a and 26b show the CAR transfection efficiencies of CD4+ and CD8+ T cells from expanded human T cells of two donors transfected with anti-CD8-targeted tLNPs encapsulating anti-CD19 CAR-encoding mRNA, respectively. Two mRNA constructs encoding anti-CD19 were used: the improved RM_61461 construct (SEQ No. 195) or the basic RM_61512 construct (SEQ No. 196). The improved construct is known to have higher expression than the basic construct, and both were used as assay controls. Groups 1 through 16 were various anti-CD8 F(ab') conjugated tLNPs with different antibody format designs and liability-engineered mutations in the variable domain. Groups 1 to 16 expressed the improved mRNA, RM_61461 (sequence number 195). The improved (RM_61461) and basic (RM_61512) control mRNAs were encapsulated in tLNPs, wherein the targeting moiety contained CBD1033.29 conjugated to the tLNP (improved control and basic control, respectively). Figures 26c and 26d show the CAR expression levels of CD4+ and CD8+ T cells, respectively, as measured by phycoerythrin fluorescence in experiments similar to Figures 26a and 26b. Figure 26e shows the CBD numbers of each group in Figures 26a through 26d, and a summary of the design and vulnerability-manipulation mutations. Figure 27a is the workflow of a cross-linked mass spectrometry study to identify the interaction site between the antibody and its antigen. This was applied to the identification of the epitope of CBD1033. Figure 27b demonstrates the identification and mapping of cross-linked amino acid positions on the existing structural model of human CD8αα homomers. Figure 27c shows the structure of a human CD8αα homomer with identified epitopes. Figure 28a shows the binding kinetics sensogram of a competitive binding experiment between CBD1033.3 and OKT8 or TRX2. After capturing CD8αα, CBD1033.3 was loaded to form a complex with CD8αα. Subsequent addition of OKT8 caused a reaction shift indicating binding to the CD8αα-CBD1033.3 complex, whereas TRX2 did not cause a reaction shift, indicating that TRX2 could not bind to the CD8αα-CBD1033.3 complex. Figure 28b shows a summary of competitive binding for human CD8αα homomers, where a spectral shift value of less than 0.7 indicates competitive binding. FIG. 28c shows two epitope bins, each containing identical or overlapping epitopes that are bound by the indicated antibody. Figure 29a shows the in vitro mCherry expression levels in primary human T cells using antibodies of CBD1033, TRX2, SK1, OKT8, humanized OKT8 variant 1 (VL and VH, taken from US11254744B2, represented by sequence numbers 229 and 230 respectively in Table 16) or humanized OKT8 variant 2 (LC and HC, taken from US11739150B2, represented by sequence numbers 231 and 232 respectively in Table 16) as the binding moiety of tLNP encapsulating mRNA-encoded mCherry. Figure 29b shows similar in vivo delivery of mCherry to CD8+ T cells in blood or spleen tissue using CBD1033 or TRX2 antibodies as binding moiety of tLNP encapsulating mRNA-encoded mCherry. Specific details for implementing the invention

[0009] The present invention provides a humanized antibody antigen-binding domain that specifically binds to CD8α (also known as CD8a and CD8 alpha, with the encoding gene known as CD8A), whole antibodies and other antibody formats comprising these antigen-binding domains, the use thereof as a targeting moiety in lipid nanoparticles (tLNPs) for delivering a payload (e.g., a nucleic acid molecule), and compositions of anti-CD8α tLNPs. The present invention also provides a composition comprising a humanized anti-CD8α antibody, an anti-CD8 tLNP encapsulating a payload, and a method of using the same. In certain embodiments, the payload is mRNA. In further embodiments, the mRNA encodes a protein that reprograms the antigen specificity of CD8+ cells. In some embodiments, the reprogramming agent encoded by the mRNA is a chimeric antigen receptor (CAR), a T cell receptor (TCR), or a T cell engager.

[0010] Specifically, the present invention provides a CD8α binding moiety comprising an immunoglobulin antigen binding domain that specifically binds to human CD8α in CD8αα homomers and CD8αβ heteromers, comprising a framework region derived from human germline heavy chain and light chain variable domain genes.

[0011] In some embodiments, the humanized anti-CD8α antibody of the present disclosure and its antigen-binding fragment specifically bind to both human and non-human primate (NHP) CD8. In some embodiments, the isolated humanized anti-CD8α monoclonal antibody of the present disclosure or its antigen-binding fragment has an agglutination temperature (T) of 60°C or higher aggIt has a melting temperature of 65°C or higher, exhibits a low propensity for self-interaction (i.e., aggregation tendency), preserves the absence of cross-reactivity of CT8, and lacks multiple reactivity to (a) double-stranded DNA and insulin; (b) baculovirus particles; or (c) human cell surface proteins and secreted proteins.

[0012] An additional sun is a tLNP containing an anti-CD8 targeting moiety, wherein the targeting moiety binds to a membrane-proximal epitope adjacent to the CD8 dimerization interface. Antibodies CT8, TRX2, and YTC182.20 compete for binding to the epitope. The epitope is a structural (i.e., non-linear) epitope comprising or adjacent to amino acids 40 to 47, 86 to 95, and 103 to 106 of CD8α. Regardless of whether such epitopes are defined by cross-competition for antibody binding, antibody-antigen cross-linking, or their position within the secondary or tertiary structure of CD8, they will be referred to herein as CT8 epitopes. Accordingly, the antigen-binding domains of CT8, TRX2, and YTC182.20 constitute means for binding to this CT8 epitope or means for targeting tLNP to CD8+ cells or CD8+ T cells. tLNP containing an antigen-binding domain in which the targeting moiety binds to the CT8 epitope transfects CD8+ cells more efficiently than tLNP containing an antigen-binding domain in which the targeting moiety recognizes some other CD8α epitope. Accordingly, tLNP containing an antigen-binding domain in which the targeting moiety binds to the CT8 epitope constitutes means for efficiently transfecting CD8+ cells or CD8+ T cells.

[0013] It should be understood that the specific embodiments of this specification described herein are not limited to the specific embodiments presented and may vary. Furthermore, it should be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting unless specifically defined herein. Additionally, as will be recognized by those skilled in the art, the specific embodiments disclosed herein may be combined without limitation with other embodiments disclosed herein.

[0014] definition

[0015] Before presenting the disclosure in more detail, providing definitions of certain terms used throughout the disclosure may aid in understanding the content. Additional definitions are provided throughout the disclosure.

[0016] Throughout this specification, unless the context specifically indicates otherwise, the terms “include” and “comprehensively” and variations thereof (e.g., “includes,” “comprehending,” “comprehensively,” and “comprehending”) are understood to indicate the inclusion of the mentioned components, features, elements, or steps or a group of components, features, elements, or steps, but not to mean the exclusion of any other components, features, elements, or steps or a group of components, features, elements, or steps. Any of the terms “comprehending,” “essentially composed of,” and “composed of” may be replaced with either of the other two terms while retaining their ordinary meanings.

[0017] As used herein, the singular forms (“a,” “an,” and “the”) include plural referents unless the context clearly indicates otherwise. As used herein, the terms “a” and “an” should be understood to refer to “one or more” of the enumerated components.

[0018] Unless otherwise indicated or otherwise evident from the context and the understanding of those skilled in the art, values ​​expressed as ranges herein may be estimated to be any specific value or sub-range within the range mentioned in different embodiments of this disclosure, and may be estimated to be up to one-tenth of the unit of the lower limit of the range unless the context clearly indicates otherwise.

[0019] As used herein and in the drawings, ranges and quantities may be expressed as "about" a specific value or range. "About" also includes an exact amount. For example, "about 5%" means "about 5%" and also "5%". The term "about" may also refer to ± 10% of a given value or a range of values. For example, "about 5%" means 4.5% to 5.5%.

[0020] As used herein, the terms “or” and “and / or” are used to describe a plurality of components, either in combination or exclusively. For example, “x, y, and / or z” may refer to “x” alone, “y” alone, “z” alone, “x, y, and z”, “(x and y) or z”, “x or (y and z)”, or “x or y or z”.

[0021] Throughout this disclosure, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the stated range and, where appropriate, its fractions (e.g., 1 / 10 and 1 / 100 of an integer). Additionally, any numeric range in this disclosure regarding any physical feature, such as polymer subunits, size, or thickness, should be understood to include any integer within the stated range unless otherwise indicated. Throughout this disclosure, numeric ranges include their stated endpoints unless specifically stated otherwise.

[0022] When followed by a list of items or elements, the phrase "at least one" refers to an open set of one or more of the elements within the list, which may include more than one of the elements but is not required to do so.

[0023] As used herein, the term “subject” refers to a warm-blooded animal that has or is likely to have one or more diseases and disorders, such as a mammal, preferably a human, or a human child.

[0024] As used herein, "derivative" refers to a chemically or biologically modified version of a compound that is structurally similar to a parent compound and can be derived (actually or theoretically) from that parent compound. Generally, "derivatives" differ from "analogs" in that the parent compound may be a starting material for generating the "derivative," whereas the parent compound does not necessarily have to be used as a starting material for generating the "analog." For example, the derivative may be more hydrophilic or hydrophobic than the parent compound, or may have altered reactivity. While derivatives may be obtained by physical (e.g., biological or chemical) modification of the parent compound, derivatives may also be conceptually derived, such as when a protein sequence is designed based on one or more known sequences, an encoding nucleic acid is constructed, and the derived protein is obtained by the expression of the encoding nucleic acid.

[0025] As used herein, "lipid nanoparticles" (LNP) refer to solid particles distinct from liposomes having an aqueous lumen. Like the lumen of a liposome, the core of an LNP is surrounded by layers of lipids that may be a continuous lipid monolayer, a lipid bilayer, or a multilayer having three or more lipid layers, but are not necessarily.

[0026] As used herein, “artificial sequence” or “synthetic sequence” refers to an amino acid or nucleotide sequence designed for a specific purpose and not derived from a specific sequence existing in nature. The purpose of such sequences may include, in particular, linkers, spacers, restriction sites, and untranslated regions.

[0027] As used herein, “transfection” or “transfecting” refers to the introduction of nucleic acids into a cell by non-viral methods. Transfection may be mediated by calcium phosphate, cationic polymers, magnetic beads, electroporation, and lipid-based reagents. In a preferred embodiment disclosed herein, transfection is mediated by solid lipid nanoparticles (LNPs) containing targeted LNPs (tLNPs). The term “transfection” is used to distinguish it from “transduction”—the transfer of genetic material from cell to cell or from virus to cell—and “transformation”—the uptake of extracellular genetic material by the natural processes of the cell. As used herein, phrases such as “delivering nucleic acids into a cell” are synonymous with transfection.

[0028] As used herein with respect to immune cells, “reprogramming” refers to altering the functionality of immune cells with respect to antigen specificity by causing the expression of exogenous T cell receptors (TCRs), chimeric antigen receptors (CARs), or immune cell engagers (collectively referred to as “reprogramming agents”). Generally, T lymphocytes and natural killer (NK) cells can be reprogrammed with TCRs, CARs, or immune cell engagers, whereas only CARs or immune cell engagers are used for the reprogramming of monocytes. In the case of immune cell engagers, immune cells that are engaged with and redirected toward the pursued antigen of the immune cell engager are reprogrammed cells, regardless of whether they express the reprogramming agent. Reprogramming may be transient or persistent depending on the nature of the engineering agent.

[0029] As used herein, the term "engineering agent" refers to an agent that confers the expression of a reprogramming agent by immune cells, particularly non-B lymphocytes or monocytes. An engineering agent may comprise a nucleic acid containing mRNA encoding the reprogramming agent. An engineering agent may also comprise a nucleic acid encoding a gene editing system, such as an RNA-induced nuclease, or a component thereof, a guide RNA, and a nucleic acid template for dissolving the reprogramming agent or knocking out an endogenous antigen receptor. Gene editing systems include base-editors, prime-editors, or gene-writers. RNA-induced nucleases include CRISPR nucleases such as Cas9, Cas12, Cas13, Cas3, CasMINI, Cas7-11, and CasX. For the transient expression of a reprogramming agent such as a CAR, mRNA encoding the reprogramming agent may be used as an engineering agent. For the sustained expression of reprogramming agents such as exogenous genes, modified genes, or correction genes (and their gene products), the engineering agent may include mRNA-encoding encoding an RNA-induced nuclease, guide RNA, a nucleic acid template, and other components of a gene / genome editing system.

[0030] Examples of gene editing components encoded by nucleic acid molecules include mRNA encoding RNA-induced nucleases, gene or base editing proteins, prime editing proteins, gene writer proteins (e.g., modified or modularized non-long terminal repeat (LTR) retrotransposons), retrotransposases, RNA writers, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, transposases, retrotransposons, reverse transcriptases (e.g., M-MLV reverse transcriptase), nicases or inactivated nucleases (e.g., Cas9, nCas9, dCas9), DNA recombinases, CRISPR nucleases (e.g., Cas9, Cas12, Cas13, Cas3, CasMINI, Cas7-11, CasX), DNA nicases, Cas9 nicases (e.g., D10A or It includes H840A), or any fusion thereof or combination thereof. Other components include guide RNA (gRNA), single guide RNA (sgRNA), prime edit guide RNA (pegRNA), clustered regularly spaced short palindromic repeat sequence (CRISPR) RNA (crRNA), trans-activated clustered regularly spaced short palindromic repeat sequence (CRISPR) RNA (tracrRNA), or DNA molecules inserted as or serving as templates for repairing double-strand breaks (DSBs) at specific genomic loci. Genome-, gene-, and base-editing technologies are reviewed in the literature [Anzalone et al., Nature Biotechnology 38:824-844, 2020], [Sakuma, Gene and Genome Editing 3-4:100017, 2022], and [Zhou et al., MedComm 3(3):e155, 2022], each of which is incorporated by reference to all that they teach regarding the components and use of such technologies to the extent that it does not conflict with the present disclosure.

[0031] As used herein, “target antigen” or “targeted antigen” refers to a surface antigen of an immune cell that can be specifically bound by the targeting moiety of tLNP.

[0032] As used herein, “trace antigen” refers to an antigen recognized by a reprogramming agent (e.g., TCR, CAR, or immune cell engager). It is common in the art to use the term “target (or targeted) antigen” in relation to any antigen bound by an antigen (or other) receptor. This may cause confusion when two distinct classes of antigen action are involved. In an effort to avoid such confusion, “target (or targeted) antigen” is used herein to refer to an antigen bound by a targeting moiety of a nanoparticle, and “trace antigen” (or cell or tissue or indication, etc.) is used to refer to an antigen bound by a reprogramming agent. (Substitutions are not used for the terms “effector versus target r,” “target cell,” “intra-target,” and “intra-target,” as they would tend to increase rather than decrease potential confusion.) In the treatment of a disease, the trace antigen will be expressed by pathogenic cells, but may also be expressed by normal cells.

[0033] As used herein, the term "conditioning agent" refers to a biological response modifier (BRM) that improves the efficiency of immune cell engineering, expands the number of immune cells that can be engineered or the number of engineered cells within a target tissue (e.g., tumor, fibrous tissue, or tissue under autoimmune attack), promotes the activity of engineered cells within a target tissue, or broadens the range of mechanisms of action contributing to a therapeutic immune response. The conditioning agent may be provided by delivering an encoding nucleic acid in a tLNP. Exemplary BRMs include cytokines such as IL-7, IL-15, or IL-18.

[0034] As used herein, the term “immune cell” may refer to any cell of the immune system. However, certain embodiments may exclude polymorphonuclear leukocytes and / or B cells, or may be limited to various forms of non-B lymphocytes such as T cells and / or NK cells, or monocytes such as dendritic cells and / or macrophages.

[0035] Unless otherwise indicated by the context, as used herein, the terms “nucleic acid” or “nucleic acid molecule” refer to RNA or DNA molecules, in particular those encoding expressible polypeptides. The description of the disclosed (t)LNP payload focuses on mRNA nucleic acid molecules having the structure of canonical mRNA. However, polypeptides may also be encoded in circular and self-amplifying (also known as self-replicating) RNA molecules and expressed therefrom. Accordingly, the sequence of any of the linear mRNA molecules disclosed herein may be incorporated into circular or self-amplifying / self-replicating RNA molecules. Similarly, each of these RNA molecules may be encoded as a DNA molecule. Each of the disclosed nucleic acid sequences, RNA, or DNA should be understood as disclosing the corresponding DNA or RNA sequence, respectively.

[0036] As used herein, "antibody" refers to a protein containing an immunoglobulin domain having a hypervariable region, referred to as the complementarity determining region (CDR), which determines the specificity of the antibody's binding to an antigen. Accordingly, the term antibody may refer to whole antibodies (also referred to as intact or full-length antibodies) as well as antibody fragments and constructs containing the antigen-binding portion of a whole antibody. Canonical natural antibodies have a pair of heavy and light chains, whereas camelids (derived from camels, alpacas, llamas, etc.) produce both antibodies with a canonical structure and antibodies containing only the heavy chain. The variable region of camelid heavy-chain-only antibodies has a distinct structure having an extended CDR3, referred to as a nanobody, when produced as a VHH or fragment. Antigen-binding fragments and constructs of antibodies include F(ab')2, F(ab'), F(ab), minibodies (sometimes equivalently denoted as Fab'2, Fab', and Fab), Fv, single-chain Fv (scFv), diabodies, and VH. By combining such elements, bispecific and multispecific reagents, such as BiTE (Bispecific T-cell Engager), can be produced. The term "monoclonal antibody" originated from hybridoma technology but is now used to refer to any single-molecule species of antibody, regardless of how it originated or was produced. Similarly, the terms F(ab), F(ab'), and Fc originated from antibody proteolysis analysis but are now used to refer to such fragments regardless of how they were obtained and whether they possess the exact end produced by historical proteolysis. Antibodies can be obtained through immunization, selection from a naive or immunized library (e.g., by phage display), modification of an isolated antibody-encoding sequence, or any combination thereof. A number of antibodies that can be used as binding moiety are known in the art.An excellent source of information regarding antibodies for International Non-proprietary Names (INNs), including sequence information, has been proposed or recommended in the literature [Wilkinson & Hale, 2022, MAbs 14(1):2123299] (including its supplementary tables), which is incorporated herein by reference for all that it teaches regarding individual antibodies and various antibody formats that may be constructed. U.S. Patent No. 11,326,182 and in particular Table 9 “Cancer, Inflammation and Immune System Antibodies” is a source of sequences and other information regarding a wide range of antibodies, including many that do not have INNs, and is incorporated herein by reference for all that it teaches regarding individual antibodies.

[0037] An antibody or its binding fragment or other binding moiety (or its fusion protein) is, while it does not significantly bind to other components present in the test sample, 10 5 M -1 Affinity or K above a "Specifically binds" to the target when binding to the target at an equilibrium binding constant of a specific binding interaction in the unit of 1 / M. Binding domains (or their fusion proteins) can be classified into "high-affinity" binding domains (or their fusion proteins) and "low-affinity" binding domains (or their fusion proteins). "High-affinity" binding domains are at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , or at least 10 13 M -1 , preferably at least 10 8 M -1or at least 10 9 M -1 of K a It refers to a binding domain having. "Low-affinity" binding domains are up to 10 8 M -1 , up to 10 7 M -1 , up to 10 6 M -1 , up to 10 5 M -1 It refers to a binding domain having Ka. Alternatively, affinity is the equilibrium dissociation constant (K) of a specific binding interaction having units of molar concentration (M). D )(e.g., 10 -5 M to 10 -13 It can be defined as M). The affinity of the binding domain polypeptide and the fusion protein according to the present disclosure can be easily determined using prior art (e.g., reference to the literature [Scatchard et al., 1949, Ann. NY Acad. Sci. 51:660]; and U.S. Patents No. 5,283,173; No. 5,468,614, or equivalents).

[0038] As used herein, “binding agent,” “binding moiety,” or “targeting moiety” refers to a protein, polypeptide, oligopeptide or peptide, carbohydrate, nucleic acid, or combination thereof that can specifically bind to a target or a number of targets. A binding agent comprises any naturally occurring, synthetic, semi-synthetic, or recombinant binding partner to a biological molecule or other target of interest. Exemplary binding moiety of the present disclosure comprises an antibody or its antigen-binding domain, Fab', F(ab')2, Fab, Fv, rIgG, scFv, hcAb (heavy chain antibody), single-domain antibody (sdAb), VHH, Variable New Antigen Receptor (VNAR), nanobody, receptor ectodomain or its ligand-binding portion, or a ligand (e.g., cytokine, chemokine). "Fab (antigen-binding fragment) is a portion of an antibody that binds to an antigen and comprises a first heavy chain constant (CH1) domain and a variable region connected to the light chain via interchain disulfide bonds. In other embodiments, the binding moiety comprises a ligand-binding domain of the receptor or a receptor ligand. In some embodiments, the binding moiety may have more than one specificity, for example, by including a bispecific or multispecific binding agent. Various assays for identifying the binding moiety of the present disclosure that specifically binds to a specific target are known, including Western blot, ELISA, biolayer interferometry, and surface plasmon resonance. The binding moiety, such as the immunoglobulin light chain and heavy chain variable domain (e.g., scFv), may be incorporated into various protein scaffolds or structures as described herein, such as antibodies or their antigen-binding fragments, scFv-Fc fusion proteins, or fusion proteins comprising two or more of these immunoglobulin binding domains."

[0039] "Framework" or "FW" refers to variable domain residues other than CDR residues. The FW of the variable domain generally consists of four FW regions: FW1, FW2, FW3, and FW4. Therefore, CDR and FR sequences are typically represented in VH or VL as the following sequences: FW1-CDR1-FW2-CDR2-FW3-CDR3-FW4.

[0040] Various systems exist for identifying hypervariability regions, and simple sequential numbering of antibody sequences is used throughout this application. In some cases, Chothia numbering is used and is specifically indicated. Several commonly used CDR numbering systems exist. The differences between Chothia numbering and Kabat numbering (also defined as Kabat, Chothia, AbM, and Contact CDRs) are described on the antibody information page of the literature [bioinf.org.uk - Prof. Andrew CR Martin's group at UCL].

[0041] "Humanized antibody" is a chimera that is a genetically modified antibody in which a CDR from an antibody, for example, a mouse antibody (donor antibody), is transplanted into a human antibody (recipient antibody) at the CDR position of the recipient sequence. Thus, the humanized antibody is an antibody having a CDR from the donor non-human antibody and, where present, a variable region framework and an invariant region from the human antibody. In a given embodiment, in an attempt to better maintain (or improve) the affinity, specificity, stability, and / or other properties of the donor antibody, the human framework sequence of the humanized antibody may be modified at a given position to contain an amino acid residue present at a given position of the donor antibody.

[0042] Although humanized antibodies are chimeras, the term "chimeric antibody" is generally used to refer to an antibody containing the variable region of the donor antibody and the constant region of the recipient antibody (e.g., the constant region of the human antibody), to distinguish it from CDR-transplanted antibodies, where the variable region itself is the chimera. Such convention is observed herein. While chimeric antibodies may be less immunogenic when administered to the species of the recipient antibody, repeated or long-term exposure usually induces immune responses that limit or eliminate clinical utility, whereas humanized antibodies avoid or reduce the occurrence of such harmful immune responses.

[0043] As used herein, the terms “monovalent” or “divalent” refer to one or two antigen-binding sites on the whole antibody or antibody fragment.

[0044] As used herein, the mouse anti-CD8α antibody clone RPA-T8 is referred to as the "CT8" antibody and is used as a donor for humanization. As expressed in human cells, CD8 is typically a dimer of two α chains or one α chain and one β chain. Most human CD8+ T cells express the αβ dimer. CT8 recognizes an epitope on the α chain. CT8 and its humanization derivatives can bind to both the α2 and αβ dimers.

[0045] The humanized anti-CD8α antigen-binding domain of the present disclosure may be incorporated into different antibody formats, e.g., antigen-binding fragments (F(ab), F(ab'), or F(ab')2), single-chain variable fragments (scFv), diabodies, minibodies, and other antibody formats described elsewhere (see [Wilkinson & Hale, 2022, Mabs[14(1): e2123299]). The term “F(ab)” refers to an antigen-binding monovalent fragment having a molecular weight of about 50,000 daltons and antigen-binding activity, consisting of VH and VL, a light chain constant domain (CL), and a first heavy chain constant domain (CH1). The term “F(ab')2” refers to an antibody divalent fragment having a molecular weight of about 100,000 daltons and antigen-binding activity, comprising two antigen-binding fragments (F(ab)) connected by disulfide crosslinking at the hinge region. F(ab') refers to a monovalent antigen-binding fragment containing some hinge region and can be generated by partial reduction of F(ab')2 or through recombinant DNA sequencing methods involving cleavage or substitution of associated hinge cysteine ​​residues. Although various Fab fragments were classically generated by proteolysis, generating them via recombinant DNA methods has become standard, particularly for monoclonal reagents. This allows for variations and modifications in their amino acid sequences and terminals, but nevertheless, the term Fab applies to such similar molecules. The term "scFv" refers to the N-terminal portion of a Fab fragment and consists of variable portions of light and heavy chains (VH and VL) connected by a short linker peptide of 10 to 25 amino acids in any order. The term "diabody" refers to a divalent fragment composed of two chains containing VH and VL domains from the same antibody or different antibodies, respectively. In the diabody format, the two variable domains (VH and VL) are connected by a short linker, typically five residues. In contrast to scFv, the linker in a diabody is generally too short for the two domains within the same polypeptide chain to bind to each other. The term "minibody" refers to a scFv-derived divalent fragment having two scFvs, each fused to an invariant heavy chain domain 3 (CH3), and in some embodiments is bispecific.

[0046] As used herein, the terms “monoclonal antibody” or “mAb” refer to an antibody molecule of a single amino acid composition that is directed toward a specific antigen and may be produced by a monoclone of B cells or hybridomas, or by recombinant methods. The use of antibody components derived from humanized monoclonal antibodies eliminates potential problems associated with murine invariance and / or immunogenicity of the framework region. Rodent monoclonal antibodies against specific antigens may be obtained by methods known to those skilled in the art (e.g., the literature [Kohler and Milstein, Nature 256: 495 (1975)] and literature [Coligan et al . (eds.), Current Protocols In Immunology, See VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991).

[0047] The various anti-human CD8α antigen-binding domains described herein are often referred to by the initial CBD followed by a four-digit number. In various experiments, these anti-CD8α antigen-binding domains are constructed as whole antibodies (e.g., human IgG1 with the LALAPA Fc silencing mutation; see Sequence No. 43 or 44), F(ab), and other antigen-binding formats. The initial CBD may also be followed by a number of the form xxxx.y or xxxx.yy, where the four-digit number again indicates the antigen-binding domain and the one or two digits following the decimal point indicate F(ab') or other antibody formats (see Table 17).

[0048] As used herein, the term “polypeptide” refers to a molecule composed of monomers (amino acids) connected linearly by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain of two or more amino acids and does not refer to a specific length of the product. Accordingly, peptide, dipeptide, tripeptide, oligopeptide, “protein,” “amino acid chain,” or any other term used to refer to a chain of two or more amino acids is included within the definition of “polypeptide,” and the term “polypeptide” may be used instead of or interchangeably with any of these terms.

[0049] As used throughout this disclosure, “identical” or “identity” refers, respectively, to similarity between a DNA, RNA, nucleotide, amino acid, or protein sequence and another DNA, RNA, nucleotide, amino acid, or protein sequence. Identity may be expressed as a percentage of the sequence identity of a first sequence with respect to a second sequence. Percentage (%) sequence identity with respect to a reference DNA sequence may be the percentage of DNA nucleotides in the candidate sequence that are identical to DNA nucleotides in the reference DNA sequence after aligning the sequences. Percentage (%) sequence identity with respect to a reference amino acid sequence may be the percentage of amino acid residues in the candidate sequence that are identical to amino acid residues in the reference amino acid sequence after aligning the sequences and introducing gaps where necessary to achieve maximum percentage sequence identity without considering any conservative substitutions as part of the sequence identity. As used throughout this disclosure, percentage sequence identity values ​​[Altschul et al., “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs,” Nucleic Acids Res. It is generated using NCBI BLAST 2.0 software (parameters are set to default values) as defined by [2007, 25, 3389-3402].

[0050] Humanized anti-CD8 binding moiety

[0051] The present disclosure provides an anti-CD8α antibody (e.g., an isolated monoclonal antibody), also referred to as an anti-CD8α antibody or its antigen-binding fragment. In some embodiments of the present disclosure, the anti-CD8α antibody or its antigen-binding fragment comprises two light-chain polypeptides (light chains) and two heavy-chain polypeptides (heavy chains) held together by covalent bonding by disulfide linkages.

[0052] In certain embodiments, VH and VL of the present disclosure may be expressed as separate polypeptides that combine with each other to form an antigen-binding fragment specific to CD8α, as in natural antibodies or various F(ab) fragments known to those skilled in the art. In other embodiments, VH and VL of the present disclosure may be included in a single polypeptide chain connected by a linker peptide. If the linker is of sufficient length, VH and VL of the same polypeptide chain may combine to form a single chain Fv (scFv) that specifically binds to CD8α. A shorter linker may be used so that VH and VL within one polypeptide chain each combine with VL and VH of a second polypeptide chain to form a diabody. Generally, antigen-binding domains may be used in a modular manner and may be combined with other protein domains.

[0053] In some embodiments, the heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region. In some embodiments, the heavy chain constant region comprises three domains: CH1, CH2, and CH3. In some embodiments, a humanized anti-CD8α variant is transplanted into all or part of the heavy chain constant region. Non-limiting exemplary heavy chain constant regions include human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, IgM, or IgD constant regions. In some embodiments, the antibody of the present disclosure comprises an IgG1 constant region. Exemplary heavy chain constant regions include a human IgG1 heavy chain constant region (SEQ No. 42) and a human IgG1 null heavy chain constant region (SEQ No. 43 or 44).

[0054] In some embodiments, the light chain comprises a light chain variable region (VL) and a light chain constant region. Humanized anti-CD8α variants of the present disclosure are transplanted into all or part of the kappa light chain constant region or the lambda light chain constant region, or part thereof. Non-limiting exemplary light chain constant regions include kappa and lambda constant regions. A non-limiting exemplary human kappa constant region is shown in SEQ ID NO. 41.

[0055] The constant domain provides a general framework for the antibody and may not be directly involved in binding the antibody to an antigen, but may be involved in various effector actions, such as antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and complement fixation, as well as binding to Fc receptors (e.g., CD16, CD32, FcRn). As used herein, “Fc” or “Fc region” refers to a heavy chain constant domain segment of the Fc fragment from the antibody (“fragment crystallizable” region or Fc region), which may comprise one or more constant domains, such as CH2, CH3, CH4, or any combination thereof. In some embodiments, the Fc region comprises the CH2 and CH3 domains of IgG, IgA, or IgD antibodies and any combination thereof, or the CH3 and CH4 domains of IgM or IgE antibodies and any combination thereof.

[0056] The Fc region can interact with different types of Fc receptors (FcRs). Different types of FcRs may include, for example, FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB, FcαRI, FcμR, FcεRI, FcεRII, and FcRn. FcRs may be located on the membranes of certain immune cells, including, for example, B lymphocytes, natural killer cells, macrophages, neutrophils, follicular dendritic cells, eosinophils, basophils, platelets, and mast cells. Once the FcR is bound by the Fc region, the FcR can initiate the various effector actions mentioned above. The FcR can transmit a signal when the FcR is agglutinated by an antibody on the cell surface. Aggregation of FcRs having an immune receptor tyrosine-based activation motif (ITAM) can sequentially activate SRC family tyrosine kinases and SYK family tyrosine kinases. SRC and SYK kinases can link the converted signals to a common activation pathway. These signals may be undesirable in the treatment of certain indications, such as autoimmune diseases.

[0057] In some embodiments, the Fc region may exhibit reduced binding affinity for one or more Fc receptors. In some embodiments, the Fc region may exhibit reduced binding affinity for one or more Fcγ receptors, FcRn receptors, or both. In some embodiments, the Fc domain is an Fc null or Fc silenced region. As used herein, the "Fc null" or "Fc silenced" region refers to a domain that exhibits weak binding or does not bind to any of the Fcγ receptors.

[0058] The Fc region or domain may have one or more, two or more, three or more, four or more, or up to five amino acid substitutions that reduce the binding of the Fc region to FcR. In some embodiments, the Fc region exhibits reduced binding to FcγRI (CD64), FcγRIIA (CD32), FcγRIIIA (CD16a), FcγRIIIB (CD16b), or any combination thereof. To reduce the binding affinity of the Fc region to FcR, the Fc region may include one or more amino acid substitutions that have the effect of reducing the affinity of the Fc region to FcR. In some embodiments, the Fc region is IgG1, and one or more substitutions within the Fc region include any one or more IgG1 heavy chain mutations corresponding to E233P, L234V, L234A, L235A, L235E, ΔG236, G237A, E318A, K320A, K322A, A327G, P329A, A330S, or P331S according to the EU index of Kabat numbering.

[0059] In some embodiments, the Fc region may contain an IgG1 isomorphic sequence modified from a wild-type IgG1 sequence. The modification may include substitutions in more than one amino acid residue, such as in two different amino acid residues including S239D / I332E (IgG1 SDIE) according to the EU index of Kabat numbering. The modification may include substitutions in more than one amino acid residue, such as in three different amino acid residues including L234A / L235A / P329A (IgG1 LALAPA) or S298A / E333A / K334A (IgG1 SAEAKA) according to the EU index of Kabat numbering. Variants may include substitutions at one or more amino acid residues, such as at five different amino acid residues including L235V / F243L / R292P / Y300L / P396L (IgG1 LVFLRPYLPL) according to the EU index of Kabat numbering. Non-restrictive exemplary human IgG1 heavy chain constant regions with Fc silencing mutations are shown in sequence numbers 43 and 44.

[0060] The Fc portion of the antibody may also mediate agonistic interactions with agents other than the Fc receptor, including the mannose receptor, complement component C1q, and TRIM21. To prevent these interactions and their agonistic effects, antibody formats lacking the Fc region, including scFv, F(ab), F(ab'), F(ab')2, and variants thereof, may be used. Table 17 presents exemplary sequences for wild-type and engineered C-kappa and F(ab') heavy chain constant domains (in some cases, cleaved to remove part or all of the hinge region found in the classical F(ab')).

[0061] In general, the dissociation constant K DThe binding affinity reported as such may be determined by kinetic or steady-state (equilibrium) analysis, i.e., from the ratio of binding off and on rates or from the binding-concentration curve, respectively. In some embodiments, the anti-CD8α antigen-binding fragment having a framework region from the human germline heavy and light chain variable domains of the present disclosure is in the form of F(ab), F(ab'), or a full-length antibody (e.g., combined with the constant domain of the Fc silenced IgG1 antibody of SEQ ID NO. 43). K of these different anti-CD8α binders D To determine this, kinetic analyses were performed for F(ab) and F(ab'); and steady-state analyses were performed for Fc-silenced IgG1 antibodies. Humanized anti-CD8 antigen-binding fragments CBD1033 to CBD1050 were tested at two temperatures (30°C and physiological 37°C) using both biolayer interferometer (BLI) kinetics and surface plasmon resonance (SPR) binding assays. The assays measured binding affinity for CD8αα homomers. In these embodiments, humanized anti-CD8α F(ab), F(ab'), or Fc null full-length antibodies were present at a K of approximately 10 nM or less. D It has. In some embodiments, CBD1032, CBD1033, CBD1035, CBD1036, CBD1037, CBD1038, CBD1039, or CBD1040 F(ab) has less than 8 nM K D It has. In some embodiments, CBD1032, CBD1033, CBD1037, or CBD1039 F(ab) has less than 5 nM of K. D It has. In some embodiments, CBD1032, CBD1033, CBD1034, CBD1035, CBD1036, CBD1037, CBD1038, CBD1039, CBD1040, CBD1042, CBD1043, CBD1045, CBD1047, CBD1048, CBD1049, or CBD1050 Fc null full-length antibody has less than 7 nM K DIt has. In some embodiments, the CBD1032, CBD1033, CBD1034, CBD1035, CBD1037, CBD1038, CBD1039, CBD1040, CBD1042, CBD1043, CBD1045, CBD1047, or CBD1049 Fc null full-length antibody has less than 5 nM K D It has. In some embodiments, CBD1033.24 or CBD1033.37 F(ab') has less than 7 nM K D It has. A widely used proxy indicator for affinity is EC 50 ..., and this is the concentration that produces half of the maximum effect. In some embodiments, the anti-CD8α Fc inactivating antibody of the present disclosure is at an EC of about 6 nM, about 3 nM, about 2 nM, about 1 nM, or about 0.5 nM. 50The humanized anti-CD8 antigen-binding fragment disclosed and tested in this application exhibited binding affinity for both CD8αα homodimers and CDαβ heterodimers, and it was found that the strength of binding to the CT8 epitope was sufficient for tLNP delivery to CD8+ cells. In particular, CBD1033 F(ab) bound to human and cyanomolgus macaque CD8αα homodimers and CD8αβ heterodimers. However, the binding affinity of CBD1033 F(ab) was 5-fold weaker than that of the parent CBD1017ch F(ab). Additionally, CBD1033 F(ab) or Fc null full-length antibodies have a binding affinity for CD8αβ heterodimers that is 6 to 10-fold weaker compared to CD8αα homodimers. Nevertheless, CBD1033-targeted tLNP exhibited transfection efficiencies similar to, or in some cases better than, in vitro CBD1017ch; and demonstrated up to 80% in vivo transfection efficiencies in CD8+ T cells known to primarily express CD8αβ heteromers. Further studies on the binding site of CD8 revealed that CBD1033 binds to a specific epitope on CD8 (referred to as the CT8 epitope) that delivers superior transfection efficiencies compared to other epitopes bound by other antibodies such as SK1 and OKT8. Thus, the binding affinity of the humanized anti-CD8 binding fragment disclosed herein to the CT8 epitope of CD8αα homomers or CD8αβ dimers is sufficient to sustain tLNP transfection activity in vivo.

[0062] In some embodiments, the anti-CD8α antibody of the present disclosure or its antigen-binding fragment specifically binds to non-human primates and human CD8αα homodimers and CDαβ heterodimers. In some cases, the humanized anti-CD8α antibody of the present disclosure or its antigen-binding fragment specifically binds to cyanomolus macaque or rhesus macaque CD8. In some embodiments, the humanized anti-CD8α antibody of the present disclosure or its antigen-binding fragment competes for binding to the same epitope on CD8α that is bound by TRX2, and vice versa, and the humanized anti-CD8α antibody is described in FIGS. 5 and FIGS. 6 of US20060002921, which is incorporated by reference to all that it teaches about TRX2. The sequence of the TRX2 antibody is shown in Table 19. In some embodiments, the humanized anti-CD8 antibody of the present disclosure or its antigen-binding fragment competes for binding to the same epitope on CD8α that is bound by YTC182.20 (as described in the literature incorporated by reference for everything it teaches about YTC182.20 [Jonker, M. et al. (1989) Reactivity of mAb specific for human CD markers with Rhesus monkey leucocytes. Leucocyte Typing IV. Oxford University Press p 1058-1063]), and vice versa. The CT8 epitope that is bound by these antibodies is located on the stem (or hinge) emerging from the cell membrane and in the membrane-proximal portion of the CD8α ectododomain near the dimer interface.Crosslinking analysis indicated that CBD1033 binds to or near amino acid residues 40, 45, 47, 86, 91, 95, 103, 105, and 106 of CD8α, indicating that CBD1033 identified the location of the CT8 epitope by interacting with the CC' loop, C' strand, turn prior to the F strand, F strand, and G strand of CD8α (as predicted by AlphaFold2). (Reference cited for his teachings on the structure of CD8 and its interaction with mAbs [Srinivasan et al., 2024. Front. Immunol. (Refer to [15:1412513]) Residues 40 and 45 are in the CC' loop, residue 47 is in the C' strand, residue 86 is in the turn before the F strand, residues 91 and 95 are in the F strand, and residues 103, 105, and 106 are in the G strand. Competitive binding analysis indicates that the anti-CD8 antibody OKT8 does not compete for binding to the same epitope. The anti-CD8 antibody SK1 also does not compete for binding to the CT8 epitope, but blocks T cell activation and competes with the anti-CD8 antibody HIT8α for binding to the same epitope. In various embodiments, the antigen-binding domain that binds to the CT8 epitope is specified as a means for binding to the CT8 epitope or a means for competing for binding to the same epitope that is bound by CT8, TRX2, and / or YTC182.20.

[0063] The variable region of an antibody contains the molecule's antigen-binding site. The variable heavy chain (VH) and variable light chain (VL) are domains of the antibody's larger and smaller polypeptide subunits, respectively, and form the antigen-binding site. The VH and VL domains of antibodies generally have similar structures, and each domain contains four conserved framework regions (FW) and three hypervariable regions. Most of the sequence variability of antibodies occurs in six hypervariable regions, each named a "Complementary Determinant Region" (CDR), with three present per VH and VL chain (VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, VL-CDR3). In a given embodiment, the antigen-recognition region of the anti-CD8 antibody variable domain of the present disclosure comprises six CDRs, or supervariable regions, located within the framework of the heavy chain variable region and the light chain variable region at the N-terminal ends of two heavy chains and two light chains. For example, the CD8 binding domain comprises a heavy chain complementarity determining region 1 (VH-CDR1), a heavy chain complementarity determining region 2 (VH-CDR2), a heavy chain complementarity determining region 3 (VH-CDR3), a light chain complementarity determining region 1 (VL-CDR1), a light chain complementarity determining region 2 (VL-CDR2), and a light chain complementarity determining region 3 (VL-CDR3).

[0064] In a given embodiment, an anti-CD8α antibody or its antigen-binding fragment having a framework region from human germline heavy chain and light chain variable domains comprises: (a) VH-CDR1 having the amino acid sequence RYTFTDYX1LH (SEQ No. 45) (where X1 is N, S, Q, or A), VH-CDR2 having the amino acid sequence FIYPYX1GGTG (SEQ No. 46) or FIYPYX2GGTG (SEQ No. 47) (where X2 is N, Q, D, S, or A), VH-CDR3 having the amino acid sequence DHRYX1EGVSFDY (SEQ No. 48), and VL-CDR1 having the amino acid sequence RASESVX3GFGX2SFMN (SEQ No. 49) (where X3 is an amino acid identified by the symbol D, E, S, or A); VL comprises VL-CDR2 having the amino acid sequence LASX2LES (SEQ No. 50), and VL-CDR3 having the amino acid sequence QQX2X2EX3PYT (SEQ No. 51). In some embodiments, VH-CDR1 has the amino acid sequence RYTFTDYNLH (SEQ No. 2). In some embodiments, VH-CDR2 has the amino acid sequence FIYPYNGGTG (SEQ No. 3), FIYPYSGGTG (SEQ No. 58), FIYPYQGGTG (SEQ No. 59), or FIYPYAGGTG (SEQ No. 60). In some embodiments, VH-CDR3 has the amino acid sequence DHRYNEGVSFDY (SEQ No. 4). In some embodiments, VL-CDR1 has the amino acid sequence RASESVDGFGNSFMN (SEQ No. 6), SEQ No. 227, or SEQ No. 228. In some embodiments, VL-CDR2 has the amino acid sequence LASNLES (Sequence No. 7). In some embodiments, VL-CDR3 has the amino acid sequence QQNNEDPYT (Sequence No. 8).

[0065] In a further embodiment, the humanized anti-CD8α antibody or its antigen-binding fragment comprises (a)(i) VH-CDR1 comprising the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprising the amino acid sequence of SEQ ID NO. 3, and VH-CDR3 comprising the amino acid sequence of SEQ ID NO. 4; (ii) VH-CDR1 comprising the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprising the amino acid sequence of SEQ ID NO. 58, and VH-CDR3 comprising the amino acid sequence of SEQ ID NO. 4; (iii) VH-CDR1 comprising the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprising the amino acid sequence of SEQ ID NO. 59, and VH-CDR3 comprising the amino acid sequence of SEQ ID NO. 4; or (iv) VH-CDR1 comprising the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprising the amino acid sequence of SEQ ID NO. 60, and VH-CDR3 comprising the amino acid sequence of SEQ ID NO. 4; and (b) VL-CDR1 containing amino acid sequence number 6, VL-CDR2 containing amino acid sequence number 7; and VL-CDR3 containing amino acid sequence number 8.

[0066] In any of the embodiments described above, (a) the recipient sequence from which the heavy chain framework region is derived is from IGHV1-46*01 / IGHJ6*01 as shown in SEQ ID NO. 9; (b) the recipient sequence from which the light chain framework region is derived is from IGKV1-39*01 / IGKJ2*01 as shown in SEQ ID NO. 15; (c) the recipient sequence from which the heavy chain framework region is derived is from a modified IGHV1-18*01 as shown in SEQ ID NO. 31; and (d) the recipient sequence from which the light chain framework region is derived is from a modified version of IGKV3D-11*01 as shown in SEQ ID NO. 37; (e) the recipient sequence from which the heavy chain framework region originates is from IGHV1-46*01 / IGHJ6*01 and the recipient sequence from which the light chain framework region originates is from IGKV1-39*01 / IGKJ2*01; (f) the recipient sequence from which the heavy chain framework region originates is from a modified IGHV1-18*01 and the recipient sequence from which the light chain framework region originates is from a modified version of IGKV3D-11*01; (g) the recipient sequence from which the heavy chain framework region originates is from IGHV1-46*01 / IGHJ6*01 and the recipient sequence from which the light chain framework region originates is from a modified version of IGKV3D-11*01; (h) The recipient sequence from which the heavy chain framework region originates is from modified IGHV1-18*01, and the recipient sequence from which the light chain framework region originates is from IGKV1-39*01 / IGKJ2*01.

[0067] In a given embodiment, the anti-CD8α antibody or the antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 66, SEQ ID NO. 67, or SEQ ID NO. 68; and includes a light chain variable region (VL) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of sequence number 16, sequence number 17, sequence number 18, sequence number 38, sequence number 39, sequence number 40, sequence number 64, or sequence number 65.

[0068] In a given embodiment, the anti-CD8α antibody or the antigen-binding fragment thereof comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 35, or SEQ ID NO. 36, wherein VH-CDR1 comprises the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprises the amino acid sequence of SEQ ID NO. 3, 58, 59, or 60, and VH-CDR3 comprises the amino acid sequence of SEQ ID NO. 4; a heavy chain variable region (VH); and includes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of sequence number 16, sequence number 17, sequence number 18, or sequence number 39, wherein VL-CDR1 includes the amino acid sequence of sequence number 6, 227, or 228, VL-CDR2 includes the amino acid sequence of sequence number 7, and VL-CDR3 includes the amino acid sequence of sequence number 8, comprising a light chain variable region (VL). In some embodiments, the anti-CD8α antibody or its antigen-binding fragment comprises VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO. 10, 11, 12, 13, or 14; and VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO. 16, provided that the amino acid sequences of VH-CDR (i.e., SEQ ID NO. 2, 3, and 4) and VL-CDR (i.e., SEQ ID NO. 6, 7, and 8) are not changed.In a further embodiment, the anti-CD8α antibody or the antigen-binding fragment thereof comprises VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO. 10, 11, 12, 13, or 14; and VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO. 17, provided that the amino acid sequences of VH-CDR and VL-CDR are not changed. In another embodiment, the anti-CD8α antibody or the antigen-binding fragment thereof comprises VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO. 10, 11, 12, 13, or 14; and VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO. 18, provided that the amino acid sequences of VH-CDR and VL-CDR are not changed. In further embodiments, the anti-CD8α antibody or its antigen-binding fragment comprises VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO. 35 or 36; and VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO. 39, provided that the amino acid sequences of VH-CDR and VL-CDR are not changed.

[0069] In some embodiments, the anti-CD8α antibody or the antigen-binding fragment thereof comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO. 33 or SEQ ID NO. 34, wherein VH-CDR1 comprises the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprises the amino acid sequence of SEQ ID NO. 3, and VH-CDR3 comprises the amino acid sequence of SEQ ID NO. 4; a heavy chain variable region (VH); and includes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO. 38 or SEQ ID NO. 40, wherein VL-CDR1 includes the amino acid sequence of SEQ ID NO. 6, VL-CDR2 includes the amino acid sequence of SEQ ID NO. 7, and VL-CDR3 includes the amino acid sequence of SEQ ID NO. 8, comprising a light chain variable region (VL).

[0070] In a given embodiment, the anti-CD8α antibody or its antigen-binding fragment comprises a VL region having an amino acid sequence of one of SEQ ID NOs 16 to 18, 38 to 40, 64, or 65. In some embodiments, the anti-CD8α antibody or its antigen-binding fragment comprises a VH region having an amino acid sequence of one of SEQ ID NOs 10 to 14, 27 to 30, 32 to 36, or 66 to 68. In further embodiments, the anti-CD8α antibody or its antigen-binding fragment comprises a VL region having an amino acid sequence of SEQ ID NO. 17 and a VH region having an amino acid sequence of one of SEQ ID NOs 11 or 27 to 30. In further embodiments, the anti-CD8α antibody or its antigen-binding fragment comprises: (a) a VH having an amino acid sequence of SEQ ID NO. 10 and a VL having an amino acid sequence of SEQ ID NO. 16; (b) a VH having an amino acid sequence of one of SEQ NOs 11 to 14, and a VL having an amino acid sequence of SEQ NO 17; or (c) a VH having an amino acid sequence of one of SEQ NOs 11 to 14, and a VL having an amino acid sequence of SEQ NO 18. In further embodiments, the anti-CD8α antibody or its antigen-binding fragment comprises a VL region having an amino acid sequence of SEQ NO 64 and a VH region having an amino acid sequence of one of SEQ NOs 11, 13, 28, 29, 67, or 68. In further embodiments, the anti-CD8α antibody or its antigen-binding fragment comprises a VL region having an amino acid sequence of SEQ NO 65 and a VH region having an amino acid sequence of one of SEQ NOs 11 and 13.

[0071] In a further embodiment, the anti-CD8α antibody or the antigen-binding fragment thereof comprises: (a) the amino acid sequence of SEQ ID NO. 10, 11, 12, 13, 14, 35, or 36, wherein VH-CDR1 comprises the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprises the amino acid sequence of SEQ ID NO. 3, VH-CDR3 comprises the amino acid sequence of SEQ ID NO. 4, and VL comprises the amino acid sequence of SEQ ID NO. 16; (b) the amino acid sequence of SEQ ID NO. 27 or 66, wherein VH-CDR1 comprises the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprises the amino acid sequence of SEQ ID NO. 58, VH-CDR3 comprises the amino acid sequence of SEQ ID NO. 4, and VL comprises the amino acid sequence of SEQ ID NO. 16; (c) VH comprising the amino acid sequence of SEQ ID NO. 28 or 67, wherein VH-CDR1 comprises the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprises the amino acid sequence of SEQ ID NO. 59, VH-CDR3 comprises the amino acid sequence of SEQ ID NO. 4, and VL comprises the amino acid sequence of SEQ ID NO. 16; (d) VH comprising the amino acid sequence of SEQ ID NO. 29 or 68, wherein VH-CDR1 comprises the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprises the amino acid sequence of SEQ ID NO. 60, VH-CDR3 comprises the amino acid sequence of SEQ ID NO. 4, and VL comprises the amino acid sequence of SEQ ID NO. 16; (e) comprising the amino acid sequence of SEQ ID NO. 10, 11, 12, 13, 14, 35, or 36, wherein VH-CDR1 comprises the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprises the amino acid sequence of SEQ ID NO. 3, VH-CDR3 comprises the amino acid sequence of SEQ ID NO. 4, and VL comprises the amino acid sequence of SEQ ID NO. 17;(f) VH comprising the amino acid sequence of SEQ ID NO. 27 or 66, wherein VH-CDR1 comprises the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprises the amino acid sequence of SEQ ID NO. 58, VH-CDR3 comprises the amino acid sequence of SEQ ID NO. 4, and VL comprises the amino acid sequence of SEQ ID NO. 17; (g) VH comprising the amino acid sequence of SEQ ID NO. 10, 11, 12, 13, 14, 35, or 36, wherein VH-CDR1 comprises the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprises the amino acid sequence of SEQ ID NO. 59, VH-CDR3 comprises the amino acid sequence of SEQ ID NO. 4, and VL comprises the amino acid sequence of SEQ ID NO. 17; (h) VH comprising the amino acid sequence of SEQ ID NO. 29 or 68, wherein VH-CDR1 comprises the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprises the amino acid sequence of SEQ ID NO. 60, VH-CDR3 comprises the amino acid sequence of SEQ ID NO. 4, and VL comprising the amino acid sequence of SEQ ID NO. 17; (i) VH comprising the amino acid sequence of SEQ ID NO. 10, 11, 12, 13, 14, 35, or 36, wherein VH-CDR1 comprises the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprises the amino acid sequence of SEQ ID NO. 3, VH-CDR3 comprises the amino acid sequence of SEQ ID NO. 4, and VL comprising the amino acid sequence of SEQ ID NO. 18; (j) comprising the amino acid sequence of SEQ ID NO. 27 or 66, wherein VH-CDR1 comprises the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprises the amino acid sequence of SEQ ID NO. 58, VH-CDR3 comprises the amino acid sequence of SEQ ID NO. 4, and VL comprises the amino acid sequence of SEQ ID NO. 18;(k) comprising the amino acid sequence of SEQ ID NO. 28 or 67, wherein VH-CDR1 comprises the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprises the amino acid sequence of SEQ ID NO. 59, VH-CDR3 comprises the amino acid sequence of SEQ ID NO. 4, and VL comprises the amino acid sequence of SEQ ID NO. 18; (l) comprising the amino acid sequence of SEQ ID NO. 29 or 68, wherein VH-CDR1 comprises the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprises the amino acid sequence of SEQ ID NO. 60, VH-CDR3 comprises the amino acid sequence of SEQ ID NO. 4, and VL comprises the amino acid sequence of SEQ ID NO. 18; (m) comprising the amino acid sequence of SEQ NO. 10, 11, 12, 13, 14, 35, or 36, wherein VH-CDR1 comprises the amino acid sequence of SEQ NO. 2, VH-CDR2 comprises the amino acid sequence of SEQ NO. 3, VH-CDR3 comprises the amino acid sequence of SEQ NO. 4, and VL comprises the amino acid sequence of SEQ NO. 39; (n) comprising the amino acid sequence of SEQ NO. 27 or 66, wherein VH-CDR1 comprises the amino acid sequence of SEQ NO. 2, VH-CDR2 comprises the amino acid sequence of SEQ NO. 58, VH-CDR3 comprises the amino acid sequence of SEQ NO. 4, and VL comprises the amino acid sequence of SEQ NO. 39; (o) comprising the amino acid sequence of SEQ ID NO. 28 or 67, wherein VH-CDR1 comprises the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprises the amino acid sequence of SEQ ID NO. 59, VH-CDR3 comprises the amino acid sequence of SEQ ID NO. 4, and VL comprises the amino acid sequence of SEQ ID NO. 39;(p) comprising the amino acid sequence of SEQ ID NO. 29 or 68, wherein VH-CDR1 comprises the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprises the amino acid sequence of SEQ ID NO. 60, VH-CDR3 comprises the amino acid sequence of SEQ ID NO. 4, and VL comprises the amino acid sequence of SEQ ID NO. 39; (q) comprising the amino acid sequence of SEQ ID NO. 10, 11, 12, 13, 14, 33, 34, 35, or 36, wherein VH-CDR1 comprises the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprises the amino acid sequence of SEQ ID NO. 3, VH-CDR3 comprises the amino acid sequence of SEQ ID NO. 4, and VL comprises the amino acid sequence of SEQ ID NO. 64; (r) comprising the amino acid sequence of SEQ ID NO. 27 or 66, wherein VH-CDR1 comprises the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprises the amino acid sequence of SEQ ID NO. 58, VH-CDR3 comprises the amino acid sequence of SEQ ID NO. 4, and VL comprises the amino acid sequence of SEQ ID NO. 64; (s) comprising the amino acid sequence of SEQ ID NO. 28 or 67, wherein VH-CDR1 comprises the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprises the amino acid sequence of SEQ ID NO. 59, VH-CDR3 comprises the amino acid sequence of SEQ ID NO. 4, and VL comprises the amino acid sequence of SEQ ID NO. 64; (t) comprising the amino acid sequence of SEQ ID NO. 29 or 68, wherein VH-CDR1 comprises the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprises the amino acid sequence of SEQ ID NO. 60, VH-CDR3 comprises the amino acid sequence of SEQ ID NO. 4, and VL comprises the amino acid sequence of SEQ ID NO. 64;(u) comprising the amino acid sequence of SEQ ID NO. 10, 11, 12, 13, 14, 33, 34, 35, or 36, wherein VH-CDR1 comprises the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprises the amino acid sequence of SEQ ID NO. 3, VH-CDR3 comprises the amino acid sequence of SEQ ID NO. 4, and VL comprises the amino acid sequence of SEQ ID NO. 65; (v) comprising the amino acid sequence of SEQ ID NO. 27 or 66, wherein VH-CDR1 comprises the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprises the amino acid sequence of SEQ ID NO. 58, VH-CDR3 comprises the amino acid sequence of SEQ ID NO. 4, and VL comprises the amino acid sequence of SEQ ID NO. 65; (w) comprising the amino acid sequence of SEQ ID NO. 28 or 67, wherein VH-CDR1 comprises the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprises the amino acid sequence of SEQ ID NO. 59, VH-CDR3 comprises the amino acid sequence of SEQ ID NO. 4, and VL comprises the amino acid sequence of SEQ ID NO. 65; (x) comprising the amino acid sequence of SEQ ID NO. 29 or 68, wherein VH-CDR1 comprises the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprises the amino acid sequence of SEQ ID NO. 60, VH-CDR3 comprises the amino acid sequence of SEQ ID NO. 4, and VL comprises the amino acid sequence of SEQ ID NO. 65.;

[0072] Examples of humanized anti-CD8α variants comprising the variable domains described in the present disclosure are prepared and are shown in Tables 3 through 5. The VH or VL domains described herein may be transplanted onto classical or engineered heavy or light chain constant regions, respectively. The constant region may be full length, F(ab), F(ab'), F(ab')2, single-chain variable fragment (scFv), diabody, minibody, or other antibody formats. Non-limiting exemplary heavy chain constant regions include human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, IgM, or IgD constant regions. Non-limiting exemplary light chain constant regions include kappa and lambda constant regions.

[0073] In the context of F(ab'), the term "manipulated" refers to a modification or mutation of an amino acid residue in the constant region. In some embodiments, the modification is a cleavage in the constant region; for example, a cleavage after proline at position 245 (P245), P240, or P241 of the IgG1 F(ab') or IgG4 F(ab') hinge region; or a cleavage after T238 of the IgG1 F(ab') hinge region (Table 17). In some embodiments, the mutation is a mutation of a cysteine ​​residue to serine or another non-cysteine ​​amino acid to remove a disulfide bond; for example, C214S on the kappa chain constant region, C233S on the IgG1 CH1 domain, or C127S on the IgG4 F(ab') CH1 domain (Table 17). In some embodiments, the mutation is a mutation of a non-cysteine ​​amino acid to cysteine ​​to support the formation of a new disulfide bond; For example, F174C of the IgG1 F(ab') or IgG4 F(ab') CH1 domain and S162C of the kappa constant region to form the CH1174-Cκ162 disulfide bond (Table 17).

[0074] The VH domain described herein may be transplanted onto a heavy chain constant region comprising the amino acid sequence of SEQ NO. 42, SEQ NO. 43, SEQ NO. 44, SEQ NO. 76, SEQ NO. 79, SEQ NO. 81, SEQ NO. 83, SEQ NO. 85, SEQ NO. 87, SEQ NO. 90, SEQ NO. 93, SEQ NO. 95, SEQ NO. 97, SEQ NO. 99, or SEQ NO. 103. The VL domain described herein may be transplanted onto a light chain constant region comprising the amino acid sequence of SEQ NO. 41, SEQ NO. 89, or SEQ NO. 100. Combinations of the VH and VL domains disclosed herein with heavy chain and light chain constant regions, respectively, are shown in Table 17.

[0075] Biophysical properties of humanized anti-CD8 binding moiety

[0076] In addition to binding affinity and specificity for the desired target molecule, therapeutic antibodies beneficially meet a set of criteria regarding their manufacturability, storage stability, and the absence of off-target binding ("stickiness"). This set of characteristics is often referred to as "manufacturability." The biophysical properties of antibodies significantly influence manufacturability. For example, the "melting temperature" (T) of a protein m ) is the temperature at which half of a protein population is in a folded state, and is therefore an indicator of thermal stability that helps determine the stability of antibodies during storage and manufacturing. Similarly, the "aggregation temperature" (T agg ) detects the initiation of agglutination, is the temperature at which molecules tend to aggregate together, and is associated with protein unfolding. Additionally, several studies have suggested that monoclonal antibodies can interact non-specifically with themselves (self-aggregating) and other serum proteins; therefore, low self-aggregation characteristics can avoid antibody agglutination, off-target effects, and rapid antibody clearance in vivo.

[0077] Binding to targets other than the CD8 antigen was measured using “off-target” evaluations, including multireactivity and cross-reactivity evaluations of the humanized anti-CD8α antibodies and their antigen-binding fragments disclosed herein (e.g., DNA and insulin multireactivity ELISA assays, baculovirus particle (BVP) multireactivity assays, and analysis of human cell membrane proteome arrays including cell surface proteins and secreted proteins). As used herein, the term “multireactivity” refers to the ability of an antibody to bind to various auto- and foreign antigens that lack structural similarity. As used herein, the term “cross-reactivity” refers to the ability of an antibody to bind to antigens other than the target antigen due to structural similarity. In particular, for humanized anti-CD8α antibodies, cross-reactivity and multireactivity assays are used to evaluate the ability of these antibodies to bind to other antigens other than CD8 that have structures similar to or different from CD8, respectively. Multiple reactivity and cross-reactivity (or off-target) effects influence various factors in vivo, including pharmacokinetics, bioavailability, clearance, and toxicity, all of which contribute to successful drug / antibody development.

[0078] In some embodiments, the anti-CD8α binders of the present disclosure have similar binding activity and binding affinity for CD8 in non-human primates as in humans. The term “interspecies binding” refers to the ability of an antibody to bind to the same or related antigens across different species (“target-specific”) while maintaining its antigenic specificity for CD8 molecules of those different species. This characteristic is particularly useful for research and clinical development because experimental data can be obtained from non-human species and reliably applied to humans.

[0079] Those skilled in the art will understand that the properties of the anti-CD8α antibody and its antigen-binding fragment disclosed herein, including any of the embodiments described above, are often unrelated to the binding affinity and specificity of the antibody. Furthermore, those skilled in the art will recognize that changes in the amino acid sequence can affect the biophysical properties of the antibody. Therefore, the preservation of affinity alone does not guarantee the feasibility of developing humanized antibodies, nor can such biophysical properties be reliably predicted from the sequence alone. However, testing of at least some variants can provide some indicators regarding the robustness of specific combinations of the framework and CDR sequences and whether any variations in the sequence might be problematic, in addition to characterizing their properties.

[0080] The thermal stability of antibodies is used as an indicator of antibody development potential, reflecting the stability of the antibody during storage and various purification steps that potentially require the use of harsh or stressful conditions. A sensitive measure of antibody thermal stability is the agglutination onset temperature (Tagg), which indicates the onset of protein denaturation. Generally, T agg >60℃ is preferable. According to this criterion, all total antibodies including the anti-CD8α antigen-binding fragments CBD1033, CBD1034, CBD1035, CBD1039, and CBD1040 are acceptable T agg While having, T for CBD1032 agg...was less desirable. The melting temperature Tm, another measure of thermal stability, represents the midpoint of protein denaturation. For potential good development, antibodies having a Tm greater than 65°C are preferred. By this criterion, all antibodies including the anti-CD8α antigen-binding fragments CBD1033, CBD1035, CBD1039, and CBD1040 had acceptable Tm. Thus, in some embodiments, the humanized CT8 antibody and its antigen-binding fragments have desirable thermal stability, and CBD1033, CBD1035, CBD1039, and CBD1040 constitute the means of humanization to bind to CD8α and have desirable thermal stability.

[0081] One problem that may be faced in CDR transplantation is that engineered antibodies become multireactive or have an increased propensity for self-interaction or self-binding. These characteristics can contribute to agglutination, off-target effects, and rapid clearance of the antibody (reducing its efficacy or potency).

[0082] Self-interaction can be evaluated by affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS) (Reference [Phan et al., 2022, MAbs 14(1): 2094750]). Full-length antibodies containing anti-CD8α antigen-binding fragments of CBD1032, CBD1033, CBD1034, CBD1035, CBD1039, or CBD1040 were evaluated by AC-SINS and all were found to have a low propensity for self-interaction. Thus, CBD1032, CBD1033, CBD1034, CBD1035, CBD1039, and CBD1040 constitute humanized means for binding to CD8α with a low propensity for self-interaction.

[0083] Several tests are available that can be used to evaluate multiple reactivity. One test evaluates reactivity with double-stranded DNA (dsDNA) and insulin. By this criterion, whole antibodies containing anti-CD8α antigen-binding fragments of CBD1032, CBD1033, CBD1034, CBD1035, CBD1039, or CBD1040 were not multiple reactive and constitute a humanized means for binding to CD8α lacking multiple reactivity to dsDNA and insulin.

[0084] Another test for multiplex reactivity evaluates the ability to bind to baculovirus particles (BVP). By this criterion, the whole antibody containing the anti-CD8α antigen-binding fragment of CBD1033 was not multiplex reactive, whereas CBD1032 was weakly multiplex reactive. Therefore, CBD1033 constitutes a humanized means for binding to CD8α that lacks multiplex reactivity to BVP.

[0085] A more comprehensive test of multiplex reactivity and cross-reactivity evaluates binding to a panel of over 6,000 human cell surface proteins and secreted proteins in the form of endogenous membrane proteins, soluble proteins, and soluble proteins tethered to the cell surface (Retrogenix Platform, Charles River, High Peak, UK). Since these are the proteins most likely to be encountered by products for in vivo use in humans (or similar), this is relevant to the potential and success of such products. By this evaluation, the CBD1017ch whole antibody containing the parent antigen-binding domain did not exhibit cross-reactivity with any of the antigens in the panel. The CBD1033, CBD1035, and CBD1039 whole antibodies preserved this lack of cross-reactivity and did not introduce any multiplex reactivity. Therefore, CBD1033, CBD1035, and CBD1039 constitute humanized means for binding to CD8α that lack multiplex reactivity and cross-reactivity to human cell surface proteins and soluble proteins.

[0086] In some embodiments of the present disclosure, certain amino acid modifications in VH-CDR (including amino acid sequence changes N33Q, N33S, N33A, N55Q, N55S, N55A, N103Q, N103S, N103A, or combinations thereof) and certain amino acid modifications in VL-CDR (including but not limited to amino acid sequence changes D30E, D30S, D30A, N34Q, N34S, N34A, N57Q, N57S, N57A, N95Q, N95S, N95A, N96Q, N96S, N96A, D98E, D98S, D98A, or combinations thereof) may be performed to remove unstable amides and prevent potential deamidation. In some embodiments, such substitutions do not affect binding affinity or only minimally, but such binding moiety may be advantageous for the purification, storage, and other processing of the antibody regardless of whether it is under high stress conditions (e.g., high pH and high temperature). As used herein, the term “vulnerable-manipulated mutation” refers to a mutation designed to remove unstable amino acid residues, such as asparagine and aspartic acid, which are at risk of post-translational modification, including during product manufacturing, by deamidation and isoasparate formation, respectively. In some embodiments, the unstable-manipulated mutation comprises one or more of the mutations in the VH-CDR and VL-CDR as mentioned above.

[0087] The term "high stress conditions" includes extreme environmental conditions that can affect molecular stability, such as high pH (pH≥8), low pH (pH≤6), high temperature (≥40℃), or a combination thereof.

[0088] Humanized anti-CD8α as a targeting moiety on LNP

[0089] Since CD8-positive T cells play an important role in adaptive immunity, the ability of humanized anti-CD8α antibodies to target CD8-positive T cells can provide therapeutic effects or diagnostic benefits in the treatment of cancer, infections, immune disorders, inflammatory diseases or pathologies, and autoimmune diseases. CD8 is also expressed on natural killer (NK) cells, which are potent and therapeutically attractive mediators of cytotoxic activity.

[0090] In some embodiments, any of the aforementioned humanized anti-CD8α antibody or its antigen-binding fragment may be used as a targeting moiety on the nanoparticle. Various nanoparticles suitable for delivering payload molecules to or into cells are known in the art and include polymer and / or lipid-containing nanoparticles to which the disclosed anti-CD8 antibody, or other polypeptides containing its antigen-binding domain, may be attached as a targeting moiety. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In certain embodiments disclosed herein, the term “tLNP” refers to an LNP comprising an anti-CD8 antibody or its antibody-binding fragment as a targeting moiety. The term “targeting moiety” refers to a component of a molecule capable of binding to another target molecule, and in particular, the targeting moiety of the LNP is an anti-CD8 antibody capable of binding to a CD8 antigen on a CD8-expressing cell.

[0091] CD8-targeted tLNPs can be used to deliver negatively charged payloads, such as nucleic acids, into CD8+ cells, particularly in the case of tLNPs containing cationic lipids. This can be performed extracorporeally (e.g., as described in PCT / US2024 / 035902, which is incorporated by reference to all teachings regarding the use of tLNPs to transfect cells in vitro or in vitro) or in vivo. Properties such as the immunogenicity of the binding moiety or its cross-reactivity with antigens other than CD8 are less important in the context of extracorporeal or in vitro use than in the context of in vivo use. Therefore, humanized antigen-binding domains of CT8 that have a larger number of mouse residues or are not characterized in terms of other measures of multiple reactivity or potential cross-reactivity with non-CD8 antigens, or even non-humanized antigen-binding domains, may be useful for providing binding specificity of targeting moiety of tLNP to be used in vitro or in vitro.

[0092] Within the range of affinities exhibited by the various antigen-binding domains disclosed herein, affinity was consequently not an effective variable for transfection efficiency by tLNPs incorporating such antigen-binding domains into their targeting moiety, nor was it an effective variable for the expression levels of transfected mRNA. However, recent data support the concept that the antibody binding site can determine whether particle-internalization is initiated. This is consistent with the observed transfection activity of tLNPs containing antigen-binding domains of the various anti-CD8 antibodies disclosed herein. When the anti-CD8 monoclonal antibodies SK1 and OKT8 provided antigen-binding domains to the targeting moiety of tLNPs, the transfection efficiency and payload expression levels observed in vitro were substantially lower than when the antigen-binding domains were provided by CBD1033 (a humanized version of the anti-CD8 monoclonal antibody CT8) or TRX2 (another humanized monoclonal anti-CD8 antibody). Payload expression levels were also similar in vivo for tLNPs having a targeting moiety containing the antigen-binding domain of CBD1033 or TRX2. CBD1033 and TRX2 compete with each other for epitope binding, similar to the anti-CD8 antibody YTC182.20, whereas OKT8 and SK1 do not compete with these antibodies. Without being bound by any specific theory, these data indicate that binding to the CT8 epitope (as defined above) enables much higher manipulation compared to binders binding elsewhere, suggesting that specific molecular stereolocation changes caused by binding to specific sites can determine cellular signaling that induces active particle uptake. Thus, CT8, TRX2, or YTC182.tLNPs that incorporate an antigen-binding domain of 20 into their targeting moiety constitute means for effective particle internalization or means for efficient transfection of payload nucleic acids such as DNA, RNA, or mRNA. In various embodiments, such means specifically include or exclude any of the antibodies, antibody formats, or antigen-binding domains disclosed herein as components of their targeting moiety.

[0093] The humanized anti-CD8-targeted tLNP of the present disclosure, comprising a cationic lipid (e.g., an ionizable cationic lipid), can deliver negatively charged cargo / payloads (e.g., nucleic acids, polypeptides, and small molecules) into cells expressing CD8. The nucleic acids introduced thereby may, in particular, encode the expression of proteins beneficial for the treatment of a diseased subject. In some embodiments, a method for delivering nucleic acids (or other negatively charged payloads) into cells is disclosed herein, comprising the step of contacting a cell with a tLNP encapsulating the nucleic acid or other payload. In some embodiments, contact is made in vitro. In some embodiments, contact is made in vivo. In some cases, in vivo contact involves intravenous, intramuscular, subcutaneous, intranodal, or lymphatic administration. Accordingly, each of the genera, subgenera, and / or species disclosed herein of the LNP or tLNP disclosed herein, comprising those based on the inclusion or exclusion of a specific lipid, a specific lipid composition, a specific payload, and / or a specific CD8-targeted moiety, comprises a payload of CD8 + It can be used to define categories of methods for delivering to cells.

[0094] The nucleic acid may include messenger (mRNA), small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotide (ASO), self-replicating RNA, or circular RNA. In some embodiments, the payload is mRNA encoding a detectable marker, for example, mCherry fluorescent protein. In some embodiments, the delivery method is a transfection method.

[0095] In some embodiments, the encapsulated nucleic acid is mRNA encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR), or an immune cell engager, such as a BiTE (bispecific T cell engager), a cytokine, a chemokine, a chemokine receptor, a dominant-negative cytokine receptor, a cell-identification protein tag, a fluorescent protein, or a molecular switch.

[0096] The encapsulated nucleic acid may also be mRNA and / or guide RNA encoding gene / genome editing enzymes or other components of the gene / genome editing system. The gene / genome editing component may be a guide RNA for an RNA-induced nuclease or other nucleic acid editing enzyme. Examples of gene editing components encoded by nucleic acid molecules include mRNA encoding RNA-induced nucleases, gene or base editing proteins, prime editing proteins, gene writer proteins (e.g., modified or modularized non-long terminal repeat (LTR) retrotransposons), retrotransposases, RNA writers, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, transposases, retrotransposons, reverse transcriptases (e.g., M-MLV reverse transcriptase), nicases or inactivated nucleases (e.g., Cas9, nCas9, dCas9), DNA recombinases, CRISPR nucleases (e.g., Cas9, Cas12, Cas13, Cas3, CasMINI, Cas7-11, CasX), DNA nicases, Cas9 nicases (e.g., D10A or It includes H840A), or any fusion thereof or combination thereof. Other components include guide RNA (gRNA), single guide RNA (sgRNA), prime edit guide RNA (pegRNA), clustered regularly spaced short palindromic repeat (CRISPR) RNA (crRNA), trans-activated clustered regularly spaced short palindromic repeat (CRISPR) RNA (tracrRNA), or DNA molecules inserted as or serving as templates for double-strand break (DSB) repair at specific genomic loci. Genome-, gene-, and base-editing technologies are described in the literature [Anzalone et al., Nature Biotechnology 38:824-844, 2020], Literature[Sakuma, Gene and Genome Editing[3-4:100017, 2022], and [Zhou et al., MedComm 3(3):e155, 2022] have been reviewed, each of which is incorporated by reference to all that they teach regarding the components and uses of such technology to the extent that it does not conflict with the present disclosure.

[0097] The delivery of mRNA into cells provides transient expression of the encoded protein (e.g., CAR, TCR, or immune cell engager) for several days. This may be sufficient and even desirable for therapeutic effects and can be repeated if somewhat longer expression is desired. While the delivery of components of a gene / genome editing system enables more permanent changes, the editing system exists only for a short time, but the changes to the cell's DNA will persist. More extensive changes are also possible using gene / genome editing systems. In addition to conferring the expression of specific proteins, gene / genome editing systems allow for altering the regulation of individual protein expression or knocking out protein expression.

[0098] In some embodiments comprising multiple agents, the nucleic acid may be multicystronic. In other embodiments comprising multiple agents or components, each agent or component is encoded or contained in a separate nucleic acid species. In some embodiments comprising multiple payload nucleic acid species, two or more nucleic acid species are packaged together in a single LNP species. In other embodiments, a subset of the payload nucleic acid species to be delivered (e.g., a single nucleic acid species) is packaged in one LNP species, while another subset of the nucleic acid species is packaged in a different LNP species. Different tLNP species may differ only by the payloads they contain. Different tLNP species may be combined within a single formulation for administration.

[0099] F(ab') and F(ab') analogs

[0100] F(ab') and F(ab')-like formats provide certain advantages as targeting moiety for tLNPs. Any antibody fragment having a structure similar to or derived from the structure of classical and proteolytically produced F(ab') is often referred to as F(ab'); however, the term "F(ab') analogue" has been adopted herein to refer to engineered sequences containing and / or cleaved amino acid substitutions, and to distinguish them from paradigmatic natural sequences. F(ab') is smaller than whole antibodies, which may be advantageous for production. When used as a targeting moiety on tLNPs, their antigen-binding domain is located further from the LNP surface than, for example, scFv, which can facilitate interaction with the target cell surface. F(ab') molecules have cysteine ​​residues that can be readily conjugated to functioned PEG-lipids (e.g., maleimide-functioned PEG-lipids) within a partial hinge region. Additionally, F(ab') can be engineered to have a unique, accessible cysteine ​​that enables site-specific splicing for product consistency. This can be achieved using recombinant DNA technology by cleaving the hinge region of F(ab'), by changing the cysteine ​​residue to another amino acid such as serine, or by both.

[0101] The hinge region cysteine ​​can form cysteine ​​with another F(ab') molecule that forms F(ab')2, which would render the cysteine ​​unavailable for conjugation to LNPs (more specifically, their functionalized lipids). This can be prevented by treating F(ab') under mild reducing conditions, but this poses a risk of destroying the interchain disulfide bond between CL and CH1. Such a risk can be eliminated by relocating the interchain bond to a less accessible region within the molecule.

[0102] Some suns combine an invariant region of F(ab') or an F(ab') analog with a humanized immunoglobulin antigen binding domain derived from the anti-CD8α antibody CT8 as disclosed herein.

[0103] Some embodiments bind an invariant region of an F(ab') analog to the antigen-binding domain of an anti-CD8 antibody. In some embodiments, the anti-CD8 antigen-binding domain recognizes a CT8 epitope. In some embodiments, the anti-CD8 antigen-binding domain is derived from YTC182.20, TRX2, or CT8. In some embodiments, the anti-CD8 antigen-binding domain comprises a humanized immunoglobulin antigen-binding domain derived from the anti-CD8α antibody CT8 disclosed herein.

[0104] In some embodiments, the F(ab') analog fabricated as disclosed herein is conjugated to an LNP, but the variable domain of the F(ab') analog and its specificity are general.

[0105] In some embodiments, the F(ab') or F(ab') analog constant region is combined with the antigen-binding domain of an anti-CD8 antibody conjugated to an LNP. In some embodiments, the anti-CD8 antigen-binding domain recognizes the CT8 epitope. In some embodiments, the anti-CD8 antigen-binding domain is derived from YTC182.20, TRX2, or CT8. In some embodiments, the anti-CD8 antigen-binding domain comprises a humanized immunoglobulin antigen-binding domain derived from the anti-CD8α antibody CT8 disclosed herein.

[0106] In relation to the aforementioned aspects, in some embodiments, the F(ab') analog comprises a Cκ S162C substitution paired with a rearranged interchain disulfide bond, e.g., IgG1 or IgG4 CH1 F174C substitution, where appropriate. In further embodiments, one, the other, or both of the cystes involved in forming the natural interchain disulfide bond are mutated, e.g., Cκ C214S, IgG1 C233S, or IgG4 CH1 C127S. In some embodiments, the Cκ domain of F(ab') has the amino acid sequence of SEQ ID NO. 41. In some embodiments, the Cκ domain retains C214 as the cysteine ​​for conjugation to the LNP, e.g., those comprising SEQ ID NO. 100. Cκ domains, such as those containing sequence number 100, are particularly suitable for pairing with heavy chains that do not retain cysteine ​​that is easily accessible to conjugate to LNPs, such as the .45 design exemplified by sequence number 99 and CBD1033.45 (see Table 17). In some embodiments, the Cκ domain does not retain C214, for example, those containing sequence number 89. Cκ domains, such as those containing sequence number 89, are particularly suitable for pairing with heavy chains that retain cysteine ​​that is easily accessible to conjugate to LNPs, for example, those containing sequence numbers 85, 87, 90, 93, 95, 97, or 103. Cκ domains such as those containing sequence number 89 are particularly suitable for pairing with heavy chains that do not retain cysteine ​​that are easily accessible for conjugation to LNPs, such as those containing sequence numbers 85, 87, 90, 93, 95, 97, or 103. In some embodiments, the F(ab') analog has a CH region cleaved at P245, for example, sequence number 81 or 83.In some embodiments, the F(ab') analog has a CH region cleaved at P241 and having substitutions P240A and P241A, e.g., SEQ NOs 85, 87, 90, 93. In some embodiments, the F(ab') analog has an IgG1 CH region cleaved at P240, e.g., SEQ NO 95. In some embodiments, the F(ab') analog has an IgG1 CH region cleaved at T238, e.g., SEQ NO 97. In some embodiments, the F(ab') analog has an IgG4 CH region cleaved at C239, e.g., SEQ NO 103. In some embodiments, the F(ab') analog cysteine ​​for conjugation to the LNP is C239, e.g., SEQ NOs 85, 87, 90, 93, 95, or 103. In some embodiments, the F(ab') analog cysteine ​​for conjugation to the LNP is C233, e.g., SEQ ID NO. 97. In some embodiments, F(ab') comprises a wild-type IgG1 constant region and has the amino acid sequence of SEQ ID NO. 76, or comprises a wild-type IgG4 constant region and has the amino acid sequence of SEQ ID NO. 79. In some embodiments, the F(ab') analog comprises an IgG1 constant region and has the amino acid sequence of SEQ ID NO. 81, SEQ ID NO. 85, SEQ ID NO. 90, SEQ ID NO. 95, SEQ ID NO. 97, or SEQ ID NO. 99. In some embodiments, the F(ab') analog comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 78, SEQ ID NO. 82, SEQ ID NO. 86, SEQ ID NO. 92, SEQ ID NO. 96, SEQ ID NO. 98, SEQ ID NO. 102, SEQ ID NO. 105, SEQ ID NO. 106, SEQ ID NO. 108, SEQ ID NO. 109, SEQ ID NO. 110, SEQ ID NO. 111, SEQ ID NO. 113, or SEQ ID NO. 114. In some embodiments, the F(ab') analog comprises an IgG4 constant region comprising the amino acid sequence of SEQ ID NO. 83, SEQ ID NO. 87, SEQ ID NO. 93, or SEQ ID NO. 103.In some embodiments, the F(ab') analog comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 84, SEQ ID NO. 88, SEQ ID NO. 94, or SEQ ID NO. 104.

[0107] The CBD1033 variable domain is linked to the invariant region in various F(ab') and F(ab') analog designs as presented in Table 17 (below). In various embodiments, the humanized CT8 variable domain or the variable domain of another antibody may be included in these designs. Accordingly, in various embodiments, the targeting moiety of the LNP may be an F(ab') and F(ab') analog of any design presented in Table 17. In some embodiments, the targeting domain has a .37 design. In some embodiments, the targeting domain has a .44 design. In some embodiments, the targeting domain has a .45 design. Lipid nanoparticles (LNP) and targeted LNPs (tLNP)

[0108] Various LNP compositions capable of serving as a basis for targeted LNPs (tLNPs) are known in the art. For in vivo use, LNPs composed of cationic lipids (particularly ionizable cationic lipids), neutral lipids (e.g., phospholipids), sterols (e.g., cholesterol), and polymer-conjugated lipids (e.g., polyethylene glycol (PEG) lipids) have exhibited advantageous properties. In certain embodiments of the present disclosure, tLNPs comprise ionizable cationic lipids, phospholipids, sterols, and PEG lipids including non-functionalized and functionalized PEG lipids. Table 14 provides a list of various LNP compositions that have been demonstrated to form LNPs encapsulating mRNA, to which a polypeptide comprising an antibody or its antigen-binding domain can be conjugated as a targeting moiety. In some embodiments, the targeting moiety is a engineered F(ab') as disclosed herein. In some embodiments, the targeting moiety comprises an antigen-binding domain specific to CD8, such as an antigen-binding domain specific to human CD8, whether the targeting moiety is a whole antibody, engineered F(ab'), or some other form of antibody. In some cases, the anti-CD8 antigen-binding domain is the humanized anti-CD8 antigen-binding domain disclosed herein. In some cases, the tLNP has the lipid content of composition F9 of Table 14. In some embodiments, composition F9 of Table 14 is used to produce a tLNP comprising an anti-CD8 binding moiety as the targeting moiety.

[0109] In any of the aforementioned tLNP embodiments, a specific embodiment comprises a tLNP comprising a targeting moiety having one of the humanized antigen-binding domains of CT8 disclosed herein, such as a VL region having the amino acid sequence of SEQ ID NO. 17 and a VH region having the amino acid sequence of SEQ ID NO. 11 or 27 to 29. In some such embodiments, the targeting moiety is a whole humanized anti-CD8 antibody having a heavy chain having a silenced Fc region, such as having the amino acid sequence of SEQ ID NO. 43 or 44. In some cases, the whole humanized anti-CD8 antibody having a heavy chain having a silenced Fc region comprises a light chain having the sequence of CBD1033HC (SEQ ID NO. 61) and / or the sequence of CBD1033LC (SEQ ID NO. 62). In some embodiments, the targeting moiety is an anti-CD8 F(ab') of classical F(ab'). In some embodiments, the targeting moiety is an anti-CD8 F(ab') of engineered F(ab'). Examples of such anti-CD8 F(ab') of classical F(ab') or engineered F(ab') are listed in Table 17. In some embodiments, the anti-CD8 F(ab') comprises a light chain having a wild-type kappa constant region, wherein the kappa constant region has the amino acid sequence number 41. In some embodiments, the anti-CD8 F(ab') comprises a light chain having an engineered kappa constant region, wherein the kappa constant region has the amino acid sequence number 89 or number 100. In some embodiments, the anti-CD8 F(ab') comprises a heavy chain having a wild-type IgG1 F(ab'), wherein the IgG1 F(ab') has the amino acid sequence number 76. In some embodiments, the anti-CD8 F(ab') comprises a heavy chain having engineered IgG1 F(ab'), wherein the IgG1 F(ab') has the amino acid sequence number 81, number 85, number 90, number 95, number 97, or number 99.In some embodiments, the anti-CD8 F(ab') comprises a heavy chain having wild-type IgG4 F(ab'), wherein the IgG4 F(ab') has the amino acid sequence of SEQ ID NO. 79. In some embodiments, the anti-CD8 F(ab') comprises a heavy chain having engineered IgG4 F(ab'), wherein the IgG4 F(ab') has the amino acid sequence SEQ ID NO. 83, SEQ ID NO. 87, SEQ ID NO. 93, or SEQ ID NO. 103. In some embodiments, the anti-CD8 F(ab') comprises a light chain having the amino acid sequence of SEQ ID NO. 77, SEQ ID NO. 91, SEQ ID NO. 101, SEQ ID NO. 107, or SEQ ID NO. 112. In some embodiments, the anti-CD8 F(ab') comprises a heavy chain having the amino acid sequence of SEQ NO. 78, SEQ NO. 82, SEQ NO. 86, SEQ NO. 92, SEQ NO. 96, SEQ NO. 98, SEQ NO. 102, SEQ NO. 105, SEQ NO. 106, SEQ NO. 108, SEQ NO. 109, SEQ NO. 110, SEQ NO. 111, SEQ NO. 113, or SEQ NO. 114. In some embodiments, the anti-CD8 F(ab') comprises a heavy chain having the amino acid sequence of SEQ NO. 80, SEQ NO. 84, SEQ NO. 88, SEQ NO. 94, or SEQ NO. 104. In any of the aforementioned tLNP embodiments for encapsulating the mRNA disclosed herein, in a given embodiment, the tLNP comprises, as its targeting moiety, an antibody comprising one of the humanized antigen-binding domains of the CT8 disclosed herein disclosed above, or an antigen-binding portion thereof.

[0110] In some embodiments, the targeting moiety of the tLNP is a classical or engineered F(ab'). In some embodiments, the targeting moiety of the tLNP includes an engineered F(ab'). Examples of such F(ab') including a wild-type or engineered constant region are listed in Table 17. In some such embodiments, F(ab') includes a light chain having a wild-type kappa constant region, wherein the kappa constant region has the amino acid sequence of SEQ ID NO. 41. In some such embodiments, F(ab') includes a light chain having an engineered kappa constant region, wherein the kappa constant region has the amino acid sequence SEQ ID NO. 89 or SEQ ID NO. 100. In some such embodiments, F(ab') includes a heavy chain having a wild-type IgG1 F(ab') constant region, wherein the IgG1 F(ab') constant region has the amino acid sequence SEQ ID NO. 76. In some such embodiments, F(ab') comprises a heavy chain having a engineered IgG1 F(ab') constant region, wherein the IgG1 F(ab') constant region has the amino acid sequence number 81, number 85, number 90, number 95, number 97, or number 99. In some such embodiments, F(ab') comprises a heavy chain having a wild-type IgG4 F(ab') constant region, wherein the IgG4 F(ab') constant region has the amino acid sequence number 79. In some such embodiments, F(ab') comprises a heavy chain having a engineered IgG4 F(ab') constant region, wherein the IgG4 F(ab') constant region has the amino acid sequence number 83, number 87, number 93, or number 103.

[0111] In some embodiments, the tLNP comprises F(ab') or anti-CD8 F(ab') comprising an S162C kappa chain substitution and an IgG1 or IgG4 CH1 F174C substitution. In some embodiments, the F(ab') or anti-CD8 F(ab') heavy chain further comprises an IgG1 CH1 C233S or IgG4 CH1 C127S substitution. In some embodiments, the F(ab') or anti-CD8 F(ab') light chain further comprises a C214S kappa chain substitution.

[0112] In some embodiments of tLNP comprising an F(ab') targeting moiety comprising an invariant region disclosed herein, the tLNP comprises one or more ionizable cationic lipids disclosed herein. In some embodiments of tLNP comprising an F(ab') targeting moiety comprising an invariant region disclosed herein, the tLNP comprises an LNP composition as disclosed in Table 14, e.g., F9.

[0113] In a given embodiment, LNP (or tLNP) is CD1, CD2* †‡ , CD3* †‡ , CD4* †‡ , CD5 †‡ , CD7 †‡ , CD8 † , CD11b ‡ , CD14 †‡ , CD16, CD25 †‡ , CD26* ‡ , CD27* †‡ , CD28* †‡ , CD30* †‡ , CD32*, CD38* †‡ , CD39 ‡ , CD40* †‡ , CD40L(CD154)* †‡ , CD44* ‡ , CD45 †‡ , CD56 †‡ , CD64* ‡ , CD62 †‡ , CD68, CD69 ‡, CD73 †‡ , CD80* ‡ , CD83 ‡ , CD86* ‡ , CD95 ‡ , CD103 ‡ , CD119 ‡ , CD126 ‡ , CD137(41BB) †‡ , CD150 ‡ , CD153 ‡ , CD161 ‡ , CD166 ‡ , CD183(CXCR3) ‡ , CD183(CXCR5) ‡ , CD223(LAG-3)* †‡ , CD254 ‡ , CD275 ‡ , CD45RA, CTLA-4* † * † , DEC205, OX40 † , PD-1* †‡ , GITR † , TIM-3* †‡ , FasL* ‡ , IL18R1, ICOS(CD278) ‡ , leu-12, TCR † , TLR1, TLR2 †‡ , TLR3* ‡ , TLR4 †‡ , TLR6, TREM2 ‡ , NKG2D ‡ , CCR, CCR1(CD191) ‡ , CCR2(CD192)* †‡ , CCR4(CD194)* †‡ , CCR6(CD196) ‡ , CCR7 ‡ , low-affinity IL-2 receptor †‡ , IL-7 receptor ‡ , IL-12 receptor ‡ , IL-15 receptor ‡ , IL-18 receptor ‡ , and IL-21 receptors ‡It comprises a binding moiety specific to an immune cell antigen selected from. In further embodiments, tLNP is CD117 † , CD34* ‡ , CD44* ‡ , CD45 †‡ , CD90(Thy1) ‡ , CD105 ‡ , CD133 ‡ , BMPR2 ‡ , and specific to HSC surface molecules selected from Sca-1; or CD70* ‡ , CD105 ‡ , CD73 ‡ , Stro-1 ‡ , SSEA-3 ‡ , SSEA-4 ‡ , CD271 ‡ , CD146 ‡ , GD2* †‡ , SUSD2, Stro-4, MSCA-1, CD56 ‡ , CD200* †‡ , PODXL ‡ , CD13 ‡ , CD29* ‡ , CD44* ‡ , and CD10 ‡ It comprises a binding moiety specific to an MSC surface molecule selected from. In various embodiments, the binding moiety is an antibody or its antigen-binding portion. (* indicates that an exemplary antibody having the indicated specificity in which the binding moiety can be induced can be found in Table 9 or 10 of U.S. Patent No. 11,326,182B2.) † This indicates that an exemplary antibody with the indicated specificity capable of inducing a binding moiety can be found in the literature [Wilkinson & Hale, 2022]. Both references are cited and referenced above. ‡This indicates that exemplary antibodies with the indicated specificity capable of inducing a binding moiety can be found in the therapeutic antibody database (TABS) (tabs.craic.com). Other suitable antibodies can be found in Appendix A.

[0114] The LNP of the present disclosure is a multicomponent composition comprising a payload and a plurality of lipid components including ionizable cationic lipids, non-functionalized and / or functionalized PEG-lipids, phospholipids, and sterols. The tLNP of the present disclosure is a multicomponent composition comprising an LNP and a binding moiety, such as a humanized anti-CD8 binder / antibody. As used herein, the term “tLNP composition” refers to the same characteristics as an LNP composition to which an anti-CD8 binding moiety acting as a targeting moiety has been added, and the density of the binding moiety on the tLNP may be expressed as a ratio to the payload on a w / w basis. Table 14 provides a list of lipid compositions shown to form the LNP. In some embodiments, the LNP or tLNP comprises a payload having a net negative charge selected from peptides, polypeptides, proteins, small molecules, or nucleic acid molecules, and combinations thereof. The payload is generally contained by or within the LNP or tLNP. As disclosed herein, the dosage always refers to the amount of payload provided.

[0115] As used herein, the term “LNP composition” refers to the lipid components present within the LNP, their molar ratios relative to one another (e.g., mol%), and the ratio of the payload to the total lipids. In certain embodiments, the payload comprises one or more species of nucleic acid molecules or other negatively charged molecules. That is, in some embodiments, the payload comprises only a single species of nucleic acid or other negatively charged molecules (or consists of such species or molecules), whereas in other embodiments, the payload comprises multiple species of nucleic acid or other negatively charged molecules, e.g., two, three, or four such species or molecules. In some embodiments where the payload comprises multiple nucleic acid species or other negatively charged molecules, more than one to at most all of the species are reactive or encode a polypeptide that is reactive to the same target.

[0116] LNP and tLNP compositions

[0117] LNP compositions contribute to the formation of stable LNPs and tLNPs, efficient encapsulation of payloads, protection of payloads from degradation until delivery into the cell, and promotion of endosome escape of payloads into the cytoplasm. These functions are independent of the specificity of binding moiety (or moiety) which serves to orient or deflect tLNPs toward specific cell type(s). Additional LNP and tLNP compositions are generally filed on May 31, 2024, and the title of the invention is ' Lipid Nanoparticle Formulations and Compositions Disclosed in PCT / US2024 / 032141, which incorporates by reference all teachings regarding the design, formation, characterization, properties, and uses of LNP and tLNP.

[0118] LNP and / or tLNP may contain various components in amounts sufficient to provide nanoparticles having the desired shape, fluidity, and bio-acceptability as described herein. With respect to the LNP or tLNP of the present disclosure, in some embodiments, the LNP (or tLNP) comprises at least one ionizable cationic lipid (e.g., as described herein) in an amount ranging from about 35 to about 65 mol% or any integer boundary sub-range thereof, e.g., about 40 to about 65 mol%, or about 40 to about 60 mol%, or about 40 mol% to about 62 mol%. In some embodiments, the LNP or tLNP comprises about 58 mol%, about 60 mol%, or 62 mol% of the ionizable cationic lipid. In some embodiments, the LNP (or tLNP) contains phospholipids in an amount ranging from about 7 to about 30 mol% or any integer boundary sub-range thereof, for example, from about 13 to about 30 mol%. In some embodiments, the LNP or tLNP contains about 10 mol% of phospholipids. In some embodiments, the LNP (or tLNP) contains sterols in an amount ranging from about 20 to about 50 mol% or any integer boundary sub-range thereof, for example, from about 20 to about 45 mol%, or from about 30 to about 50 mol%, or from about 30 to about 45 mol%. In some embodiments, the LNP or tLNP contains about 30.5, 26.5, or 23.5 mol% of sterols. In some embodiments, the LNP (or tLNP) contains at least one auxiliary lipid in an amount ranging from about 1 to about 30 mol%. In some embodiments, LNP or tLNP is a total PEG-lipid in the range of about 1 mol% to about 5 mol% or any integer thereof × 10 -1It contains an amount in a sub-boundary range, for example, an amount of total PEG-lipid in the range of about 1 mol% to about 2 mol%. In some embodiments, the LNP (or tLNP) contains at least one unfunctionalized PEG-lipid in the range of 0 to about 5 mol% or any integer thereof × 10 -1 It contains an amount in a boundary sub-range, e.g., 0 to about 3 mol%, or about 0.1 to about 5 mol%, or about 0.5 to about 5 mol%, or about 0.5 to about 3 mol%. In some embodiments, the LNP or tLNP contains about 1.4 mol% of unfunctionalized PEG-lipid. In some embodiments, the LNP or tLNP contains at least one functionalized PEG-lipid in an amount of about 0.1 to about 5 mol% or any integer thereof × 10 -1 It is included in an amount in the boundary sub-range, e.g., in the range of about 0.1 to 0.3 mol%. In certain embodiments, the LNP or tLNP comprises about 0.1 mol%, about 0.2 mol%, or about 0.3 mol% of functionalized PEG-lipid. In some embodiments, the LNP or tLNP comprises about 0.1 mol% of functionalized PEG-lipid. In some embodiments, the functionalized PEG-lipid is conjugated to a binding moiety. In some embodiments, the binding moiety is a modified F(ab') as disclosed herein. In some embodiments, the binding moiety comprises an anti-CD8 antigen-binding domain, e.g., the humanized anti-CD8α antigen-binding domain disclosed herein. In certain cases, the tLNP comprises an anti-CD8α total antibody as a binding moiety, which is present at an antibody:mRNA ratio (w / w) of about 0.3 to about 1.0.

[0119] In certain embodiments, the present disclosure provides an LNP or tLNP, wherein the LNP or tLNP comprises about 35 mol% to about 65 mol% of ionizable cationic lipids, about 0.5 mol% to about 3 mol% of PEG-lipids (including non-functionalized PEG-lipids and optionally functionalized PEG-lipids), about 7 mol% to about 13 mol% of phospholipids, and about 30 mol% to about 50 mol% of sterols. In some embodiments, the LNP or tLNP comprises a payload having a net negative charge, e.g., a peptide, polypeptide, protein, small molecule, or nucleic acid molecule, and combinations thereof. The payload is generally contained by or within the LNP or tLNP. As disclosed herein, the dosage always refers to the amount of payload provided. In some embodiments, the payload comprises one or more species of nucleic acid molecules. For tLNPs encapsulating mRNA, the dosage is typically in the range of 0.05 to 5 mg / kg regardless of the recipient species. In some embodiments, the dosage is in the range of 0.1 to 1 mg / kg.

[0120] The payload-to-total lipid ratio may be expressed on a w / w basis or, in the case of nucleic acid molecules, as an N / P ratio. In relation to the LNP or tLNP of the present disclosure, in some embodiments, the total lipid-to-nucleic acid ratio is about 10:1 to about 50:1 by weight. In some embodiments, the total lipid-to-nucleic acid ratio is about 10:1, about 20:1, about 30:1, or about 40:1 to about 50:1, or any range limited by 10:1 to 20:1, 30:1, 40:1, or 50:1, or a pair of these ratios. The lipid-to-nucleic acid ratio may also be reported as an N / P ratio, which is the ratio of a positively charged lipid amine (N = nitrogen) group to a negatively charged nucleic acid molecule phosphate (P) group. In some embodiments, the N / P ratio is about 3 to about 9, about 3 to about 7, about 3 to about 6, about 4 to about 6, about 5 to about 6, or about 6. In some embodiments, the N / P ratio is 3 to 9, 3 to 7, 3 to 6, 4 to 6, 5 to 6, or 6.

[0121] Due to physiological and manufacturing constraints, LNP or tLNP particles having a hydrodynamic diameter of about 50 to about 150 nm are preferred for in vivo use. Accordingly, in some embodiments, the LNP or tLNP has a hydrodynamic diameter of 50 to 150 nm, and in some embodiments, the hydrodynamic diameter is ≤120, ≤110, ≤100, or ≤90 nm. Uniformity of particle size is also preferred, and a polydispersity index (PDI) of ≤0.2 (on a scale from 0 to 1) is acceptable. Both the hydrodynamic diameter and the polydispersity index are measured by dynamic light scattering (DLS). The particle diameter measured by cryo-transmission electron microscopy (Cryo-TEM) may be smaller than the value measured by DLS.

[0122] The density of binder moiety on tLNP can be defined based on the amount of antibody input in the conjugation reaction, or according to the ratio of antibody (binder) to mRNA (w / w) as measured in tLNP. For an intact antibody (e.g., full IgG), in some embodiments, the preferred ratios are about 0.3 to about 1.0, about 0.3 to about 0.7, about 0.3 to about 0.5, about 0.5 to about 1.0, and about 0.5 to about 0.7 with respect to the input or finally measured binder ratio. In certain embodiments, the tLNP has antibody ratios of 0.3 to 1.0, 0.3 to 0.7, 0.3 to 0.5, 0.5 to 1.0, and 0.5 to 0.7 with respect to the input or finally measured binder moiety density ratio. In some embodiments, when the size of the binder differs from that of the intact antibody (e.g., scFv, diabody, or minibody, etc.), the w / w ratio is adjusted for different sizes of the binding moiety.

[0123] ionizable cationic lipids

[0124] In certain embodiments, ionizable cationic lipids are described in US20230320995A1; international patent applications PCT / US2024 / 049627 and PCT / US2024 / 049649; and U.S. provisional patent applications 63 / 632,944 and 63 / 632,940; the disclosures thereof are incorporated herein by reference in their entirety. Ionizable cationic lipids are useful components for forming complexes with negatively charged payloads and facilitating the delivery of payloads into the cytoplasm of the cell after endocytosis. Accordingly, each genus and species of ionizable cationic lipids disclosed herein may be used in the LNP and tLNP formulations and compositions of the present disclosure and in methods of use thereof. In a given embodiment, the ionizable cationic lipid(s) of the LNP having a measured pKa of 6 to 7 are maintained in an essentially neutral state in the bloodstream and interstitial space, but may become ionized as the endosome becomes acidified after absorption into the cell. Upon acidification in the endosome space, the lipid is protonated and associates more strongly with the phosphate backbone of the nucleic acid, which destabilizes the structure of the LNP and promotes the release of nucleic acid from the LNP into the cytoplasm of the cell (also referred to as endosome escape). Thus, the ionizable cationic lipid disclosed herein constitutes a means for destabilizing the structure of the LNP (when ionized) or a means for promoting nucleic acid release or endosome escape.

[0125] For simplicity, chemical moiety is generally defined and referred to as a monovalent chemical moiety (e.g., alkyl, aryl, etc.). Nevertheless, such terms may also be used to denote a corresponding multivalent moiety under appropriate structural circumstances that are clear to those skilled in the art. For example, the "alkyl" moiety generally refers to a monovalent radical (e.g., CH3-CH2-), but under certain circumstances, a divalent linked moiety may be "alkyl," in which case those skilled in the art will understand that the alkyl is a divalent radical (e.g., -CH2-CH2-) equivalent to the term "alkylene." (Likewise, in situations where a divalent moiety is required and referred to as “aryl,” those skilled in the art will understand that the term “aryl” refers to arylene, which is the corresponding divalent moiety.) All atoms are understood to have their normal valence number for bond formation (i.e., 4 for carbon, 3 for nitrogen, 2 for oxygen, and 2, 4, or 6 for sulfur, depending on the oxidation state of the sulfur atom).

[0126] As used herein, the term "alkyl" refers to a saturated straight-chain and branched-chain aliphatic group having 1 to 12 carbon atoms. Accordingly, "alkyl" refers to C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 and C 12 Includes energy.

[0127] As used herein, the term "alkenyl" means an unsaturated straight-chain or branched-chain aliphatic group having 2 to 12 carbon atoms and one or more carbon-carbon double bonds. Accordingly, "alkenyl" refers to C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 and C 12 Includes energy.

[0128] In some embodiments, the hydrocarbon chain is unsubstituted. In other embodiments, one or more hydrogens of the alkyl or alkenyl group may be substituted with the same or different substituents.

[0129] Aryl refers to an aromatic or heteroaromatic ring that lacks one hydrogen, leaving a bond that connects to another part of an organic molecule. Examples of aryls include, without limitation, phenyl, naphthalenyl, pyridine, pyrimidine, pyrazine, pyrrole, furan, thiophene, imidazole, thiazole, oxazole, etc.

[0130] Aryl-alkyl refers to a moiety comprising one or more aryl rings and one or more alkyl moietyes. The position of one or more aryl rings may vary within the alkyl portion of the moiety. For example, one or more aryl rings may be at the ends of one or more alkyl moietyes, fused into the carbon chains of one or more alkyl moietyes, or substitute one or more hydrogens of one or more alkyl moietyes; or one or more alkyl moietyes may substitute one or more hydrogens of one or more aryl rings. In some embodiments, a single ring is present; whereas in other embodiments, it is a multiple ring.

[0131] Branched alkyls are saturated alkyl moietys in which the alkyl group is not a straight chain. An alkyl portion, such as methyl, ethyl, propyl, butyl, etc., may be attached to a variable position of the main alkyl chain. In some embodiments, a single branch exists; whereas in other embodiments, multiple branches exist.

[0132] A branched alkenyl refers to an alkenyl group comprising at least one branch extending out of the main chain, which can be formed by substituting one or more hydrogens of the main chain with the same or different alkyl groups, e.g., methyl, ethyl, propyl, butyl, etc., without limitation. In some embodiments, the branched alkenyl has a single branched structure, whereas in other embodiments, the branched alkenyl may have multiple branches.

[0133] Straight-chain alkyls are non-branched, non-cyclic versions of the alkyl moiety described above.

[0134] Linear alkenyls are non-branched, non-cyclic versions of the aforementioned alkenyl moiety.

[0135] In a specific embodiment, the ionizable cationic lipid of the present disclosure has the structure of formula M5:

[0136]

[0137] In the above formula,

[0138] Each R 1 is independently C7-C 11 alkyl or C7-C 11 Selected from alkenyl;

[0139] A 1 Silver (CH2) 1-2 And,

[0140] A 2 is O and,

[0141] A 3 Silver (CH2) 1-5 And, where X is N, A 3 It is not CH2,

[0142] X is N, CH, or C-CH3, and

[0143] A 4 is CH2, C=O, NH, NCH3, or O, and

[0144] A 4 In the case where C=O, A 5 is absent, or is O, S, NH, or NCH3, or A 4 If C=O, A 5 is C=O, and

[0145] A 6 is O, S, NH, NCH3, or (CH2) 0-2 And,

[0146] A 7 Silver (CH2) 0-6and, here A 6 In the case where this is O, S, NH, NCH3, A 7 Silver (CH2) 2-4 And,

[0147] Y is

[0148]

[0149] , or is,

[0150] Here, Z is a combination;

[0151] R 2 is O, and R 3 is C=O, and W is CH or N, or R 2 is C=O, and R 3 is O, and W is CH;

[0152] Here, A 5 Unless C=O, A 6 and A 7 Neither of these is (CH2)0;

[0153] Here

[0154] a), A 1 is CH2, and A 3 Silver (CH2) 2-5 And, X is N, and A 4 is C=O, and A 5 is O, S, NH, NCH3, and A 6 Silver (CH2) 1-2 and, A 7 Silver (CH2) 1-4 This or,

[0155] b) A 1 is CH2, and A 3 Silver (CH2) 1-4 and, X is CH and A 4 is CH2, NH, NCH3, O, and A 5 is C=O, and A 6 is O, NH, NCH3, and A 7 Silver (CH) 2-6This or,

[0156] c) A 1 is (CH2)2, and A 3 Silver (CH2) 1-4 and, X is C-CH3, and A 4 is C=O, and A 5 is O, NH, NCH3, and A 6 Silver (CH2) 1-2 and, A 7 Silver (CH2) 1-4 This or,

[0157] d) A 1 is CH2, and A 3 Silver (CH2) 2-5 And, X is N, and A 4 is C=O, and A 5 is absent, and A 6 is (CH2)0, and A 7 is (CH2)0, and Y is This or,

[0158] e) A 1 is CH2, and A 3 Silver (CH2) 1-5 and, X is CH and A 4 is CH2, NH, NCH3, or O, and A 5 is C=O, and A 6 is (CH2)0, and A 7 is (CH2)0, and Y is This or,

[0159] f) A 1 is (CH2)2, and A 3 Silver (CH2) 1-5 and, X is CCH3, and A 4 is C=O, and A 5 is absent, and A 6 is (CH2)0, and A 7 is (CH2)0, and Y is And;

[0160] Here

[0161] span:

[0162] The number of adjacent atoms in is in the range of 7 to 17.

[0163] As used herein, if the subscript has a value of "0", it is absent. For example, A 6 If this is (CH2)0, A 6 is absent.

[0164] In a specific embodiment of chemical formula M5, R 2 is O, and R 3 is C=O, and W is CH or N. For example, in a specific embodiment of formula M5, R 2 is O, and R 3 C=O and W is CH.

[0165] In a specific embodiment of chemical formula M5, R 2 is C=O, and R 3 is O, and W is CH.

[0166] In a specific embodiment of chemical formula M5, A 1 is CH2, and A 3 Silver (CH2) 2-5 And, X is N, and A 4 is C=O, and A 5 is O, S, NH, NCH3, and A 6 Silver (CH2) 1-2 and, A 7 Silver (CH2) 1-4 is. For example, in a given embodiment, A 1 is CH2, and A 3 Silver (CH2) 2-5 And, X is N, and A 4 is C=O, and A 5 is O and A 6 Silver (CH2) 1-2 and, A 7 Silver (CH2) 1-4 am.

[0167] In a specific embodiment of chemical formula M5, A 1 is CH2, and A 3 Silver (CH2) 1-4and, X is CH and A 4 is CH2, NH, NCH3, O, and A 5 is C=O, and A 6 is O, NH, NCH3, or CH2, and A 7 Silver (CH) 2-6 is. In a predetermined embodiment as described herein, A 1 is CH2, and A 3 Silver (CH2) 1-4 and, X is CH and A 4 is NH, and A 5 is C=O, and A 6 is O, NH, NCH3, or CH2, and A 7 Silver (CH) 2-6 is. For example, in a predetermined embodiment of chemical formula M5, A 1 is CH2, and A 3 Silver (CH2) 1-4 and, X is CH and A 4 is NH, and A 5 is C=O, and A 6 is O and A 7 Silver (CH) 2-6 is. For example, in a predetermined embodiment of chemical formula M5, A 1 is CH2, and A 3 Silver (CH2) 1-4 and, X is CH and A 4 is NH, and A 5 is C=O, and A 6 is CH2, and A 7 Silver (CH) 2-6 is. In a predetermined embodiment as described herein, A 1 is CH2 or A 3 Silver (CH2) 1-4 Either or X is CH or A 4 is CH2 or A 5 is C=O or A 6 is O, NH, NCH3, or CH2, or A 7 Silver (CH) 2-6is. For example, in a predetermined embodiment of chemical formula M5, A 1 is CH2, and A 3 Silver (CH2) 1-4 and, X is CH and A 4 is CH2, and A 5 is C=O, and A 6 is O and A 7 Silver (CH) 2-6 is. In a predetermined embodiment as described herein, A 1 is CH2, and A 3 Silver (CH2) 1-4 and, X is CH and A 4 is O and A 5 is C=O, and A 6 is O, NH, NCH3, or CH2, and A 7 Silver (CH) 2-6 is. For example, in a predetermined embodiment of chemical formula M5, A 1 is CH2, and A 3 Silver (CH2) 1-4 and, X is CH and A 4 is O and A 5 is C=O, and A 6 is CH2, and A 7 Silver (CH) 2-6 is. In a predetermined embodiment as described herein, A 1 is CH2, and A 3 Silver (CH2) 1-4 and, X is CH and A 4 is an NCH3, and A 5 is C=O, and A 6 is O, NH, NCH3, or CH2, and A 7 Silver (CH) 2-6 is. For example, in a predetermined embodiment of chemical formula M5, A 1 is CH2, and A 3 Silver (CH2) 1-4 and, X is CH and A 4 is an NCH3, and A 5 is C=O, and A 6is CH2, and A 7 Silver (CH) 2-6 am.

[0168] In a specific embodiment of chemical formula M5, A 1 is (CH2)2 or A 3 Silver (CH2) 1-4 Or, X is C-CH3 or A 4 is C=O or A 5 is O, NH, NCH3 or A 6 Silver (CH2) 1-2 Or, A 7 Silver (CH2) 1-4 is. For example, in a given embodiment, A 1 is (CH2)2 or A 3 Silver (CH2) 1-4 Or, X is C-CH3 or A 4 is C=O or A 5 is O or A 6 Silver (CH2) 1-2 Or, A 7 Silver (CH2) 1-4 am.

[0169] In a specific embodiment of chemical formula M5, span: The number of adjacent bonded atoms present in is in the range of 7 to 17. For example, in a given embodiment, span: The number of adjacent bonded atoms present in is in the range of 7 to 11 or 7 to 10. In a predetermined embodiment, span: The number of adjacent bonded atoms present in is in the range of 10 to 17 (e.g., 10 to 16, or 10 to 14, or 10 to 12). For example, in a given embodiment, span: The number of adjacent bonded atoms present in is 10. For example, in a given embodiment, span: The number of adjacent bonded atoms in is 7. The inventors have discovered that changing the number of adjacent bonded atoms in each span can enable adjustment of the pKa of cationic lipids.

[0170] In some embodiments of the formula M5, Y is And Z is a bond. In some embodiments of chemical formula M5, Y is And Z is a bond. For example, in some embodiments of formula M5, Y is And Z is a combination.

[0171] In some embodiments of the formula M5, Y is And Z is a bond. For example, for some embodiments of chemical formula M5, Y is And Z is a combination.

[0172] In some embodiments of the formula M5, Y is And Z is a bond. For example, in some embodiments of formula M5, Y is And Z is a combination.

[0173] In some embodiments of the formula M5, Y is And Z is a combination.

[0174] In some embodiments of the formula M5, Y is And Z is a combination.

[0175] In some embodiments of the formula M5, Y is And Z is a combination.

[0176] In some embodiments of the formula M5, Y is And Z is a combination.

[0177] In some embodiments of the formula M5, Y is And Z is a combination.

[0178] In some embodiments of the formula M5, Y is And Z is a combination.

[0179] In some embodiments of the formula M5, Y is And Z is a combination.

[0180] In some embodiments of the formula M5, Y is And Z is a combination.

[0181] In some embodiments of the formula M5, Y is And Z is a combination.

[0182] In some embodiments of the formula M5, Y is And Z is a combination.

[0183] In some embodiments of the formula M5, Y is And Z is a combination.

[0184] In some embodiments of the formula M5, Y is And Z is a combination.

[0185] In some embodiments of the formula M5, Y is And Z is a combination.

[0186] In some embodiments of the formula M5, Y is And Z is a combination.

[0187] In some embodiments of the formula M5, Y is And Z is a combination.

[0188] In some embodiments of the formula M5, Y is And Z is a combination.

[0189] In some embodiments of the formula M5, Y is And Z is a combination.

[0190] In some embodiments of the formula M5, Y is And Z is a combination.

[0191] In some embodiments of the formula M5, Y is And Z is a combination.

[0192] In some embodiments as described herein, ionizable cationic lipids have the structure of Formula 1:

[0193] [Chemical Formula 1]

[0194]

[0195] In the above formula,

[0196] Y is O, NH, N-CH3, or CH2, and

[0197] n is an integer from 0 to 4, and

[0198] X is or And,

[0199] m is an integer from 1 to 3, and

[0200] o is an integer from 1 to 4, and

[0201] p is an integer from 1 to 4, and

[0202] Here, when p = 1:

[0203] Each R is independently C6 to C 16 Straight-chain alkyl; C6 to C 16 Branched alkyl; C6 to C 16 Straight-chain alkenyl; C6 to C 16 Branched alkenyl; C9 to C 16 Cycloalkyl-alkyl (wherein the cycloalkyl is a C3 to C8 cycloalkyl located inside or at both ends of the alkyl chain); or C8 to C 18 aryl-alkyl (wherein aryl is phenyl or naphthalenyl and is located inside or at both ends of the alkyl chain);

[0204] Here, the case where p = 2:

[0205] Each R is independently C6 to C 14 Straight-chain alkyl; C6 to C 14 Straight-chain alkenyl; C6 to C 14 Branched alkyl; C6 to C 14 Branched alkenyl; C9 to C 14 Cycloalkyl-alkyl (wherein the cycloalkyl is a C3 to C8 cycloalkyl located inside or at both ends of the alkyl chain); or C8 to C 16aryl-alkyl (wherein aryl is phenyl or naphthalenyl and is located inside or at both ends of the alkyl chain);

[0206] Here, the case where p = 3:

[0207] Each R is independently C6 to C 12 Straight-chain alkyl; C6 to C 12 Straight-chain alkenyl; C6 to C 12 Branched alkyl; C6 to C 12 Branched alkenyl; C9 to C 12 Cycloalkyl-alkyl (wherein the cycloalkyl is a C3 to C8 cycloalkyl located inside or at both ends of the alkyl chain); or C8 to C 14 aryl-alkyl (wherein aryl is phenyl or naphthalenyl and is located inside or at both ends of the alkyl chain);

[0208] Here, the case where p = 4:

[0209] Each R is independently C6 to C 10 Straight-chain alkyl; C6 to C 10 Straight-chain alkenyl; C6 to C 10 Branched alkyl; C6 to C 10 Branched alkenyl; C9 to C 10 Cycloalkyl-alkyl (wherein the cycloalkyl is a C3 to C8 cycloalkyl located inside or at both ends of the alkyl group); or C8 to C 12 It is an aryl-alky (where aryl is phenyl or naphthalenyl and is located inside or at both ends of the alkyl chain).

[0210] In some embodiments, the ionizable cationic lipid has the following structure CICL:

[0211] (CICL),

[0212] In the above equation, R is , or am.

[0213] In a given embodiment, the ionizable cationic lipid of CICL is R In this case, it is referred to as CICL1, which is am.

[0214] In a given embodiment, the ionizable cationic lipid of CICL is R In this case, it is referred to as CICL2, which is am.

[0215] In a given embodiment, the ionizable cationic lipid of CICL is R In this case, it is referred to as CICL3, which is am.

[0216] In a given embodiment, the ionizable cationic lipid of CICL is R In this case, it is referred to as CICL4, which is am.

[0217] In some embodiments, the ionizable cationic lipid has the following structure CICL-IE:

[0218] (CICL-IE),

[0219] In the above equation, R is , or am.

[0220] In a given embodiment, the ionizable cationic lipid of CICL-IE is R In this case, it is referred to as CICL250, which is am.

[0221] In a given embodiment, the ionizable cationic lipid of CICL-IE is R In this case, it is referred to as CICL250.2, which is am.

[0222] In a given embodiment, the ionizable cationic lipid of CICL-IE is R In this case, it is referred to as CICL250.3, which is am.

[0223] In a given embodiment, the ionizable cationic lipid of CICL-IE is R In this case, it is referred to as CICL250.4, which is am.

[0224] In a specific embodiment, the restricted ionizable cationic lipid of the present disclosure has the structure of formula M6:

[0225]

[0226] In the above equation, X is

[0227]

[0228] , or And;

[0229] Y is O, S, NH, or NCH3;

[0230] Z is O, NH, or NCH3;

[0231] R 2 is O, and R 3 is C=O, and W is CH or N, or R 2 is C=O, and R 3 is O, and W is CH;

[0232] Each R 1 is independently C7-C 11 alkyl or C7-C 11 Selected from alkenyl;

[0233] Each A- 1 , A 2 , A 3 , and A 4 is independently selected from (CH2)0 and (CH2)1, and

[0234] A 5 is (CH2) 0-4 Selected from , CH=CH, and CH2-CH=CH-CH2;

[0235] Wavy bonding indicates that any relative or absolute stereochemical configuration of the corresponding ring atoms, or a mixture of stereochemical configurations, can be assumed.

[0236] As used herein, if the subscript has a value of "0", it is absent. For example, A 1 If this is (CH2)0, A 1 is absent.

[0237] In a specific embodiment of chemical formula M6, R 2 is O, and R 3 is C=O, and W is CH or N. For example, in a specific embodiment of formula M6, R 2 is O, and R 3 C=O and W is CH.

[0238] In a specific embodiment of chemical formula M6, R 2 is C=O, and R 3 is O, and W is CH.

[0239] In various embodiments of M6, A 1 To A 4 It is selected so that there are only two main chain atoms between the nearest ester oxygen and the ring nitrogen in each of the nearest tail groups.

[0240] In a specific embodiment of M6, A 1 is (CH2)0, and A 2 is (CH2)0, and A 3 is (CH2)1, and A 4 is (CH2)1, and A 5 is (CH2) 1-4 Or CH2-CH=CH-CH2.

[0241] In a specific embodiment of M6, A 1 is (CH2)0, and A 2 is (CH2)1, and A 3 is (CH2)1, and A 4 is (CH2)0, and A 5 is (CH2)1.

[0242] In a specific embodiment of M6, A 1 is (CH2)1, and A 2 is (CH2)1, and A3 is (CH2)0, and A 4 is (CH2)0, and A 5 is (CH2)0.

[0243] In a specific embodiment of M6, A 1 is (CH2)1, and A 2 is (CH2)1, and A 3 is (CH2)0, and A 4 is (CH2)0, and A 5 is (CH2)1.

[0244] In a specific embodiment of M6, A 1 is (CH2)1, and A 2 is (CH2)1, and A 3 is (CH2)0, and A 4 is (CH2)0, and A 5 is (CH2)2 or CH=CH.

[0245] In some embodiments of the formula M6 as described herein, X is is. For example, in some embodiments of chemical formula M6, X is am.

[0246] In some embodiments of the formula M6 as described herein, X is am.

[0247] In some embodiments of the formula M6 as described herein, X is am.

[0248] In some embodiments of the formula M6 as described herein, X is is. In some embodiments, X is am.

[0249] In some embodiments of the formula M6 as described herein, X is am.

[0250] In some embodiments of the formula M6 as described herein, X is am.

[0251] In some embodiments of the formula M6 as described herein, X is am.

[0252] In some embodiments of the formula M6 as described herein, X is am.

[0253] In some embodiments of the formula M6 as described herein, X is am.

[0254] In some embodiments of the formula M6 as described herein, X is am.

[0255] In some embodiments of the formula M6 as described herein, X is am.

[0256] In some embodiments of the formula M6 as described herein, X is am.

[0257] In some embodiments of the formula M6 as described herein, X is am.

[0258] In some embodiments of the formula M6, X is is. In some embodiments of chemical formula M6, X is is. In some embodiments of chemical formula M6, X is am.

[0259] In some embodiments of the formula M6, X is is. In some embodiments of chemical formula M6, X is am.

[0260] In some embodiments of the formula M6, X is is. In some embodiments of chemical formula M6, X is is. In some embodiments of chemical formula M6, X is am.

[0261] In some embodiments of the formula M6, X is is. In some embodiments of chemical formula M6, X is is. In some embodiments of chemical formula M6, X is is. In some embodiments of chemical formula M6, X is am.

[0262] In some embodiments of the formula M6, X is is. In some embodiments of chemical formula M6, X is is. In some embodiments of chemical formula M6, X is am.

[0263] In some embodiments of the formula M6, X is is. In some embodiments of chemical formula M6, X is is. In some embodiments of chemical formula M6, X is am.

[0264] In some embodiments of the formula M6, X is is. In some embodiments of chemical formula M6, X is is. In some embodiments of chemical formula M6, X is am.

[0265] In some embodiments of the formula M6, X is is. In some embodiments of chemical formula M6, X is is. In some embodiments of chemical formula M6, X is am.

[0266] In some embodiments of the formula M6, X is is. In some embodiments of chemical formula M6, X is am.

[0267] In some embodiments of the formula M6, X is is. In some embodiments of chemical formula M6, X is am.

[0268] As described above, in some embodiments of formula M6, Y may be selected from O, S, NH, or NCH3. In some embodiments of formula M6, Y is O. In some other embodiments of formula M6, Y is S.

[0269] In some embodiments of the formula M6, X is And Y is O. In some embodiments of chemical formula M6, X is And Y is S.

[0270] As described above, Z can be selected from O, NH, or NCH3. In some embodiments, Z is O.

[0271] In some embodiments of the formula M6, X is and Z is O. In some embodiments of chemical formula M6, X is and Z is O. In some embodiments of chemical formula M6, X is And Z is O.

[0272] In some embodiments of the formula M6, X is and Z is O. In some embodiments of chemical formula M6, X is And Z is O.

[0273] In some embodiments of the formula M6, X is and Z is O. In some embodiments of chemical formula M6, X is and Z is O. In some embodiments of chemical formula M6, X is And Z is O.

[0274] In some embodiments of the formula M6, X is and Z is O. In some embodiments of chemical formula M6, X is and Z is O. In some embodiments of chemical formula M6, X is and Z is O. In some embodiments of chemical formula M6, X is And Z is O.

[0275] In some embodiments of the formula M6, X is and Z is O. In some embodiments of chemical formula M6, X is and Z is O. In some embodiments of chemical formula M6, X is And Z is O.

[0276] In some embodiments of the formula M6, X is and Z is O. In some embodiments of chemical formula M6, X is and Z is O. In some embodiments of chemical formula M6, X is And Z is O.

[0277] In some embodiments of the formula M6, X is and Z is O. In some embodiments of chemical formula M6, X is and Z is O. In some embodiments of chemical formula M6, X is And Z is O.

[0278] In some embodiments of the formula M6, X is and Z is O. In some embodiments of chemical formula M6, X is And Y is O. In some embodiments of chemical formula M6, X is And Z is O.

[0279] In some embodiments of the formula M6, X is and Z is O. In some embodiments of chemical formula M6, X is And Z is O.

[0280] In some embodiments of the formula M6, X is and Z is O. In some embodiments of chemical formula M6, X is And Z is O.

[0281] As described above, for both chemical formulas M5 and M6, each R 1 C independently 7- -C 11 alkyl or C7-C 11 It is selected from alkenil. In some embodiments of formulas M5 and / or M6, each R- 1 is independently C7-C 11 Alkyl, e.g., C-7-C 10Selected from alkyl, or C7-C9 alkyl. In a given embodiment of formula M5 and / or M6, each R 1 is independently linear C7-C 11 Alkyl, e.g., linear C-7-C 10 It is selected from alkyl, or linear C7-C9 alkyl. In some embodiments of formulas M5 and / or M6 as described herein, each R 1 is independently (CH2) 6-8 It is selected from CH3. In these and some of the other embodiments, R 1 It is (CH2)7CH3. In some embodiments of chemical formula M5 and / or M6, each R- 1 is independently linear C7-C 11 Alkenyls, e.g., linear C-7-C 10 It is selected from alkenyls, or linear C7-C9 alkenyls. For example, in some embodiments of formulas M5 and / or M6, each R 1 is a linear C-8 alkenyl. In certain other embodiments of formulas M5 and / or M6, each R 1 is independently branched C7-C 11 Alkyl, e.g., C-7-C 10 It is selected from alkyl, or C7-C9 alkyl. For example, in some embodiments of formula M5 and / or M6, each R- 1 is a branched C8 alkyl. In a given embodiment of formula M5 and / or M6, each R 1 is independently branched C7-C 11 Alkenyl, e.g., C-7-C 10 It is selected from alkenyls, or C7-C9 alkenyls. For example, in some embodiments of formulas M5 and / or M6, each R- 1 is a branched C8 alkenyl. R 1In some embodiments of formulas M5 and / or M6, which are branched alkyl or alkenyls, the branching point is positioned such that the ester carbonyl is not at the α position relative to the branching point, for example, so that they are at the β position relative to the branching point.

[0282] In a given embodiment of the formulas M5 and / or M6 as described herein, each R 1 is identical. In a given embodiment of formula M5 and / or M6, each R closest to the common branch point 1 Although they are identical, those closest to the first common branch point are different from those closest to the second common branch point. In a given embodiment of formula M5 and / or M6, each R closest to the common branch point 1 Although they differ, R closest to the first common branch point 1 The pair is identical to the pair closest to the second common branch point.

[0283] In a specific embodiment of Formula M6, the ionizable cationic lipid is substantially enantiomerically pure (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%). In a specific embodiment of Formula M6, the ionizable cationic lipid is a racemic mixture. In a specific embodiment of Formula M6, the ionizable cationic lipid is a mixture of two or more stereoisomers. In a specific embodiment of Formula M6, at least two of the two or more stereoisomers are diastereomers. In a specific embodiment of Formula M6, at least two of the two or more stereoisomers are enantiomers.

[0284] In some embodiments as described herein, the ionizable cationic lipid has the structure of Formula 2:

[0285] [Chemical Formula 2]

[0286]

[0287] In the above formula,

[0288] Y is O, NH, N-CH3, or CH2, and

[0289] n is an integer from 0 to 4, and

[0290] X is , or And,

[0291] m is an integer from 1 to 3, and

[0292] o is an integer from 1 to 4, and

[0293] p is an integer from 1 to 4, and

[0294] Here, when p = 1:

[0295] Each R is independently C6 to C 16 Straight-chain alkyl; C6 to C 16 Branched alkyl; C6 to C 16 Straight-chain alkenyl; C6 to C 16 Branched alkenyl; C9 to C 16 Cycloalkyl-alkyl (wherein the cycloalkyl is a C3 to C8 cycloalkyl located inside or at both ends of the alkyl chain); or C8 to C 18 aryl-alkyl (wherein aryl is phenyl or naphthalenyl and is located inside or at both ends of the alkyl chain);,

[0296] Here, the case where p = 2:

[0297] Each R is independently C6 to C 14 Straight-chain alkyl; C6 to C 14 Straight-chain alkenyl; C6 to C 14 Branched alkyl; C6 to C 14 Branched alkenyl; C9 to C 14 Cycloalkyl-alkyl (wherein the cycloalkyl is a C3 to C8 cycloalkyl located inside or at both ends of the alkyl chain); or C8 to C 16It is an aryl-alkyl (wherein aryl is phenyl or naphthalenyl and is located inside or at both ends of the alkyl chain), and

[0298] Here, the case where p = 3:

[0299] Each R is independently C6 to C 12 Straight-chain alkyl; C6 to C 12 Straight-chain alkenyl; C6 to C 12 Branched alkyl; C6 to C 12 Branched alkenyl; C9 to C 12 Cycloalkyl-alkyl (wherein the cycloalkyl is a C3 to C8 cycloalkyl located inside or at both ends of the alkyl chain); or C8 to C 14 aryl-alkyl (wherein aryl is phenyl or naphthalenyl and is located inside or at both ends of the alkyl chain);

[0300] Here, the case where p = 4:

[0301] Each R is independently C6 to C 10 Straight-chain alkyl; C6 to C 10 Straight-chain alkenyl; C6 to C 10 Branched alkyl; C6 to C 10 Branched alkenyl; C9 to C 10 Cycloalkyl-alkyl (wherein the cycloalkyl is a C3 to C8 cycloalkyl located inside or at both ends of the alkyl group); or C8 to C 12 It is an aryl-alky (where aryl is phenyl or naphthalenyl and is located inside or at both ends of the alkyl chain).

[0302] In some embodiments as described herein, the ionizable cationic lipid has the structure of Formula 3:

[0303] [Chemical Formula 3]

[0304]

[0305] In the above formula,

[0306] W is C=O or CH2, and

[0307] n is an integer from 0 to 4, and

[0308] X is , or And,

[0309] m is an integer from 1 to 3, and

[0310] o is an integer from 1 to 4, and

[0311] p is an integer from 1 to 4, and

[0312] Here, when p = 1:

[0313] Each R c is independently C8 to C 18 Straight-chain alkyl; C8 to C 18 Straight-chain alkenyl; C8 to C 18 Branched alkyl; C8 to C 18 Branched alkenyl; C 11 to C 18 Cycloalkyl-alkyl (wherein the cycloalkyl is a C3 to C8 cycloalkyl located inside or at both ends of the alkyl chain); or C 10 to C 20 It is an aryl-alkyl (wherein aryl is phenyl or naphthalenyl and is located inside or at both ends of the alkyl chain), and

[0314] Here, the case where p = 2:

[0315] Each R c is independently C8 to C 16 Straight-chain alkyl; C8 to C 16 Straight-chain alkenyl; C8 to C 16 Branched alkyl; C8 to C 16 Branched alkenyl; C 11 to C 16 Cycloalkyl-alkyl (wherein the cycloalkyl is a C3 to C8 cycloalkyl located inside or at both ends of the alkyl chain); or C 10 to C 18 It is an aryl-alkyl (wherein aryl is phenyl or naphthalenyl and is located inside or at both ends of the alkyl chain), and

[0316] Here, the case where p = 3:

[0317] Each R c is independently C8 to C 14 Straight-chain alkyl; C8 to C 14 Straight-chain alkenyl; C8 to C 14 Branched alkyl; C8 to C 14 Branched alkenyl; C 11 to C 14 Cycloalkyl-alkyl (wherein the cycloalkyl is a C3 to C8 cycloalkyl located inside or at both ends of the alkyl chain); or C 10 to C 16 It is an aryl-alkyl (wherein aryl is phenyl or naphthalenyl and is located inside or at both ends of the alkyl chain), and

[0318] Here, the case where p = 4:

[0319] Each R c is independently C8 to C 12 Straight-chain alkyl; C8 to C 12 Straight-chain alkenyl; C8 to C 12 Branched alkyl; C8 to C 12 Branched alkenyl; C 11 to C 12 Cycloalkyl-alkyl (wherein the cycloalkyl is a C3 to C8 cycloalkyl located inside or at both ends of the alkyl group); or C 10 to C 14 It is an aryl-alky (where aryl is phenyl or naphthalenyl and is located inside or at both ends of the alkyl chain).

[0320] R in text c and R in the chemical structure c is equivalent.

[0321] The ionizable cationic lipids of the present disclosure have a branched structure to provide the lipids with a conical shape rather than a cylindrical shape, and this structure helps to promote endosome lytic activity. The greater the endosome lytic activity, the more efficient the release of a biologically active payload (e.g., one or more species of nucleic acid molecules).

[0322] The ionizable cationic lipids described herein may be useful as components of lipid nanoparticles for delivering nucleic acids, including DNA, mRNA, or siRNA, into cells. The ionizable cationic lipids may have a c-pKa (calculated pKa) in the range of about 6, 7, or 8 to about 9, 10, or 11. For example, in various embodiments described herein, the ionizable cationic lipids have a c-pKa in the range of about 6 to about 10, about 7 to about 10, about 8 to about 10, about 8 to about 9, about 8 to about 9, 6 to 10, 7 to 10, 8 to 10, or 8 to 9. In certain embodiments, the ionizable cationic lipids have a c-pKa in the range of about 8.4 to about 8.7, or 8.4 to 8.7. The ionizable cationic lipids described herein may have a cLogD in the range of about 9 to about 18, for example, about 10 to about 18, about 10 to about 16, about 10 to about 14, about 11 to about 18, about 11 to about 15, or about 11 to about 14. The ionizable cationic lipids described herein may have a cLogD in the range of 9 to 18, for example, 10 to 18, 10 to 16, 10 to 14, 11 to 18, 11 to 15, or 11 to 14. In certain embodiments, the ionizable cationic lipids have a cLogD in the range of about 13.6 to about 14.4, or 13.6 to 14.4. In certain embodiments, the ionizable cationic lipids described herein may have a c-pKa in the range of about 8 to about 11, or 8 to 11, and a cLogD in the range of about 9 to about 18, or 9 to 18. For example, in certain embodiments, the ionizable cationic lipids may have a c-pKa in the range of about 8.4 to about 8.7, or 8.4 to 8.7, and a cLogD in the range of about 13.6 to about 14.4, or 13.6 to 14.4.These ranges can result in pKas measured in LNPs of about 6 to about 7 or 6 to 7, which promote ionization in endosomes after delivery into the cell.

[0323] In some embodiments, a somewhat greater basicity may be desirable and may be obtained from ionizable cationic lipids having c-pKa and cLogD in the ranges disclosed herein. In some embodiments, the cLogD of the ionizable cationic lipids of the disclosure is about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, or within a range limited by any pair of these values. The lipid design also describes potential biodegradable pathways of target lipids, such as those by esterases in plasma, the liver, and other tissues. Another consideration of the lipid design is the processing pathway of fragments of ionizable lipids resulting from degradation, such as after esterase cleavage(s). Preferably, the resulting fragments are rapidly removed from the body without the need for hepatic oxidative metabolism.

[0324] The synthesis of lipids having the structures of M5, CICL, CICL-IE, or M6 is described in U.S. Patent Applications No. 63 / 632,931 (partially M6), No. 63 / 632,937 (partially M5), No. 63 / 632,940 (CICL-IE, partly M5), and No. 63 / 632,944 (partially M6), and U.S. Patent Application Publication No. 2023 / 0320995 (CICL), each of which is incorporated by reference in its entirety for all that it teaches not only the synthesis of such lipids but also for specific subgenuses and individual species.

[0325] Additional ionizable cationic lipids and LNP compositions comprising them are WO 2017 / 049245, WO 2022 / 112855, WO2013 / 185116, WO2015074085, WO2016081029, WO2017 / 117530, WO2018 / 118102, WO2022 / 235935, WO 2023 / 086514, WO2024 / 044728, WO2023 / 196931, WO2023 / 044333A1, WO2013089151, WO2023 / 183616, WO2013 / 065825, WO2013 / 089152, WO2015 / 186770, WO2022 / 166213, WO2023 / 045366, WO2019 / 131580, WO 2005 / 007196, WO 2006 / 053430, WO 2007 / 086883, WO 2009 / 129387, WO 2010 / 048536, U.S. Patents No. 9,868,692, No. 10,435,616, No. 11,246,933, No. 11,382,979, No. 8,058,069, No. 8,492,359, No. 8,722,082, No. 8,822,668, No. 9,364,435, Disclosures 9,408,914, 9,504,651, 10,526,284, 10,961,188, 11,141,378, and 11,241,493, each of which is incorporated herein by reference with respect to cationic ionizable lipids, LNPs comprising them, and all that it teaches regarding nucleic acid delivery mediated by such LNPs that is not in conflict with the present disclosure.In a given embodiment, the tLNP comprises, as its targeting moiety, an antibody comprising the humanized antigen-binding domain of CT8 of the present disclosure or an antigen-binding portion thereof, WO 2017 / 049245, WO 2022 / 112855, WO 2005 / 007196, WO 2006 / 053430, WO 2007 / 086883, WO 2009 / 129387, WO 2010 / 048536, U.S. Patents 9,868,692, 10,435,616, 11,246,933, 11,382,979, 8,058,069, 8,492,359, and 8,822,668. It further includes cationic ionizable lipids from any one of headings 9,364,435, 9,504,651, 11,141,378, and 11,241,493.

[0326] In some embodiments, the LNP or tLNP comprises about 35 mol% to about 65 mol%, about 40 mol% to about 62 mol%, or about 54 mol% to about 60 mol% of ionizable cationic lipids. In some embodiments, the lipid composition comprises 40 mol% or more and / or 62 mol% or less of ionizable cationic lipids. In a given embodiment, the LNP of the tLNP comprises ionizable cationic lipids in a range defined by about 54 mol%, about 58 mol%, or about 62 mol%, or any pair of these values. In a further embodiment, the LNP comprises 35 mol% to 65 mol%, 40 mol% to 62 mol%, or 54 mol% to 60 mol% of ionizable cationic lipids. In another further embodiment, the LNP has 40 mol% or more or 62 mol% or less of ionizable cationic lipids. In a given embodiment, the LNP comprises an ionizable cationic lipid in a range limited by 54 mol%, 58 mol%, or 62 mol%, or any pair of these values.

[0327] phospholipids

[0328] As described above, in various embodiments, LNP and tLNP comprise phospholipids. As understood by those skilled in the art, phospholipids are amphiphilic molecules. Due to the amphiphilic properties of phospholipids, these molecules are known to form bilayers and, by being incorporated into LNP and tLNP as described herein, can provide membrane formation, stability, and rigidity. As used herein, phospholipids comprise a hydrophilic head group comprising a functionalized phosphate group and two hydrophobic tail groups derived from fatty acids. For example, in various embodiments as described herein, phospholipids comprise a phosphate group functionalized with ethanolamine, choline, glycerol, serine, or inositol. As described above, phospholipids comprise two hydrophobic tail groups derived from fatty acids. These hydrophobic tail groups may be derived from unsaturated or saturated fatty acids. For example, the hydrophobic tail groups may be derived from C12-C20 fatty acids.

[0329] With respect to the LNP or tLNP of the present disclosure, in various embodiments, the phospholipid comprises dimyristoylphosphatidylglycerol (DMPG), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), distearoyl-glycero-phosphate (18:0 PA, DSGP), dioleoylphosphatidylethanolamine (DOPE), dioleoyl-glycero-phosphate (18:1 PA, DOGP) or diarachidoylphosphatidylcholine (DAPC), or a combination thereof. In various embodiments, the phospholipid is dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylcholine (DPPC), or 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC). In some embodiments, the phospholipid is distearoylphosphatidylcholine (DSPC). The phospholipid can contribute to the formation of a monolayer, bilayer, or multilayer membrane surrounding the core of an LNP or tLNP. Additionally, phospholipids, such as DSPC, DMPC, DPPC, and DAPC, impart stability and rigidity to the membrane structure. Phospholipids such as DOPE impart fusion properties. Additionally, phospholipids such as DMPG, which achieve a negative charge at physiological pH, facilitate charge regulation. Accordingly, the phospholipid comprises means for facilitating membrane formation, means for imparting membrane stability and rigidity, means for imparting fusion properties, and means for charge regulation. Some embodiments specifically include one or more of the above phospholipids, while other embodiments specifically exclude one or more of the above phospholipids.

[0330] In some embodiments, the LNP or tLNP has about 7 mol% to about 13 mol% of phospholipids, about 7 mol% to about 10 mol% of phospholipids, or about 10 mol% to about 13 mol% of phospholipids. In certain embodiments, the LNP has about 7 mol%, about 10 mol%, or about 13 mol% of phospholipids. In certain cases, the phospholipid is DSPC. In certain cases, the phospholipid is DAPC.

[0331] Sterol

[0332] In a given embodiment, the disclosed LNP and tLNP comprise a sterol. A sterol refers to a subgroup of steroids containing at least one hydroxyl (OH) group. More specifically, it is a steroid derivative in which the H at the 3rd position is substituted with an OH group, or, in other words, but equally, a steroid in which the H at the 3rd position is substituted with an OH group. Examples of sterols include, but are not limited to, cholesterol, ergosterol, β-sitosterol, stigmasterol, stigmasterol, 20-hydroxycholesterol, 22-hydroxycholesterol, etc. With respect to the LNP or tLNP of the present disclosure, in various embodiments, the sterol is cholesterol, 20-hydroxycholesterol, 20(S)-hydroxycholesterol, 22-hydroxycholesterol, or phytosterol, or a combination thereof. In additional embodiments, the phytosterol comprises campesterol, sitosterol, or stigmasterol, or a combination thereof. In certain embodiments, cholesterol is not derived from animals but is obtained by synthesizing it using plant sterols as a starting point. LNPs containing C-24 alkyl (e.g., methyl or ethyl) phytosterols have been reported to provide enhanced gene transfection. The length of the alkyl tail, the flexibility of the sterol ring, and the polarity associated with the retained C-3 -OH group are important for achieving high transfection efficiency. Although β-sitosterol and stigmasterol have demonstrated excellent performance, vitamins D2, D3, and calcipotriol (an analogue lacking the whole body of cholesterol), as well as betulin, lupeol ursolic acid, and olenolic acid (containing a fifth ring), should be avoided. Sterols play a role in filling the space between other lipids in the LNP or tLNP and influencing the shape of the LNP or tLNP. Sterols also control the fluidity of lipid compositions, thereby reducing temperature dependence.Accordingly, sterols such as cholesterol, ergosterol, 20-hydroxycholesterol, 22-hydroxycholesterol, campesterol, fucosterol, β-sitosterol, and stigmasterol constitute means for controlling LNP shape and fluidity or sterol means for increasing transfection efficiency. Some embodiments specifically include one or more of the above sterols, while other embodiments specifically exclude one or more of the above sterols. When designing a lipid composition for LNP or tLNP, in some embodiments, the sterol content may be selected to compensate for various amounts of other types of lipids, e.g., ionizable cationic lipids or phospholipids.

[0333] In some embodiments, LNP or tLNP has about 27 mol% or about 30 mol% to about 50 mol% of sterols, or about 30 mol% to about 38 mol% of sterols. In certain embodiments, LNP or tLNP has about 30.5 mol%, about 33.5 mol%, or about 37.5 mol% of sterols. In certain embodiments, LNP or tLNP has 27 mol% or 30 mol% to 50 mol% of sterols or 30 mol% to 38 mol% of sterols. In further embodiments, LNP or tLNP has 30.5 mol%, 33.5 mol%, or 37.5 mol% of sterols. In certain cases, the sterol is cholesterol. In certain embodiments, the sterol is a mixture of sterols, for example, a mixture of cholesterol and β-sitosterol, or a mixture of cholesterol and 20-hydroxycholesterol. In some cases, the sterol component consists of about 25 mol% 20-hydroxycholesterol and about 75 mol% cholesterol. In some cases, the sterol component consists of about 25 mol% β-sitosterol and about 75 mol% cholesterol. In some cases, the sterol component consists of about 50 mol% β-sitosterol and about 50 mol% cholesterol. In some cases, the sterol component consists of 25 mol% 20-hydroxycholesterol and 75 mol% cholesterol. In additional cases, the sterol component consists of 25 mol% β-sitosterol and 75 mol% cholesterol. In other additional cases, the sterol component consists of 50 mol% β-sitosterol and 50 mol% cholesterol.

[0334] auxiliary lipids

[0335] In relation to LNP or tLNP, in some embodiments, the known lipid is absent or comprises an ionizable lipid. In some embodiments, the ionizable lipid is cholesterol hemisuccinate (CHEMS). In some embodiments, the auxiliary lipid is a charged lipid, such as a quaternary ammonium head group-containing lipid. In some cases, the quaternary ammonium head group-containing lipid comprises 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), N-(1-(2,3-dioleyyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), or 3β-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol), or a combination thereof. In addition to the chloride salt of the quaternary ammonium head group containing the lipid, in additional cases, bromide, mesylate, and tosylate salts are included.

[0336] PEG-lipid

[0337] In relation to the LNP or tLNP of the present disclosure, the PEG-lipid is a lipid conjugated to polyethylene glycol (PEG). In some embodiments as described herein, the PEG-lipid is a C14-C20 lipid conjugated to PEG. For example, in various embodiments as described herein, the PEG-lipid is a C14-C20 lipid conjugated to PEG, or a C14-C18 lipid conjugated to PEG, or a C14-C16 lipid conjugated to PEG. In certain embodiments as described herein, the PEG-lipid is a fatty acid conjugated to PEG. The fatty acid of the PEG-lipid may have various chain lengths. In each case, in some embodiments, the PEG-lipid is a fatty acid conjugated to PEG, wherein the fatty acid chain length is in the C14-C20 range (e.g., in the C14-C18 or C14-C16 range). PEG-lipids with fatty acid chain lengths of less than C14 are lost too quickly from LNP or tLNP, whereas those with chain lengths greater than C20 are prone to difficulties in formulation.

[0338] PEG can be manufactured in various sizes. In certain embodiments, the PEG of the LNP and tLNP disclosed herein is PEG-1000 to PEG-5000. It should be understood that polyethylene formulations of these sizes are polydisperse, and that the nominal size represents the approximate average molecular weight of the distribution. (OCH2CH2) n Assuming the molecular weight of an individual repeating unit is 44, the PEG molecule with n=22 will have a molecular weight of 986, that with n=45 will have a molecular weight of 1998, and that with n=113 will have a molecular weight of 4990. 22 to 113 are used to denote PEG lipids containing PEG moiety in the range of PEG-1000 to PEG-5000, such as PEG-1000, PEG-1500, PEG-2000, PEG-2500, PEG-3000, PEG-3500, PEG-4000, PEG-4500, and PEG-5000; however, some molecules prepared at the average molecular weight boundary will have n outside that range. For individual formulations, n 22 is used to denote PEG-lipids containing the PEG moiety of PEG-1000, and n 45 is used to denote PEG-lipids containing the PEG moiety of PEG-2000, and n 67 is used to denote PEG-lipids containing the PEG moiety of PEG-3000, and n 90 is used to denote PEG-lipids containing the PEG moiety of PEG-4000, and n 113 is used to denote a PEG-lipid comprising a PEG moiety of PEG-5000. Some embodiments comprise a PEG moiety in a range limited by any pair of the previously mentioned n values ​​or average molecular weights. In some embodiments of the PEG-lipid, the PEG has a molecular weight (MW) of 500 to 5000 or 1000 to 5000 Da. For example, in some embodiments, the PEG of the PEG-lipid has a molecular weight in the range of 1500 to 5000 Da or 2000 to 5000 Da. In some embodiments as described herein, the PEG-lipid has a molecular weight in the range of 500 to 4000 Da, or 500 to 3000 Da, or 1000 to 4000 Da, or 1000 to 3000, or 1000 to 2500, or 1500 to 4000, or 1500 to 3000, or 1500 to 2500 Da. In some embodiments, the PEG moiety is PEG-500, PEG-1000, PEG-1500, PEG-2000, PEG-2500, PEG-3000, PEG-3500, PEG-4000, PEG-4500, and PEG-5000. In some embodiments, the PEG unit has an MW of 2000 Da (sometimes abbreviated as PEG(2k)). Some embodiments include a PEG moiety of PEG-1000, PEG-2000, or PEG-5000. In some cases, the PEG moiety is PEG-2000. Certain embodiments include DSG-PEG, e.g., DSG-PEG-2000. Certain embodiments include DSPE-PEG, e.g., DSPE-PEG-2000. Certain embodiments include both DSG-PEG-2000 and / or DSPE-PEG2000.

[0339] Conventional PEG lipids are classified into two classes: diacylglycerol and diacyl phospholipids. Examples of diacylglycerol PEG lipids include DMG-PEG (1,2-dimyristoyl-glycero-3-methoxypolyethylene glycol), DPG-PEG (1,2-dipalmitoyl-glycero-3-methoxypolyethylene glycol), DSG-PEG (1,2-distearoyl-glycero-3-methoxypolyethylene glycol) and DOG-PEG (1,2-dioleoyl-glycero-3-methoxypolyethylene glycol). Examples of diacyl phospholipids include DMPE-PEG (1,2-dimyristoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol), DPPE-PEG (1,2-dipalmitoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol), DSPE-PEG (1,2-distearoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol) and DOPE-PEG (1,2-dioleoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol).

[0340] In some embodiments, MW2000 PEG-lipids (e.g., PEG-lipids comprising PEG with a molecular weight of 2000 Da) are DMG-PEG2000 (1,2-dimyristoyl-glycero-3-methoxypolyethylene glycol-2000), DPG-PEG2000 (1,2-dipalmitoyl-glycero-3-methoxypolyethylene glycol-2000), DSG-PEG2000 (1,2-distearoyl-glycero-3-methoxypolyethylene glycol-2000), DOG-PEG2000 (1,2-dioleoyl-glycero-3-methoxypolyethylene glycol-2000), DMPE-PEG200 (1,2-dimyristoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), It comprises DPPE-PEG2000 (1,2-dipalmitoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), DSPE-PEG2000 (1,2-distearoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), DOPE-PEG2000 (1,2-dioleoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000) or a combination thereof. In some embodiments, the PEG unit has an MW of 2000 Da.In some embodiments, the MW2000 PEG-lipid is DMrG-PEG2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), DPrG-PEG2000 (1,2-dipalmitoyl-rac-glycero-3-methoxypolyethylene glycol-2000), DSrG-PEG2000 (1,2-distearoyl-rac-glycero-3-methoxypolyethylene glycol-2000), DorG-PEG2000 (1,2-dioleoyl-glycero-3-methoxypolyethylene-rac-glycol-2000), DMPEr-PEG200 (1,2-dimyristoyl-rac-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), It includes DPPEr-PEG2000 (1,2-dipalmitoyl-rac-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), DSPEr-PEG2000 (1,2-distearoyl-rac-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), DOPEr-PEG2000 (1,2-dioleoyl-rac-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), or combinations thereof. The glycerol of these lipids is chiral. Thus, in some embodiments, the PEG-phospholipid is a racemic mixture. Alternatively, an optically pure opposite of the glycerol moiety may be used, i.e., the glycerol moiety is homochiral. As used herein with respect to glycerol moiety, optically pure means ≥95% of a single enantiomer (D or L). In some embodiments, the enantiomer excess rate is ≥98%. In some embodiments, the enantiomer excess rate is ≥99%.Additional PEG-lipids comprising achiral PEG-lipids constructed on a symmetric dihydroxyacetone scaffold, a symmetric 2-(hydroxymethyl)butane-1,4-diol, or a symmetric glycerol scaffold are disclosed in U.S. provisional patent application No. 63 / 362,502 filed April 5, 2022, and PCT / US 2023 / 017648 (WO 2023 / 196445) filed April 5, 2023, both of which are titled 'PEG-lipids and lipid nanoparticles', and are incorporated by reference in their entirety.

[0341] The above example of a PEG-lipid is presented as methoxypolyethylene glycol, but the terminus does not necessarily have to be methoxyl. For any of the non-functionalized PEG-lipids, in an alternative embodiment, the PEG moiety of the PEG-lipid may be terminated by a methoxyl, benzyloxyl, 4-methoxybenzyloxyl, or hydroxyl group (i.e., an alcohol). The terminal hydroxyl facilitates functionalization. Methoxyl, benzyloxyl, and 4-methoxybenzyloxyl groups are advantageously provided to the PEG-lipid to be used as a component of LNP without functionalization. However, all four of these alternatives are useful as (non-functionalized) PEG-lipid components of LNP. The 4-methoxybenzyloxyl group, often used as a protecting group during the synthesis of PEG-lipids, is easily removed to produce the corresponding hydroxyl group. Thus, the 4-methoxybenzyloxyl group provides a convenient route to an alcohol when not synthesized directly. Alcohol is useful for functionalizing PEG-lipids before incorporating them into LNPs, so that a binding moiety, e.g., a fabricated F(ab') binding moiety or a humanized anti-CD8α binding moiety as disclosed herein, can be conjugated to the LNP as a targeting moiety (making it a tLNP). As used herein, the end of a PEG moiety, and similar constructs refer to the end of a PEG moiety not attached to a lipid.

[0342] PEG moiety provides a hydrophilic surface to LNPs, contributing to their stability and reducing polydispersity by inhibiting their aggregation or merging; that is, it reduces the heterogeneity of LNP dispersion. Additionally, PEG moiety can interfere with LNP binding, including binding to plasma proteins. These plasma proteins include apoE, which is known to mediate the uptake of LNPs by the liver; therefore, inhibition of binding can lead to an increased proportion of LNPs reaching other tissues. These plasma proteins also include opsonins, and inhibition of binding reduces recognition by the reticuloendothelial system. PEG moiety can also be functionalized to function as an attachment site for targeting moiety. Conjugation of a binding moiety to a PEG moiety, for example, a engineered F(ab') binding moiety as disclosed herein or a humanized anti-CD8α binding moiety, enables tLNP to bypass the liver and bind to a target tissue or cell type, thereby significantly increasing the proportion of LNP reaching the targeted tissue or cell type. Thus, PEG lipids can serve as a means to inhibit LNP binding, and PEG lipids conjugated to anti-CD8 can serve as a means for LNP targeting of CD8+ cells.

[0343] As used herein, the terms "functionalized PEG-lipids" and similar compositions generally refer to both unreacted and reactive entities. The lipid composition of the LNP may be described with reference to the reactive species even after conjugation has occurred (forming the tLNP). For example, the lipid composition may be described as comprising DSPE-PEG-maleimide and may be referred to as further comprising an anti-CD8 binding moiety or a engineered F(ab') binding moiety without explicitly noting that the maleimide will be converted to succinimide (or hydrolyzed succinimide) during the reaction to form the conjugate. Similarly, if the reactive group is bromomaleimide, it will become maleimide after conjugation. This difference in chemical nomenclature for unreacted and reactive species should be understood implicitly, even if not explicitly stated. Certain embodiments include DSG-PEG, e.g., DSG-PEG-2000. Certain embodiments include functionalized DSPE-PEG, e.g., functionalized DSPE-PEG-2000. Certain embodiments include both DSG-PEG-2000 and functionalized DSPE-PEG-2000. In some cases, the functionalized PEG-lipid is functionalized with a maleimide moiety, e.g., DSPE-PEG-2000-MAL.

[0344] In certain embodiments, the LNP comprises one or more PEG-lipids and / or functionalized PEG-lipids—where both functionalized and non-functionalized PEG-lipids are present, the PEG-lipids may be the same or different—and one or more ionizable cationic lipids; the LNP may further comprise phospholipids, sterols, auxiliary lipids, or any combination thereof. The term “functionalized PEG-lipid” refers to a PEG-lipid in which a PEG moiety is derivatized with a chemically reactive group that can be used to conjugate a targeting moiety to the PEG-lipid. The functionalized PEG-lipid may be reacted with an anti-CD8 binding moiety or a engineered F(ab') binding moiety so that an anti-CD8 binding moiety or an engineered F(ab') binding moiety is conjugated to the PEG portion of the lipid. Accordingly, the conjugated anti-CD8 binding moiety can form a tLNP by acting as a moiety that targets the LNP to CD8+ cells. In some embodiments, the anti-CD8 binding moiety or the engineered F(ab') binding moiety is conjugated to the functionalized PEG-lipid after the LNP containing the functionalized PEG-lipid is formed. In other embodiments, the anti-CD8 binding moiety or the engineered F(ab') binding moiety is conjugated to the PEG-lipid, and then the conjugate is inserted into the previously formed LNP.

[0345] In a specific embodiment, the LNP is a tLNP comprising one or more functionalized PEG lipids conjugated to an anti-CD8 binding moiety or a engineered F(ab') binding moiety. In a specific embodiment, the tLNP also comprises a PEG lipid that is not functionalized or is not conjugated to a binding moiety. In some embodiments, the functionalization is a maleimide. In some embodiments, the functionalization is a bromomaleimide or bromomaleimide amide, alkynylamide, or alkynylimide moiety located at the terminal hydroxyl end of the PEG moiety. In some embodiments, the anti-CD8 binding moiety comprises an anti-CD8α antibody or its anti-CD8α binding portion, for example, an engineered F(ab') as disclosed herein. In some embodiments, the binding moiety is a polypeptide comprising an N- or C-terminal extension comprising a binding domain and an accessible thiol group. In some embodiments, the conjugation linkage comprises a reaction product of a thiol within an anti-CD8 binding moiety or a engineered F(ab') binding moiety and a functionalized PEG-lipid. In some embodiments, the functionalization is maleimide, azide, alkyne, dibenzocyclooctine (DBCO), bromomaleimide or bromomaleimide amide, alkynylamide, or alkynylimide. In some embodiments, the anti-CD8 binding moiety comprises an anti-CD8 antibody or an anti-CD8 binding portion thereof. In some embodiments, the anti-CD8 binding moiety is a polypeptide comprising an N- or C-terminal extension comprising a binding domain and an accessible thiol group, e.g., an engineered F(ab') as disclosed herein.

[0346] In certain embodiments, the PEG-lipid and / or functionalized PEG-lipid comprises a scaffold selected from the following formulas S1, S2, S3, or S4:

[0347]

[0348]

[0349] In the above formula, represents the ester bond site with a fatty acid, and indicates a site for the formation of an ester (S1) or ether (S2, S3, S4) with a PEG moiety. In some embodiments, the fatty acid ester is C 14 -C 20 It is a straight-chain alkyl fatty acid. In some embodiments, the PEG moiety is functionalized, and the fatty acid ester is C 16 -C 20 It is a straight-chain alkyl fatty acid. For example, straight-chain alkyl fatty acids are C 14 , C 15 , C 16 , C 17 , C 18 , C 19 or C 20 is. In some embodiments, the fatty acid ester is C 14 -C 20 It is a symmetric branched-chain alkyl fatty acid. For example, branched-chain alkyl fatty acids are C 14 , C 15 , C 16 , C 17 , C 18 , C 19 or C 20 Symmetry means that each alkyl branch has the same number of carbons. In some embodiments, the branch is located at position 3, 4, 5, 6, or 7 of the fatty acid ester. The synthesis and use of PEG-lipids constructed on scaffolds S1 to S4 are disclosed in WO 2023 / 196445 A1, which is incorporated by reference to all teachings regarding PEG-lipids and their use.

[0350] Some embodiments of the disclosed ionizable cationic lipids have head groups having small (<250 Da) PEG moiety. As used herein, the term PEG-lipid does not refer to these lipids. These small PEG moiety is generally too small to hinder binding to a similar degree as the larger PEG moiety of the PEG-lipid disclosed above, but will affect the lipophilicity of the ionizable cationic lipid. Furthermore, PEG-lipids are primarily located within outward-facing lamellae, whereas most ionizable cationic lipids are understood to exist within LNPs.

[0351] In a given embodiment, the functionalized PEG-lipid of the LNP or tLNP of the present disclosure comprises one or more fatty acid tails, each of which, in the case of straight-chain fatty acids, is not shorter than C16 and not longer than C20. In the case of branched-chain fatty acids, a tail that is not shorter than C14 fatty acids and not longer than C20 is acceptable. In some embodiments, the fatty acid tail is C16. In some embodiments, the fatty acid tail is C18. In some embodiments, the functionalized PEG-lipid comprises dipalmitoyl lipid. In some embodiments, the functionalized PEG-lipid comprises distearoyl lipid. The fatty acid tail serves to anchor the PEG-lipid to the tLNP to reduce or eliminate the detachment of the PEG-lipid from the tLNP. This is a useful property whether the PEG-lipid is functionalized or not, but it is more significant in the case of functionalized PEG-lipids, because a targeting moiety will be attached to it, and if the PEG-lipid (having a conjugated anti-CD8α binding moiety, e.g., an antibody) detaches from the tLNP, the targeting action may be impaired.

[0352] In some embodiments, LNP or tLNP comprises PEG lipids comprising about 0.5 mol% to about 3 mol% or 0.5 mol% to 3 mol%, which include functionalized PEG lipids and non-functionalized PEG lipids. In certain embodiments, LNP or tLNP comprises DSG-PEG. In other embodiments, LNP or tLNP comprises DMG-PEG or DPG-PEG. In certain embodiments, LNP or tLNP comprises DSPE-PEG. In some embodiments, the functionalized PEG lipid and the non-functionalized PEG lipid are not the same PEG lipid, for example, the non-functionalized PEG lipid may be diacylglycerol and the functionalized PEG lipid may be a diacyl phospholipid. tLNP having such a mixture has reduced expression in the liver, possibly due to reduced absorption. In certain embodiments, the functionalized PEG-lipid is DSPE-PEG, and the non-functionalized PEG-lipid is DSG-PEG. In some embodiments, LNP or tLNP comprises about 0.4 mol% to about 2.9 mol% or about 0.9 mol% to about 1.4 mol% of non-functionalized PEG lipid. In certain embodiments, LNP or tLNP comprises about 1.4 mol% or 1.4 mol% of non-functionalized PEG lipid. In some embodiments, LNP or tLNP comprises about 0.1 mol% to about 0.3 mol% or 0.1 mol% to 0.3 mol% of functionalized lipid. In some cases, the functionalized lipid is DSPE-PEG. In certain cases, LNP or tLNP comprises about 0.1 mol%, about 0.2 mol%, or about 0.3 mol% of DSPE-PEG. In certain cases, LNP or tLNP comprises 0.1 mol%, 0.2 mol%, or 0.3 mol% DSPE-PEG. In certain cases, the functionalized PEG-lipid is conjugated to an anti-CD8α binding moiety or a engineered F(ab') as disclosed herein.As used herein, phrases "joined to" and similar constructions are intended to convey a state of existence, namely a structure, rather than a process, unless the context indicates otherwise.

[0353] junction

[0354] To conjugate an anti-CD8α binding moiety to the PEG of PEG-lipids, any suitable chemistry including maleimide (see [Parhiz et al., Journal of Controlled Release 291:106-115, 2018]) and cyclo (see [Kolb et al., Angewandte Chemie International Edition 40(11):2004–2021, 2001]; and [Evans, Australian Journal of Chemistry 60(6):384–395, 2007]) chemistry may be used. Reagents for this reaction include lipid-PEG-maleimide, lipid-PEG-cysteine, lipid-PEG-alkyne, lipid-PEG-dibenzocyclooctine (DBCO), and lipid-PEG-azide. Additionally, the conjugation reaction utilizes lipid-PEG-bromomaleimide, lipid-PEG-alkylnoic amide, lipid-PEG-alkynoic imide, and lipid-PEG-alkyne reactions as disclosed in PCT / US23 / 17648, titled "PEG-Lipids and Lipid Nanoparticles," which incorporates all teachings regarding conjugation chemistry and alternative PEG-lipids. On the anti-CD8α binding moiety side of the reaction, proteins may be derivatized by using conventional cysteine ​​sulfhydryl or by adding a sulfur-containing carboxylic acid, for example, to the epsilon-amino of lysine, and reacting it with maleimide, bromomaleimide (collectively referred to as "maleimide"), alkylnoic amide, or alkynoic imide. Alternatively, alkynes may be added to the sulfhydryl or epsilon-amino of lysine to participate in the click chemistry reaction.

[0355] To modify the epsilon-amino group of the anti-CD8α binding moiety lysine and react it with maleimide-functionalized PEG-lipids, the anti-CD8α binding moiety (e.g., antibody) can be reacted with N-succinimidyl S-acetylthioacetate (SATA). Then, the SATA is deprotected, for example, using 0.5 M hydroxylamine, and subsequently unreacted components are removed by a G-25 Sephadex Quick Spin Protein column (Roche Applied Science, Indianapolis, Indiana, USA). Subsequently, the reactive sulfhydryl group on the anti-CD8α binding moiety is conjugated to the maleimide moiety on the LNP of the present disclosure using thioether conjugation chemistry. Purification can be performed using a Sepharose CL-4B gel filtration column (Sigma-Aldrich). The tLNP (LNP conjugated with the targeting antibody) can be stored frozen at -80°C until needed. Others conjugated antibodies to free functioning PEG-lipids and then incorporated the conjugated lipids into pre-formed LNPs. However, it was found that incorporating functioning PEG-lipids into the LNP during LNP formation and subsequently conjugating an anti-CD8α binding moiety to the functioning PEG-lipids within the LNP produces more controllable and consistent results.

[0356] In addition, there are several approaches for site-specific conjugation. While not suitable only for truncated forms of antibodies, C-terminal extensions of natural or artificial sequences containing particularly accessible cysteine ​​residues are commonly used. For example, partial reduction of cystine bonds within antibodies using tris(2-carboxy)phosphine (TCEP) can also generate thiol groups for conjugation, which can be site-specific under defined conditions for processable antibody fragments. Potential cysteine ​​residues, particularly within F(ab'), that can be reduced by TCEP for conjugation to LNPs are shown in Table 17. Cysteine, glutathione (GSH), mercaptoethylamine (MEA), and dithiobutylamine (DTBA) can also be used for reduction instead of TCEP. The use of the latter two is [Crivianu-Gaita] et al. , Biochem Biphys Rep. It is described in [2: 23-28, (2015)]. If conditions are sufficiently controlled, β-mercaptoethanol and dithiothreitol (DTT) may also be used. Various engineered F(ab') structures disclosed herein may form F(ab')2 to at least some extent. However, they are generally referred to as F(ab') to be consistent with their use as non-dimeric molecules that are conjugated to LNPs and serve as targeting moiety for the tLNP thus formed.

[0357] Alternatively, the C-terminal extension may contain the sorbase A substrate sequence LPXTG (Sequence No. 197) (where X is any amino acid), which can then be functionalized in a reaction catalyzed by sorbase A and, for example, conjugated to PEG lipids via a click chemical reaction (e.g., [Moliner-Morro et al[Refer to ., 2020, Biomolecules 10(12):1661], which is incorporated herein by reference to everything it teaches regarding antibody conjugation mediated by sorbase A reactions and / or click chemistry). The use of click chemistry for the conjugation of targeting moiety, such as various forms of antibodies, is disclosed, for example, in WO 2024 / 102,770, which is incorporated herein by reference in its entirety to everything it teaches regarding the conjugation of targeting moiety to LNPs that does not conflict with the present disclosure.

[0358] For the whole antibody and other forms including the Fc region, site-specific conjugation to either (or both) of the two specific lysine residues (Lys248 and Lys288) can be achieved without any alteration or extension of the natural antibody sequence by using one of the AJICAP® reagents (e.g., the literature [Matsuda et al. , 2021, Molecular Pharmaceutics 18:4058-4066]; Literature[Fujii et al. , 2023, Bioconjugate ChemistryRefer to 34(4):728-738 [https: / / doi.org / 10.1021 / acs.bioconjchem.3c00040]], and WO2019 / 240287, which are incorporated herein by reference to everything they teach regarding the conjugation of antibodies and AJICAP reagents). The AJICAP reagent is a modified affinity peptide that binds to a specific locus on Fc and reacts with an adjacent lysine residue to form an affinity peptide conjugate of the antibody. The peptide is then cleaved with a base to leave a thiol-functionalized lysine residue, which can then undergo conjugation, for example, via a maleimide or haloamide reaction. Functionation using azide or dibenzocyclooctine (DBCO) is also possible for conjugation by click chemistry. These and similar technologies are further described in US20200190165 (corresponding to WO2018199337), US20210139549 (corresponding to WO2019 / 240287), and US20230248842 (corresponding to WO2020184944), which are incorporated by reference in their entirety regarding such modified affinity peptides and their uses.

[0359] The term "affinity peptide" refers to a peptide having the ability to bind specifically and high affinity for other molecules. In a specific embodiment, the affinity peptide binds to a specific locus on the Fc region of an antibody. In a specific embodiment, the affinity peptide is modified into a chemically reactive group that enables it to form a covalent bond with an adjacent amino acid residue in the antibody, for example, a specific lysine residue such as Lys248 or Lys288 in IgG1.

[0360] Accordingly, in some embodiments, the anti-CD8α binding moiety is conjugated to the PEG moiety of the PEG-lipid via a thiol-modified lysine residue. In some embodiments, conjugation is performed via a cysteine ​​residue in the natural or added antibody sequence. In such embodiments, whether using a thiol of cysteine ​​or thiolized lysine, the thiol in the antibody may be conjugated to the maleimide group of the maleimide-modified PEG-lipid in the LNP using a maleimide-thiol reaction. In other embodiments, conjugation is performed via a sorbase A substrate sequence. In yet another embodiment, conjugation is performed via a specific lysine residue (Lys248 or Lys288) in the Fc region. In certain embodiments of such embodiments, the humanized anti-CD8 antibody is linked to the LNP using N-succinimidyl S-acetylthioacetate (SATA)-maleimide conjugation chemistry to form a targeted LNP (tLNP). The antibody is first beneficially modified with SATA to introduce sulfhydryl groups to accessible lysine residues, thereby enabling conjugation to maleimide. (Some lysine residues may be embedded within the protein and inaccessible to SATA reagents.) Tris(2-carboxy)phosphine (TCEP) is used to partially reduce cysteine ​​bonds within the antibody first, thereby generating thiol groups for conjugation through the maleimide moiety of the LNP, which can then conjugate the diabody to F(ab')2.

[0361] nucleic acid molecules

[0362] In a given embodiment, the disclosed LNP and tLNP comprise one or more species of nucleic acid molecules or comprise a payload made thereof. In some embodiments, the LNP or tLNP payload comprises only one nucleic acid species, whereas in other embodiments, the LNP or tLNP payload comprises multiple nucleic acid species, for example, two, three, or four nucleic acid species. For example, in an embodiment in which the payload comprises a nucleic acid encoding a CAR or an immune cell engager (ICE), the payload may comprise or be composed of the following: 1) a single nucleic acid species encoding a single species of CAR or ICE; 2) a single nucleic acid species encoding two or more species of CAR or ICE (or a mixture of CAR and ICE), e.g., a tracer antigen; 3) a single nucleic acid species encoding two or more species of CAR or ICE (or a mixture of CAR and ICE), e.g., bicistronic or multicistronic mRNA in which at least one CAR and / or ICE is specific to a tracer antigen different from the other(s); 4) two or more nucleic acid species encoding two or more species of CAR or ICE (or a mixture of CAR and ICE), each having specificity to the same tracer antigen; or 5) two or more species in which at least one CAR and / or ICE is specific to a target antigen different from the other(s). Two or more nucleic acid species encoding a CAR or ICE (or a mixture of CAR and ICE). Where two or more CARs and / or ICEs have specificity for the same target antigen, they may have specificity for the same or different epitopes of the same trace antigen. Further modifications will be obvious to those skilled in the art (e.g., multiple bicystonic or multicystonic nucleic acids, nucleic acids encoding a TCR, etc.). The nucleic acid may be RNA or DNA. The nucleic acid may be multicystonic, e.g., bicystonic.

[0363] In some embodiments, the nucleic acid molecule is mRNA, self-replicating RNA, circular RNA, siRNA, miRNA, DNA, a gene editing component (e.g., guide RNA, tracr RNA, sgRNA), a gene writing component, mRNA encoding a gene or base editing protein, zinc-finger nuclease, TALEN, CRISPR nuclease, e.g., Cas9, a DNA molecule to serve as a template for insertion or repair), or a combination thereof. In some embodiments, the nucleic acid comprises small interfering RNA (siRNA), microRNA (miRNA), or antisense oligonucleotides (ASO). In some embodiments, the nucleic acid comprises self-replicating RNA or circular RNA. In some embodiments, the mRNA encodes a reprogramming agent, or includes or encodes a conditioning agent. In some embodiments, the mRNA (linear, circular, or self-replicating) includes a miRNA binding site. In some embodiments, the mRNA encodes a chimeric antigen receptor (CAR). In other embodiments, the mRNA encodes a gene editing or base editing or gene writing protein. In some embodiments, the nucleic acid is a guide RNA. In some embodiments, the LNP or tLNP comprises both the gene-editing or base-editing or gene-recording protein-encoding mRNA and one or more guide RNAs. The activity of the CRISPR nuclease may be altered, for example, by modifying the nuclease to become a nicase instead of inducing a double-strand break, or to bind to the sequence specified by the guide RNA but without enzymatic activity. The base-editing protein is often a fusion protein comprising a deaminase domain and a sequence-specific DNA binding domain (e.g., an inactive CRISPR nuclease).

[0364] In some embodiments, the reprogramming agent comprises an immune receptor (e.g., a chimeric antigen receptor or a T cell receptor) or an immune cell engager (e.g., a bispecific T cell engager (BiTE), a bispecific killer cell engager (BiKE), a trispecific killer cell engager (TriKE), a biaffinity retargeting antibody (DART), TRIDENT (linking two DART units or a DART unit and a Fab domain), a macrophage engager (e.g., BiME), an innate immune cell engager, etc.).

[0365] In some embodiments, the nucleic acid is RNA, e.g., mRNA, and the RNA comprises one or more modified nucleosides. In some embodiments, the modified nucleosides are pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methyluridine, N6-methyladenosine, 2'-O-methyluridine, or 2-thiouridine. In certain embodiments, all uridines are substituted with modified nucleosides. Further disclosures regarding modified nucleosides and their uses can be found in U.S. Patent No. 8,278,036, which is incorporated herein by reference to these teachings.

[0366] In some embodiments, the reprogramming agent is a gene / genome editing component or encodes it. In some embodiments, the gene / genome editing component is a guide RNA for an RNA-induced nuclease or other nucleic acid editing enzyme, a clustered regularly spaced short palindromic repeat RNA (crisprRNA), or a trans-activated clustered regularly spaced short palindromic repeat RNA (tracrRNA). In some embodiments, the gene / genome editing component is a nucleic acid-encoded enzyme, such as an RNA-induced nuclease, a gene or base editing protein, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a transposase, or a CRISPR nuclease (e.g., Cas9 or Cas12, etc.). In some embodiments, the gene / genome editing component is DNA to be inserted or DNA acting as a template in gene or genome editing, for example, as a template for repairing double-strand breaks.

[0367] In some embodiments comprising multiple agents, the nucleic acid may be multicystronic. In other embodiments comprising multiple agents or components, each agent or component is encoded or contained in a separate nucleic acid species. In some embodiments comprising multiple payload nucleic acid species, two or more nucleic acid species are packaged together in a single LNP species. In other embodiments, a subset of the payload nucleic acid species to be delivered (e.g., a single nucleic acid species) is packaged in one LNP or tLNP species, while another subset of the nucleic acid species is packaged in a different LNP or tLNP species. Different (t)LNP species may differ only in the payload they contain. Different (t)LNP species may be combined into a single formulation or pharmaceutical composition for administration.

[0368] In any of the embodiments described above, the nucleic acid molecule payload of the LNP or tLNP of the present disclosure is a specific antigen, e.g., a B cell maturation agent (BCMA) †‡ , CA9 †‡ , CD1, CD2* †‡ , CD3* †‡ , CD4* †‡ , CD5 †‡ , CD7 †‡ , CD11b ‡ , CD14 †‡ , CD16, CD19* †‡ , CD20(MS4A1)* †‡ , CD22* †‡ , CD23* †‡ , CD25 †‡ , CD26* ‡ , CD27* †‡ , CD28* †‡ , CD30(TNFRSF8)* †‡ , CD32*, CD33* †‡ , CD38* †‡ , CD39 ‡ , CD40* †‡ , CD40L(CD154)* †‡ , CD44* ‡ , CD45 †‡ , CD45RA, CD56(NCAM1)* †‡ , CD64* ‡ , CD62 †‡ , CD68, CD69 ‡ , CD70* †‡ , CD73 †‡ , CD80* ‡ , CD83 ‡ , CD86* ‡ , CD95 ‡ , CD103 ‡ , CD119 ‡ , CD126 ‡ , CD133 ‡ , CD137(41BB) †‡ , CD138(SDC1)* ‡ , CD150 ‡ , CD153 ‡ , CD161‡ , CD166 ‡ , CD174, CD183(CXCR3) ‡ , CD185(CXCR5) ‡ , CD223(LAG-3)* †‡ , CD254 ‡ , CD267(TACI) ‡ , CD274(PD-L1)* †‡ , CD275 ‡ , CD276(B7-H3) †‡ , ADAM12 ‡ , CTLA-4* † * † , DEC205, OX40 † , PD-1* †‡ , GITR † , TIM-3* †‡ , FasL* ‡ , IL18R1, ICOS(CD278) ‡ , leu-12, TCR † , TLR1, TLR2 †‡ , TLR3* ‡ , TLR4 †‡ , TLR6, TREM2 ‡ , NKG2D ‡ , CCR, CCR1(CD191) ‡ , CCR2(CD192)* †‡ , CCR4(CD194)* †‡ , CCR6(CD196) ‡ , CCR7 ‡ , low-affinity IL-2 receptor †‡ , IL-7 receptor ‡ , IL-12 receptor ‡ , IL-15 receptor ‡ , IL-18 receptor ‡ , and IL-21 receptors ‡ CEACAM5* †‡ , CLL1 ‡ , CSPG4* ‡ , Kappa*, Lambda*, FCRL5 †‡ , GPRC5D †‡ , CTSK, PD-1(CD279) †‡, CD319(SLAMF7)* †‡ , CD248(TEM1) ‡ , ULBP1, ULBP2; CD319(SLAMF7)* †‡ , GPRC5D †‡ , Claudine 6 (CLDN6), Claudine 18.2 (CLDN18.2), GD2* †‡ , HER2* †‡ , ITGA11, EGFR* †‡ , EGFRvIII*, CD276(B7H3) †‡ , PSMA* †‡ , PSCA ‡ , CAIX(CA9) †‡ , CD171(L1-CAM)* ‡ , CEA* ‡ , CSPG4* ‡ , DLL3, EPHA2* ‡ , FAP* †‡ , LRRC15 †‡ , FOLR1* †‡ , IL-13Rα* †‡ , mesothelin (MSLN)* †‡ , MUC1* †‡ , MUC16* †‡ , Nectin-4 †‡ , NOX4, SGCD, SYNDIG1, ​​CDH11 ‡ , PLPP4, SLC24A2, PDGFRB* ‡ , THY1 ‡ , ANTXR1 ‡ , GAS1, CALHM5, SDC1EPCAM* †‡ , ERBB2* ‡ , FOLH1, GPC3* †‡ , GPNMB* ‡ , IL1RAP †‡ , IL3RA* ‡ , IL13RA2(IL13Rα2)* ‡ , KDR(VEGFR2)* ‡ , CD171(L1CAM)* ‡ , MET* ‡ , TROP2* †‡ , and ROR1†‡ Encodes a CAR, TCR, or ICE specific to.

[0369] Tolerance

[0370] Conventional LNPs are primarily delivered to the liver. Hepatotoxicity has been a major dose-limiting parameter observed with LNP-containing agents. For example, ONPATTRO®, containing the ionizable lipid MC3, has a NOAEL (No Adverse Effect Level) of only 0.3 mg / kg for multiple administrations in rats. The benchmark LNP, containing the ionizable cationic lipid ALC-0315 used in the SARS-CoV-2 vaccine COMIRNATY®, elevates levels of liver enzymes and acute-phase proteins in rats at a single dose of ≥1 mg / kg. Attaching an antibody to the benchmark LNP alone partially reverses this elevation, and the reversal is greater when the antibody directs the LNP to some other tissue (i.e., tLNP). However, the use of CICL-1, a highly biodegradable ionizable cationic lipid (its catabolism should be similar to that disclosed herein), reduced the delivery to the liver and associated liver enzyme and acute phase protein levels of LNP, antibody-conjugated LNP, and tLNP to a greater extent.

[0371] Method for manufacturing LNP or tLNP

[0372] In some embodiments, the present disclosure provides a method for preparing LNP or tLNP, comprising the step of mixing an aqueous solution of nucleic acid (or other negatively charged payload) and an alcoholic solution of lipids in the ratios disclosed herein. In certain embodiments, the mixing is rapid.

[0373] The aqueous solution is buffered with citrate or acetate, for example, without limitation, at a pH of about 3 to about 5. In various embodiments, the alcohol may be ethanol, isopropanol, t-butanol, or a combination thereof. In some embodiments, rapid mixing is achieved by pumping the two solutions through a T-junction or using an impingement jet mixer. Microfluidic mixing through a staggered herringbone mixer (SHM) or a hydrodynamic mixer (hydrodynamic focusing), a microfluidic branch mixer, and a microfluidic baffle mixer may also be used. After the LNPs are formed, they are diluted with a buffer, for example, phosphate, HEPES, or Tris, in a pH range of 6 to 8.5 to reduce the alcohol (ethanol) concentration. The diluted LNP is purified by dialysis or ultrafiltration or dialysis filtration using tangential flow filtration (TFF) on a buffer (e.g., phosphate, HEPES, or Tris) in a pH range of 6 to 8.5 to remove alcohol. Alternatively, size exclusion chromatography may be used. Once the alcohol is completely removed, the buffer is replaced with the same buffer containing a cryoprotectant (e.g., glycerol or a sugar, such as sucrose, trehalose, or mannose). The LNP is concentrated to a desired concentration, subsequently filtered through a 0.2 μm filter, for example, polyethersulfone (PES) or a modified PES filter, filled into a glass vial, plugged, capped, and frozen for storage. In an alternative embodiment, a cryoprotectant is used, and the LNP is freeze-dried for storage instead of being frozen liquid. Further methodologies for manufacturing LNPs can be found, for example, in U.S. Patent Application Publication No. US 2020 / 0297634, US 2013 / 0115274, and International Patent Application Publication No. WO 2017 / 048770, each of which is incorporated by reference for all that they teach regarding the generation of LNPs.

[0374] Some embodiments are methods for preparing tLNPs comprising the step of rapidly mixing an aqueous solution of nucleic acid (or other negatively charged payload) with an alcoholic solution of lipids as disclosed for LNPs. In some embodiments, the lipid mixture comprises functionalized PEG-lipids for subsequent conjugation to a targeting moiety. As used herein, functionalized PEG-lipids refer to PEG-lipids in which the PEG moiety is derivatized with chemically reactive groups (e.g., maleimide, N-hydroxysuccinimide (NHS) ester, Cys, azide, alkyne, etc.) that can be used to conjugate a targeting moiety to a PEG-lipid and, accordingly, to an LNP containing PEG-lipids. In other embodiments, the functionalized PEG-lipids are inserted into the LNP following the initial formation of the LNP from other components. In any type of embodiment, the targeting moiety is conjugated to the functionalized PEG-lipids after the functionalized PEG-lipid-containing LNP has been formed. Protocols for conjugation can be found, for example, in the literature [Parhiz et al. 2018, J. Controlled Release 291:106-115] and [Tombacz et al., 2021, Molecular Therapy 29(11):3293-3304], each of which is included for reference regarding everything it teaches about the conjugation of PEG-lipids to binding moiety. Alternatively, the targeting moiety can be conjugated to PEG-lipids before being inserted into a pre-formed LNP.

[0375] In a specific embodiment of the tLNP manufacturing method, the method comprises the following:

[0376] i) a step of forming an initial LNP by mixing all components of tLNP in the proportions disclosed herein, excluding one or more functionalized PEG-lipids and one or more targeting moiety;

[0377] ii) a step of mixing an initial LNP with one or more functionalized PEG-lipids to form a pre-conjugated tLNP; and

[0378] iii) A step of forming a tLNP by conjugating a pre-conjugated tLNP with one or more targeting moiety.

[0379] In a specific embodiment of the tLNP manufacturing method, the method comprises the following:

[0380] i) a step of forming a pre-conjugated tLNP by mixing all components of the tLNP in the proportions disclosed herein, excluding one or more targeting moiety and including one or more functionalized PEG-lipids; and

[0381] ii) a step of forming a tLNP by conjugating the pre-conjugated tLNP with one or more targeting moiety.

[0382] In a specific embodiment of the tLNP manufacturing method, the method comprises the following:

[0383] i) forming one or more conjugated functionalized PEG-lipids by conjugating one or more functionalized PEG-lipids with one or more targeting moiety; and

[0384] ii) a step of forming tLNP by mixing all components of tLNP in the proportions disclosed herein, including one or more conjugated functionalized PEG-lipids.

[0385] In a specific embodiment of the tLNP manufacturing method, the method comprises the following:

[0386] i) a step of forming one or more conjugated functionalized PEG-lipids by conjugating one or more functionalized PEG-lipids with one or more targeting moiety;

[0387] ii) a step of forming an LNP by mixing all components of the tLNP, excluding one or more conjugated functionalized PEG-lipids; and

[0388] iii) A step of forming tLNP by mixing the initial LNP with one or more conjugated functionalized PEG-lipids.

[0389] After conjugation, tLNP is purified and stored by dialysis, tangential flow filtration, or size exclusion chromatography as disclosed above for LNP.

[0390] The encapsulation efficiency of nucleic acids by LNP or tLNP is typically determined by using a nucleic acid-binding fluorescent dye added to the intact aliquot and dissolved aliquot of the final LNP or tLNP formulation to determine the amounts of unencapsulated nucleic acids and total nucleic acids, respectively. The encapsulation efficiency is typically expressed as a percentage and calculated as 100 × (TU) / T, where T is the total amount of nucleic acids and U is the amount of unencapsulated nucleic acids. In various embodiments, the encapsulation efficiency is 80% or more, 85% or more, 90% or more, or 95% or more.

[0391] Anti-CD8 tLNP

[0392] The present disclosure considers any of the aforementioned embodiments of an anti-CD8α binder conjugated to a tLNP composition disclosed herein. For example, in a given embodiment, the targeted lipid nanoparticle (tLNP) comprises (a) a lipid formulation (e.g., any of those listed in Table 14) comprising an ionizable cationic lipid (e.g., CICL of the present disclosure or a variant thereof), a phospholipid, a sterol, a functionalized PEG-lipid, and a non-functionalized PEG-lipid, and (b) a humanized anti-CD8α antibody or an antigen-binding fragment thereof conjugated to the lipid, wherein the humanized anti-CD8α antibody or the antigen-binding fragment thereof comprises (a)(i) VH-CDR1 comprising the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprising the amino acid sequence of SEQ ID NO. 3, and VH-CDR3 comprising the amino acid sequence of SEQ ID NO. 4; (ii) VH-CDR1 containing the amino acid sequence of sequence number 2, VH-CDR2 containing the amino acid sequence of sequence number 58, and VH-CDR3 containing the amino acid sequence of sequence number 4; (iii) VH-CDR1 containing the amino acid sequence of sequence number 2, VH-CDR2 containing the amino acid sequence of sequence number 59, and VH-CDR3 containing the amino acid sequence of sequence number 4; or (iv) VH-CDR1 containing the amino acid sequence of sequence number 2, VH-CDR2 containing the amino acid sequence of sequence number 60, and VH-CDR3 containing the amino acid sequence of sequence number 4; and (b) VL-CDR1 containing the amino acid sequence of sequence number 6, VL-CDR2 containing the amino acid sequence of sequence number 7; and VL-CDR3 containing the amino acid sequence of sequence number 8.

[0393] In a further embodiment, the targeted lipid nanoparticle (tLNP) comprises (a) a lipid formulation (e.g., any of those listed in Table 14) comprising an ionizable cationic lipid (e.g., CICL of the present disclosure or a variant thereof), a phospholipid, a sterol, a functionalized PEG-lipid, and a non-functionalized PEG-lipid, and (b) an anti-CD8α antibody or an antigen-binding fragment thereof conjugated to the lipid, wherein the anti-CD8α antibody or the antigen-binding fragment thereof comprises a heavy chain variable region (VH) having an amino acid sequence that is at least 90% and up to 100% identical to the amino acid sequence of SEQ ID NO. 10, 11, 12, 13, 14, 27, 28, 29, 35, or 36 (wherein VH-CDR1 comprises the amino acid sequence of SEQ ID NO. 2, and VH-CDR2 comprises the amino acid sequence of SEQ ID NO. 3, 58, 59, or 60). It includes, wherein VH-CDR3 includes the amino acid sequence of SEQ ID NO. 4); and a light chain variable region (VL) that includes an amino acid sequence identical to at least 90% and up to 100% of the amino acid sequence of SEQ ID NO. 16, 17, 18, or 39 (wherein VL-CDR1 includes the amino acid sequence of SEQ ID NO. 6, VL-CDR2 includes the amino acid sequence of SEQ ID NO. 7, and VL-CDR3 includes the amino acid sequence of SEQ ID NO. 8).

[0394] As used herein, “LNP formulation” or “tLNP formulation” refers to each complete composition (e.g., all lipids constituting the LNP or all lipids together with the targeting moiety constituting the tLNP, each optionally comprising a payload such as a nucleic acid molecule) and further comprises a buffer, a carrier, a solvent, or other excipient. In some embodiments, the humanized anti-CD8α antibody of the present disclosure or its antigen-binding fragment, or the targeted LNP (tLNP) of the present disclosure, is conjugated to such anti-CD8α antibody or antigen-binding fragment, and such anti-CD8α conjugate or CD8-targeted tLNP may be formulated with a pharmaceutically acceptable carrier, excipient, or stabilizer as a composition or pharmaceutical composition.

[0395] In certain embodiments, such compositions are suitable for administration to humans or non-human animals via one or more routes of administration using methods known in the art. The term “pharmaceuticalally acceptable carrier” means one or more non-toxic materials that do not interfere with the efficacy of the biological activity of the active ingredient. Such formulations may routinely contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. Such pharmaceutically acceptable formulations may also contain compatible solid or liquid fillers, diluents, or encapsulating materials suitable for administration to humans. Other carriers, excipients, and / or additives considered for use in the formulations described herein include, for example, antimicrobial agents, antioxidants, antistatic agents, lipids, protein excipients such as serum albumin, gelatin, casein, salt-forming counterions such as sodium, etc. These and additional pharmaceutical carriers, excipients, and / or additives suitable for use in the formulations described herein are known in the art, for example, as listed in the literature ["Remington: The Science & Practice of Pharmacy," 23rd ed., Lippincott Williams & Wilkins, (2005)] and the literature ["Physician's Desk Reference," 71st ed., Medical Economics, Montvale, NJ (2005)]. Pharmaceutically acceptable carriers may be selected to suit the desired or required mode of administration, solubility, and / or stability.

[0396] In some embodiments, the humanized anti-CD8α antibody or its antigen-binding fragment disclosed herein may be delivered via various routes of administration, such as intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or epidural administration. Administration may be local or systemic. The mode of administration may be at the discretion of the operator and depends in part on the site of the medical condition. In most cases, administration causes the release into the bloodstream of the humanized anti-CD8α antibody or polypeptide containing its antigen-binding domain as described herein.

[0397] In other embodiments, CD8-targeted tLNP conjugated to the humanized anti-CD8α antibody antigen-binding fragment of the present disclosure is administered parenterally, for example, by intravenous infusion. Other embodiments use other routes of administration, including subcutaneous, intraperitoneal, intranodal, and intratumoral. In most cases, administration causes the binding of tLNP to CD8-positive cells (e.g., T cells) and the release of a payload (e.g., a nucleic acid molecule such as RNA) encapsulated by tLNP into the cell.

[0398] Method for delivering payloads into cells using anti-CD8 tLNP

[0399] In some embodiments, a method for delivering nucleic acid (or other negatively charged payload) into CD8+ expressing cells (CD8+ cells) is disclosed herein, comprising the step of contacting CD8+ cells with a CD8-targeted tLNP of any of the aforementioned embodiments. Various embodiments of the method for delivering the payload to CD8+ cells are limited to one or the other. Each of the various genera, subgenuses, and / or species of the LNP or tLNP disclosed herein, including those based on specific lipids, specific lipid compositions, and / or the inclusion or exclusion of specific payloads, may be used to define the category of the method for delivering the payload to CD8+ cells. In some embodiments, contact is performed in vitro. In some embodiments, contact is performed in vivo. In some cases, in vivo contact involves intravenous, intramuscular, subcutaneous, intranodal, or lymphatic administration. In additional cases, transfection of hepatocytes is reduced compared to tLNPs containing conventional ionizable cationic lipids, such as ALC-0315 (Table 14). In some embodiments, LNP or tLNP is administered 1 to 3 times per week for 1, 2, 3, or 4 weeks. In some embodiments, toxicity is limited (or largely limited) to Grade 0, Grade 1, or Grade 2 as discussed above.

[0400] The LNP and tLNP compositions and formulations disclosed herein have reduced toxicity compared to widely used conventional LNP compositions, such as those containing ALC-0315. In various embodiments, toxicity may be described as observable toxicity, substantial toxicity, severe toxicity, or acceptable toxicity, or dose-limiting toxicity (e.g., the maximum tolerated dose (MTD) without limitation). Observable toxicity means that changes are observed but the effects are negligible or mild. Substantial toxicity means that there is a negative impact on the patient's overall health or quality of life. In some cases, substantial toxicity may be mitigated or resolved by other ongoing medical interventions. Severe toxicity means that the effects require acute medical intervention and / or dose reduction or discontinuation of treatment. The acceptability of toxicity will be influenced by the specific disease being treated and its severity, and the availability of palliative medical interventions. In some embodiments, toxicity is limited (or largely limited) to observable toxicity. In some embodiments, toxicity is limited (or largely limited) to Grade 0, Grade 1, or Grade 2.

[0401] In some embodiments, the payload is a nucleic acid, and the delivery method is a method of transfecting CD8+ cells. In some embodiments, the nucleic acid payload comprises mRNA, circular RNA, self-amplifying RNA, or guide RNA. Nucleic acid structures suitable for delivery by LNP or tLNP, and in particular mRNA structures as well as individual RNA molecules encoding specific polypeptides, are disclosed in U.S. Patent Application No. 18 / 934,237 (Agent Administration No. 23-1871-US), filed November 1, 2024, each of which is incorporated by reference to all that it teaches regarding nucleic acid payloads for in vivo transfection and their design.

[0402] In some embodiments, the payload comprises a nucleic acid encoding an immune receptor or an immune cell engager, and the delivery method is also a method for reprogramming immune cells to express a CD8+ surface molecule. In some embodiments, the payload is a BRM or comprises a nucleic acid encoding a BRM, and the delivery method is also a method for providing a conditioning agent. In various embodiments, the BRM or conditioning agent is a gamma chain receptor cytokine, e.g., IL-2, IL-7, IL-15, IL-15 / 15R-alpha, IL-21; an immunomodulatory cytokine, e.g., IL-12, IL-18; a chemokine, e.g., RANTES, IP10, MIG; or another BRM, e.g., Flt3, GM-CSF, and G-CSF.

[0403] In some embodiments, the payload comprises a nucleic acid encoding a gene / genome editing enzyme and / or a guide RNA or other components of a gene / genome editing system, and the delivery method is also a method for reprogramming a cell. In some cases, the cell is an immune cell expressing a CD8+ surface molecule. In some cases, the cell is a hematopoietic stem cell (HSC). In some cases, the cell is a mesenchymal stem cell (MSC). In a given embodiment comprising the step of delivering the payload into an immune cell, the anti-CD8 binding moiety binds to the lymphocyte CD8+ surface molecule.

[0404] In a predetermined embodiment comprising the step of delivering a payload into an immune cell, the anti-CD8 tLNP binds to a CD8+ expressing lymphocyte.

[0405] Treatment methods

[0406] The anti-CD8 binder and tLNP conjugated to such anti-CD8 binder of the present disclosure are useful for treating diseases (e.g., CBD1032, CBD1033, CBD1035, CBD1037, CBD1039, CBD1047, CBD1049, etc., and conjugates of such binder to LNP). Such anti-CD8 binder and CD8-specific tLNP disclosed herein provide a targeted approach for drug delivery strategies. Accordingly, a specific embodiment provides a method for treating a disease (or its symptoms), comprising the step of administering a therapeutically effective amount of the anti-CD8 binder or CD8-specific tLNP or a composition containing the same to a mammal (e.g., human) requiring treatment of the disease (or its symptoms).

[0407] "Treat" and / or "treating" refers to any indication of successful treatment or improvement of a disease or condition. Treatment may include, for example, reducing, delaying, or alleviating the severity of one or more symptoms of a disease or condition, or it may include reducing the frequency with which a patient experiences symptoms of a disease, defect, disability, or harmful condition. The term "treatment" may be used herein to refer to a method that brings about some level of treatment or improvement of a disease or condition, and may consider various outcomes aimed at such goals, including the complete prevention of the condition.

[0408] Terms such as "prevent" and "preventing" refer to the prevention of a disease or pathological condition in a patient, e.g., the production of autoimmune antibodies. For example, if an individual at risk of developing an autoimmune flare-up or other related symptoms is treated by the method of the present disclosure and subsequently no autoimmune-related flare-up or other related symptoms occur, the disease is prevented in that individual for at least a predetermined period. Prevention may also refer to preventing the recurrence of a disease or pathological condition in a patient who has previously been treated for the disease or pathological condition, e.g., by preventing a recurrence.

[0409] The therapeutically effective dose (also referred to as the effective dose) may be an amount of a composition containing an anti-CD8 binder or a CD8-specific tLNP sufficient to provide a beneficial effect to the individual to whom the composition is administered or to otherwise reduce harmful non-beneficial events. The therapeutically effective dose may be a dose that produces one or more desired or desirable (e.g., beneficial) effects when administered, and such administration is made one or more times over a given period. The exact dose may vary depending on the purpose of treatment and can be determined by a person skilled in the art using known techniques and the teachings provided herein.

[0410] The anti-CD8 binder or CD8-specific tLNP of the present disclosure that may be used in therapy may be formulated and dosage established in a manner consistent with good medical practice, taking into account the disease or condition to be treated, the individual patient's condition, the delivery site of the composition, the method of administration, and other factors known to the operator. The composition may be prepared according to the manufacturing description set forth herein.

[0411] The composition may be used in the method described herein and may be administered to a subject in need using techniques known to those skilled in the art that may be suitable as a therapy for a disease or condition affecting the subject. Those skilled in the art will understand that the amount, duration, and frequency of administration of the pharmaceutical composition to a subject in need may depend on several factors, including, for example, the subject's health, the patient's specific disease or condition, the grade or level of the patient's specific disease or condition, and any additional treatments the subject is or has received.

[0412] The anti-CD8 binder or CD8-specific tLNP, composition, and method of the present disclosure are useful as a single agent for the treatment or prevention of diseases, such as autoimmune disorders (e.g., idiopathic inflammatory myopathy such as antisynthetase syndrome) and cancer. Alternatively, the anti-CD8 binder or CD8-specific tLNP, composition, and method of the present disclosure may be used in combination therapy with a second therapeutic agent for the treatment or prevention of diseases such as autoimmune disorders and cancer.

[0413] In some embodiments, the present disclosure provides a method for treating a disease or disorder, comprising the step of administering the anti-CD8 binder or CD8-specific tLNP of the present disclosure to a subject who requires treatment of the disease or disorder. In some embodiments, the subject is a human. In some embodiments, the antibody or tLNP of the present disclosure is administered systemically. In some embodiments, the antibody or tLNP of the present disclosure is administered by intravenous or subcutaneous infusion or injection. In some embodiments, the antibody or tLNP of the present disclosure is administered topically. In some embodiments, the antibody or tLNP of the present disclosure is administered by intraperitoneal or intralesional infusion injection. Some embodiments of the LNP and tLNP disclosed herein may treat a disease or disorder as set forth in paragraphs

[0307] through

[0312] .

[0414] In some embodiments, the disease or disorder is an autoimmune disease. Examples of autoimmune diseases are, without limitation, myocarditis, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, chorea Sydnam, myasthenia gravis, systemic lupus erythematosus, fibrous pneumonia, multiple sclerosis, rheumatic fever, polysomnolitis syndrome, agranulocytosis, autoimmune hemolytic anemia, bullous pemphigus-like disease, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, spinal cord, giant cell arteritis / polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, IgA nephropathy, polyarteritis nodosum, ankylosing spondylitis, allergic reactions, insulin-resistant diabetes mellitus, psoriasis, diabetes mellitus, Addison's disease, Graves' disease, endometriosis, celiac disease, Crohn's disease, Henoch-Schönlein purpura, ulcerative colitis, Goodpasture Includes syndrome, ductal thromboangiitis, Sjögren's syndrome, aplastic anemia, rheumatoid arthritis, sarcoidosis, scleritis, T cell-mediated or B cell-mediated autoimmunity, B cell-mediated (antibody-mediated) autoimmune disease, necrotizing myopathy, chronic inflammatory demyelinating polyneuropathy (CIDP), nerve root myositis (NMO) myositis, nerve root myositis spectrum disorder, pemphigus vulgaris, systemic sclerosis, antisynthetic enzyme syndrome (idiopathic inflammatory myopathy), lupus nephritis, membranous nephropathy, Fanconi anemia, and vasculitis.

[0415] In some embodiments, the autoimmune disease is T cell-mediated autoimmunity or B cell-mediated autoimmunity. In some cases, B-cell-mediated autoimmune diseases include myositis (e.g., anti-synthetase myositis), lupus nephritis, membranous nephropathy, systemic lupus erythematosus, anti-neutrophil cytoplasmic antibody (ANCA) vasculitis, nerve root canal syndrome spectrum disorder (NMOSD), myasthenia gravis, pemphigus vulgaris, rheumatoid arthritis, dermatomyositis, immune-mediated necrotizing myopathy (IMNM), anti-synthetase syndrome, polymyositis, systemic sclerosis, extensive cutaneous systemic sclerosis, limited cutaneous systemic sclerosis, anti-synthetase syndrome (idiopathic inflammatory myopathy), rigid human syndrome, myeloid oligodendrocyte glycoprotein autoantibody-associated disease (MOGAD), multiple sclerosis, relapsing-remitting multiple sclerosis, secondary progressive multiple sclerosis, primary progressive multiple sclerosis, non-active secondary progressive multiple sclerosis, Sjögren's syndrome, IgA nephropathy, It is an IgG4-related disease, or Fanconi anemia. In certain embodiments, the B cell-mediated autoimmune disease is myositis, lupus nephritis, membranous neuropathy, scleroderma, systemic lupus erythematosus, myasthenia gravis, ANCA vasculitis, multiple sclerosis, or pemphigus vulgaris. In certain embodiments, the B cell-mediated autoimmune disease is myositis, lupus nephritis, membranous neuropathy, or scleroderma. In certain embodiments, the B cell-mediated autoimmune disease is myositis. In some cases, the myositis is anti-synthetase myositis. In certain embodiments, the B cell-mediated autoimmune disease is systemic lupus erythematosus, myasthenia gravis, ANCA vasculitis, multiple sclerosis, or pemphigus vulgaris.

[0416] In some embodiments, the disease or disorder is rejection of an allogeneic organ or tissue graft. Existing antibodies and / or B cells, in their role as antigen-presenting cells, can promote rapid immune rejection through known mechanisms, and thus depleting a large number of B cells may help prevent allogeneic rejection.

[0417] In some embodiments, the disease or disorder is cancer. Examples of cancer include, but are not limited to, carcinomas, sarcomas, and blood cancers. In some embodiments, the blood cancer is lymphoma, leukemia, or myeloma. In some cases, the blood cancer is a B-line or T-line cancer. In some cases, the B-line cancer is multiple myeloma, diffuse large B-cell lymphoma, acute myeloid leukemia, mantle cell lymphoma, follicular lymphoma, acute lymphoblastic leukemia B, chronic lymphocytic leukemia, or myelodysplastic syndrome. In some embodiments, the cancer is a sarcoma. In some embodiments, the cancer is a carcinoma, such as breast cancer, colon cancer, ovarian cancer, lung cancer, testicular cancer, or pancreatic cancer. In some embodiments, the cancer is melanoma.

[0418] In some embodiments, the disease or disorder is a genetic disease or disorder, e.g., a monogenetic disease. In some cases, the genetic disease or disorder is a hemoglobinopathy, e.g., sickle cell disease or β-thalassemia.

[0419] In some embodiments, the disease or disorder is a fibrotic disease or disorder. In some cases, the fibrotic disease is cardiac fibrosis, arthritis, idiopathic pulmonary fibrosis, and non-alcoholic steatohepatitis (also known as metabolic dysfunction-associated steatohepatitis). In other cases, the disorder is associated with tumor-associated fibroblasts.

[0420] A treatment method for tLNP comprising a nucleic acid encoding a chimeric antigen receptor (CAR).

[0421] In some embodiments, the tLNP of the present disclosure comprises a nucleic acid encoding a chimeric antigen receptor (CAR). The receptor is chimeric because it combines both antigen-binding and T cell-activating functions into a single receptor. There are five generally recognized generations of CARs. The “first generation” CAR for use in the present invention comprises an antigen-binding domain, e.g., a single-chain variable fragment (scFv) or VHH, fused to a transmembrane domain, which is fused to a cytoplasmic / intracellular domain of the T cell receptor chain. The “first generation” CAR typically has an intracellular signaling (or activation) domain from the CD3ζ chain, which is the primary messenger of signals from the endogenous T cell receptor (TCR). The “first generation” CAR provides neoantigen recognition and can induce activation of both CD4+ and CD8+ T cells via the CD3ζ chain signaling domain in the single fusion molecule, independent of HLA-mediated antigen presentation. The use of an intracellular signaling domain of CD3ζ in which one or two of the three ITAM motifs are destroyed can regulate the balance between effectors and memory programs (Reference [Feucht et al., 2019 Nat Med 25(1):82-88]). An intracellular signaling domain of FcyRIIIA (CD16A), a low-affinity receptor for CD3ε or IgG, can be used as a substitute for CD3ζ. In some embodiments, the intracellular signaling domain of CD3ε comprises the sequence KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (Sequence No. 115). In some embodiments, the intracellular signaling domain of FcγRIIIA (CD16A) comprises the sequence of FcγRIIIA: KTNIRSSTRDWKDHKFKWRKDPQDK (Sequence No. 116). In some embodiments, these intracellular signaling domains constitute means for signaling or means for activation.

[0422] The "second-generation" CAR for use in the present invention comprises an antigen-binding domain, e.g., scFv or VHH, fused to a transmembrane domain, fused to an intracellular signaling domain capable of activating T cells, and to a co-stimulatory domain designed to enhance T cell efficacy and persistence (Reference [Sadelain et al., 2013, Cancer Discov. 3:388-398]). Thus, the CAR design can combine antigen recognition and signaling, which are two actions physiologically performed by two separate complexes: the TCR heterodimer and the CD3 complex. The "second-generation" CAR includes intracellular domains from various co-stimulatory molecules, e.g., CD28, 4-1BB, ICOS, OX40, CD27, etc., in the cytoplasmic tail of the CAR to provide additional signals to the cell. "Second-generation" CARs provide both co-stimulation by, for example, CD28 or 4-1BB domains and activation by, for example, CD3ζ signaling domains. Preclinical studies have indicated that "second-generation" CARs can improve the antitumor activity of CAR-T cells. For example, the robust efficacy of "second-generation" CAR-modified T cells was demonstrated in clinical trials targeting the CD19 molecule in patients with chronic lymphoblastic leukemia (CLL) and acute lymphoblastic leukemia (ALL) (Davila et al., 2012, Oncoimmunol. 1(9):1577-1583). In some embodiments, these co-stimulation domains constitute means for co-stimulation.

[0423] "Third-generation" CARs provide multiple co-stimulations by including both CD28 and 4-1BB domains, for example, and provide activation by including a CD3ζ activation domain, for example.

[0424] "Fourth generation" CARs provide, for example, co-stimulation by CD28 or 4-1BB domains and, for example, activation by CD3ζ signaling domains in addition to constitutive or inducible chemokine components.

[0425] "Fifth generation" CARs provide, for example, co-stimulation by CD28 or 4-1BB domains and, for example, activation by CD3ζ signaling domains, constitutive or inducible chemokine components, and intracellular domains of cytokine receptors, for example, IL-2Rβ.

[0426] Further variations of the basic CAR structure and source for various domains are described in the literature [Zabel et al., Immunol Lett 2019 212:53-69], which is incorporated by reference to everything it teaches regarding the CAR structure and its functional domains to the extent that it is consistent with the present disclosure.

[0427] a) Signal peptide

[0428] In a given embodiment, the CAR may include a signal peptide at the N-terminus. Non-limiting examples of the signal peptide include CD8α signal peptide, IgK signal peptide, and granulocyte-macrophage colony-stimulating factor receptor subunit alpha (GMCSFR-α, also known as colony-stimulating factor 2 receptor subunit alpha (CSF2RA)) signal peptide, and variants thereof, the amino acid sequences of which are provided in Table 1 below.

[0429] [Table 1]

[0430]

[0431] b) Extracellular binding domain

[0432] The CAR comprises an extracellular binding domain, also referred to as a binder or binding moiety. In a specific embodiment, the extracellular binding domain may comprise one or more antibodies specific to a single tracer antigen or a plurality of tracer antigens. The antibody may be an antibody fragment, e.g., scFv, or a single-domain antibody fragment, e.g., VHH. In a specific embodiment, the scFv is a variable region of the heavy chain of the antibody (V) linked by a linker. H ) and light chain variable region (V L May include ). V H and V L is in any order, that is, V H -Linker-V L or V L -Linker-V H It may be connected to. Non-limiting examples of linkers include the Whitlow linker, (G4S)n (SEQ No. 123, where n is a positive integer, e.g., 1, 2, 3, 4, 5, 6, etc.), and variants thereof. In a given embodiment, the antigen may be an antigen that is exclusively or preferentially expressed on tumor cells, or an antigen that is characteristic of an autoimmune disease or an inflammatory disease.

[0433] An exemplary tracer antigen to which CAR, TCR, or ICE can have specificity is B cell maturation agent (BCMA) †‡ , CA9 †‡ , CD4 †‡ , CD5 †‡ , CD19* †‡ , CD20(MS4A1)* †‡ , CD22* †‡ , FCRL5 †‡ , GPRC5D †‡ , CD23* †‡ , CD30(TNFRSF8)* †‡ , CD33* †‡ , CD38* †‡ , CD44* ‡ , CD70* †‡ , CD133 ‡, CD174, CD274(PD-L1)* †‡ , CD276(B7-H3) †‡ , CEACAM5* †‡ , CLL1 ‡ , CSPG4* ‡ , Kappa*, Lambda*, NCAM1(CD56)* ‡ , PD-1(CD279) †‡ , ROR1 †‡ , CD138(SDC1)* ‡ , CD319(SLAMF7)* †‡ , CD248(TEM1) ‡ , ULBP1, and ULBP2 (associated with leukemia); CD319 (SLAMF7)* †‡ , CD38* †‡ , CD138 †‡ , GPRC5D †‡ , CD267(TACI) ‡ and BCMA †‡ (associated with multiple myeloma); and Claudin 6 (CLDN6), Claudin 18.2 (CLDN18.2), GD2* †‡ , HER2* †‡ , EGFR* †‡ , EGFRvIII*, CD276(B7H3) †‡ , PSMA* †‡ , PSCA ‡ , CAIX(CA9) †‡ , CD171(L1-CAM)* ‡ , CEA* ‡ , CSPG4* ‡ , DLL3, EPHA2* ‡ , FAP* †‡ , LRRC15 †‡ , FOLR1* †‡ , IL-13Rα* †‡ , mesothelin (MSLN)* †‡ , MUC1* †‡ , MUC16* †‡ , EPCAM* †‡ , ERBB2* ‡ , FOLH1, GPC3* †‡ , GPNMB* ‡, IL1RAP †‡ , IL3RA* ‡ , IL13RA2(IL13Rα2)* ‡ , KDR(VEGFR2)* ‡ , CD171(L1CAM)* ‡ , MET* ‡ , TROP2* †‡ , and ROR1 †‡ (Includes, but is not limited to) (associated with solid tumors). Antigens associated with B-cell leukemia may also be useful for B-cell depletion in non-oncological applications, but CD19 (present on progenitor-B cells, pre-B cells, immature, inexperienced, germinal, and memory B cells, and short-lived plasma cells (sometimes referred to as short-lived plasma cells)) and BCMA (present on memory B cells, short-lived plasma cells, and long-lived plasma cells) are of particular interest. (* indicates that exemplary antibodies having the indicated specificities capable of inducing binding moiety can be found in Table 9 or 10 of U.S. Patent No. 11,326,182B2.) † This indicates that an exemplary antibody with the indicated specificity capable of inducing a binding moiety can be found in the literature [Wilkinson & Hale, 2022]. Both references are cited and referenced above. ‡(This indicates that exemplary antibodies having the indicated specificity from which a binding moiety can be induced can be found in the Therapeutic Antibody Database (TABS) (tabs.craic.com). Other suitable antibodies can be found in Appendix A. Many of these trace antigens are receptors themselves capable of binding to their ligands when expressed on immune cells. Accordingly, in some embodiments, the extracellular binding domain of the CAR comprises a ligand of the receptor expressed on the target cell. In other additional embodiments, the extracellular binding domain of the CAR comprises a ligand binding domain of the receptor to a ligand expressed on the target cell. In any of these embodiments, the extracellular binding domain of the CAR may be codon-optimized for expression in host cells or may have variant sequences to enhance the action of the extracellular binding domain. The benefits of the embodiments and embodiments disclosed herein are independent of the specificity of the binding moiety. As such, the disclosed embodiments and embodiments are generally agnostic regarding binding specificity. In certain embodiments, specific binding specificity may be required. A more extensive discussion of antibodies recognizing the numerous individual antigens listed above can be found in WIPO application publication WO2024040195A1 and U.S. patent application No. 18 / 731,223, which are respectively incorporated by reference to all that they teach regarding antibodies and related molecules that can be used to provide a binding moiety that recognizes a target antigen.

[0434] c) Hinge domain

[0435] In a given embodiment, the CAR may include a hinge domain also referred to as a spacer. The terms “hinge” and “spacer” may be used interchangeably in this disclosure. Non-limiting examples of hinge domains include CD8α hinge domains, CD28 hinge domains, IgG4 hinge domains, IgG4 hinge-CH2-CH3 domains, and variants thereof, the amino acid sequences of which are provided in Table 2 below.

[0436] [Table 2]

[0437]

[0438] d) Membrane-penetrating domain

[0439] In a given embodiment, the CAR may include a transmembrane domain. In another embodiment, the transmembrane domain may include the transmembrane regions of CD3ζ, CD3ε, CD3γ, CD3δ, CD4, CD5, CD8α, CD8β, CD9, CD16, CD22, CD28, CD32, CD33, CD34, CD37, CD40, CD45, CD64, CD80, CD86, OX40 / CD134, 4-1BB / CD137, CD40L / CD154, FAS, FcεRIγ, FGFR2B, TCRα, TCRβ, or VEGFR2, or functional variants thereof comprising human versions of each of these sequences. Table 3 provides amino acid sequences of some exemplary transmembrane domains.

[0440] [Table 3]

[0441]

[0442] e) Intracellular domain

[0443] In a given embodiment, the CAR may include an intracellular signaling domain. Various generations of CARs include an intracellular domain that provides an activating or stimulating action from, for example, CD3ζ, CD3ε, or CD16A. Second and third generation CARs each added one or more intracellular domains to provide a co-stimulating function from, for example, particularly CD28 or 4-1BB. In a specific embodiment, the intracellular signaling domain is B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7-DC, PDCD6, 4-1BB / TNFRSF9 / CD137, 4-1BB ligand / TNFSF9, BAFF / BLyS / TNFSF13B, BAFF R / TNFRSF13C, CD27 / TNFRSF7, CD27 ligand / TNFSF7, CD30 / TNFRSF8, CD30 ligand / TNFSF8, CD40 / TNFRSF5, CD40 / TNFSF5, CD40 ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, Lymphotoxin-alpha / TNFβ, OX40 / TNFRSF4 / CD134, OX40 ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1A / TNFSF15, TNFα, TNF RII / TNFRSF1B, 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, SLAM / CD150, CD2, CD7, CD53, CD82 / Kai-1, CD90 / Thy1, CD96, CD160, CD200, CD300a / LMIR1, HLA Class I, HLA-DR, Icarus,It may comprise one or more signaling domains selected from integrin alpha 4 / CD49d, integrin alpha 4 beta 1, integrin alpha 4 beta 7 / LPAM-1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, dectin-1 / CLEC7A, DPPIV / CD26, EphB6, TIM-1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLP R, lymphocyte action-associated antigen-1 (LFA-1), NKG2C, CD3ζ, immune receptor tyrosine-based activation motif (ITAM), ligands that specifically bind to CD83, and functional variants thereof including human versions of each of these domains. In some embodiments, the intracellular signaling domain comprises one or more signaling domains selected from the CD3ζ domain, ITAM, CD28 domain, 4-1BB domain, or functional variants thereof. Table 4 provides amino acid sequences for several exemplary intracellular signaling domains. 4-1BB, also known as CD137, delivers a potent co-stimulatory signal to T cells to promote differentiation and enhance the long-term survival of T lymphocytes. CD28 is another co-stimulatory molecule on T cells. CD3 zeta(ζ) binds to the T cell receptor (TCR) to generate a signal and contains an immune receptor tyrosine-based activation motif (ITAM). The CD3ζ signaling domain refers to amino acid residues from the cytoplasmic domain of the zeta chain sufficient to functionally deliver the initial signal required for T cell activation. In certain embodiments, as in the case of tisagenlecleucel as described below, the CD3ζ signaling domain of SEQ No. 129 may have a mutation at amino acid position 14, for example, a mutation from glutamine (Q) to lysine (K) (see SEQ No. 130).

[0444] [Table 4]

[0445]

[0446] f) Exemplary CAR works

[0447] In certain embodiments, the CAR is used to treat a disease or pathology associated with a tracer cell expressing an antigen tracked by the CAR as described in the uses and treatment methods disclosed herein. For example, in some embodiments, an anti-CD19 or anti-CD20 or anti-BCMA CAR may be used to track and treat B-cell malignancies or B-cell-mediated autoimmune pathologies or diseases. In other embodiments, an anti-FAP CAR may be used to track and treat solid tumors or fibrosis (e.g., cardiac fibrosis, cancer-associated fibroblasts). Examples of CARs that may be used according to the embodiments described herein include those disclosed in US 7,446,190 (claim-CD19), US 10,287,350 (claim-CD19), US 2021 / 0363245 (claim-CD19 and claim-CD20), US 10,543,263 (claim-CD22), US 10,426,797 (claim-CD33), US 10,844,128 (claim-CD123), US 10,428,141 (claim-ROR1), and US 2021 / 0087295 (claim-FAP), each of which is incorporated by reference to all that it teaches regarding the structure and action of the CAR and regarding the antigen specificity and indications for follow-up of the CAR, to the extent that it does not conflict with the present disclosure.

[0448] In a given embodiment, a binding domain from an antibody may be used to construct a CAR for tracking and treating solid tumors or fibrosis. An exemplary binding domain is an antibody, e.g., anti-LRRC15 (WO 2021 / 102332), anti-FAP (US 2012 / 0128591; US ​​2012 / 0128591; US ​​2012 / 0128591; US ​​2003 / 0103968; US 6,455,677; US 2009 / 0304718; US 2009 / 0304718; US 2012 / 0258119); anti-ADAM12 (WO 2015 / 028027; WO 2020 / 191293); and can be obtained from the ITGA11 (WO 2008 / 075038; US 2011 / 0256061). Other antibodies that can be used to construct CARs to track and treat solid tumors or fibrosis are anti-CTSK, anti-NOX4, anti-SGCD, anti-SYNDIG1, ​​anti-CDH11, anti-PLPP4, anti-SLC24A2, anti-PDGFRB, anti-THY1, anti-ANTXR1, anti-GAS1, anti-CALHM5, anti-COL11A1, anti-COL1A2, anti-FBN1, anti-COL10A1, anti-COL3A1, anti-COL5A2, anti-COL1A1, anti-COL8A2, anti-COL6A3, anti-GLT8D2, anti-SULF1, anti-COL12A1, anti-GXYLT2, anti-NID2, anti-THBS2, anti-COL5A1, anti-FN1, anti-COL6A1, and anti-C3orf80. Includes.

[0449] The mRNA disclosed herein encoding a CAR comprises both a mature CAR and a signal peptide. The mature CAR comprises at least an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the CAR further comprises one or more co-stimulatory domains in the intracellular portion of the CAR. In some embodiments, the CAR further comprises an extracellular hinge or extension domain between the transmembrane domain and the antigen-binding domain; this domain may be derived from the same protein as the transmembrane domain. In some embodiments, the CAR may comprise a plurality of antigen-binding domains. In certain embodiments of the mRNA disclosed herein, the CAR is an anti-CD19 CAR, an anti-CD20 CAR, an anti-BCMA CAR, or an anti-FAP CAR.

[0450] i) Anti-CD19 CAR

[0451] In a given embodiment, two CAR configurations are used for the anti-CD19 CAR: CAR1 and CAR2. The CAR1 mRNA encodes an amino acid sequence consisting of the following domains in order from the N-terminus to the C-terminus: a CD8α signaling peptide (SP), an anti-CD19 scFv derived from mAb 47G4 (light chain variable domain, VL; linker, L; heavy chain variable domain, VH; 47G4 is disclosed in US2010 / 0104509), a CD8α hinge, a CD8α transmembrane domain (TM), a CD28 co-stimulatory domain (co-stim), and a CD3ζ signaling domain (stim). The CAR1 amino acid sequence is originally disclosed in U.S. Patent No. 10,287,350 (WO2015 / 187528) as Sequence No. 199, from which the CAR1 amino acid sequence and its synthesis are incorporated herein by reference. The amino acid sequence of a mature CAR1 protein (i.e., without signal peptides) is provided as Sequence No. 198. The incorporation of the CD8α hinge and transmembrane domain in CAR1 helps reduce cytokine release syndrome (cytokine storm) compared to similar anti-CD19 CAR molecules containing the CD28 hinge and transmembrane domain instead, but in vivo, the CAR may benefit from the stronger signal provided by the CD28 hinge and transmembrane domain.

[0452] In a predetermined embodiment comprising an anti-CD19 CAR, the anti-CD19 CAR comprises an anti-CD19 binding domain. Some embodiments of the anti-CD19 CAR comprising the anti-CD19 binding domain further comprise a CD28 hinge, a transmembrane domain, a co-stimulation domain, and a CD3ζ signaling domain. Some embodiments of the anti-CD19 CAR comprising the anti-CD19 binding domain further comprise a hinge from CD8α and a transmembrane domain, a CD28 co-stimulation domain, and a CD3ζ-chain signaling domain. In a predetermined embodiment, the anti-CD19 binding domain comprises 47G4 scFv. In a given embodiment, a CAR-T cell comprising an anti-CD19 CAR comprising a CD28 hinge, a transmembrane domain, and a co-stimulation domain exhibits more active cell killing than a CAR-T cell comprising an anti-CD19 CAR comprising a CD8α hinge and a transmembrane domain, and a CD28 co-stimulation domain.

[0453] In a given embodiment, the CAR2 mRNA used encodes an amino acid sequence (SEQ No. 201) consisting of the following domains in order from the N-terminus to the C-terminus: a CD8α signal peptide (SP), an anti-CD19 scFv derived from mAb 47G4 (light chain variable domain, VL; linker, L; heavy chain variable domain, VH), a CD28 hinge, a CD28 transmembrane (TM), a CD28 co-stimulatory domain (co-stim), and a CD3ζ signaling domain (stim). The amino acid sequence of an immature CAR2 protein (i.e., having the signal peptide) is disclosed in Genbank: QHQ73565.1 and is provided as SEQ No. 201. The combination of the CD28 hinge and transmembrane domains, as well as the 47G4 scFv, provides CAR2 with an advantage for transient in vivo transfection, in contrast to traditional CAR-T cells containing an integrated DNA sequence encoding the CAR. CAR2 is expressed at higher levels than CAR1 from mRNA using the same UTR and codon optimization methods, and T cells expressing CAR2 have more CD19 + Remove cells.

[0454] Further examples of anti-CD19 CARs include those comprising a CD19 binding moiety derived from the mouse antibody FMC63. FMC63 and the derived scFv are [Nicholson] et al. [, 1997, Mol. Immun. 34(16-17):1157-1165], and PCT applications published WO 2018 / 213337 and WO 2015 / 187528, the full contents of each of which are incorporated herein by reference to all that they teach regarding claim-CD19 CAR and their uses.

[0455] [Table 5]

[0456]

[0457] In some cases, anti-CD19 CARs are tisagenlecleucel (Literature [Vairy et al. , 2018, Drug Des Devel Ther. 12: 3885-3898]), lysocaptagen marareucel, or axicaptagen ciloleucel and brexucaptagen autoleucel (Literature [Cappell et al. , 2023, Nat Rev Clin Oncol It is a CAR found in [20: 359-371]), and they use the same CAR. In this paragraph, the full contents of each of the aforementioned references are incorporated by reference for everything teaching about the design, structure, and activity of the anti-CD19 CAR.

[0458] [Table 6]

[0459]

[0460]

[0461] [Table 7]

[0462]

[0463] [Table 8]

[0464]

[0465] [Table 9]

[0466]

[0467] A CAR based on 47G4 is disclosed in U.S. Patent No. 10,287,350, which incorporates by reference all that teaches anti-CD19 CAR and its uses. In some embodiments, the extracellular binding domain of the CD19 CAR is derived from an antibody specific to CD19, which is, for example, SJ25C1 (document [Bejcek et al. , 1995, Cancer Res. 55:2346-2351]), HD37(Literature [Pezutto et al. , 1987, J. Immunol. 138(9):2793-2799]), 4G7(literature[Meeker et al., 1984, Hybridoma 3:305-320]), B43(Reference [Bejcek et al., 1995 Cancer Res 55(11):2346-2351]), BLY3(Reference [Bejcek et al., 1995 Cancer Res 55(11):2346-2351]), B4(Reference [Freedman et al. , 1987, Blood 70:418-427]), B4 HB12b (Literature [Kansas & Tedder, 1991, J. Immunol. 147:4094-4102]; Literature [Yazawa et al. , 2005, Proc. Natl. Acad. Sci. USA 102:15178-15183]; Literature[Herbst et al. , J. Pharmacol. Exp. Ther. 335:213-222 (2010)]), BU12(Literature[Callard et al. [ , 1992, J. Immunology, 148(10): 2983-2987]), and CLB-CD19 ([De Rie, 1989, Cell. Immunol. 118:368-381]). In any of these embodiments, the extracellular binding domain of the CD19 CAR is V H , V L It may include one or more CDRs of , and / or any antibody.

[0468] [Table 10]

[0469]

[0470] ii) Anti-CD20 CAR

[0471] CD20 is an antigen found early on the surface of B cells from the pro-B stage and at levels that gradually increase until B cell maturation, as well as being found on the cells of most B cell neoplasms. CD20-positive cells are sometimes found in cases of Hodgkin's disease, multiple myeloma, and thymoma. Examples of anti-CD20 CARs include those containing a CD20-binding moiety derived from an antibody specific to CD20, which, for example, MB-106 (Fred Hutchinson Cancer Research Center, literature [Shadman et al. [ , 2019, Blood 134(Suppl.1):3235] see reference), UCART20(Cellectis, www.cellbiomedgroup.com), or C-CAR066(Cellular Biomedicine Group, literature [Liang et al. [See , 2021, J. Clin. Oncol. 39(15) suppl:2508]) It includes Leu16 and 2.1.2. In some embodiments, the extracellular binding domain of the CD20 CAR is a heavy chain variable region (V) of Leu16 linked by a linker, such as the CAR22 and CAR25 described herein. H ) and light chain variable region (V L Includes scFv derived from a Leu16 monoclonal antibody, comprising ) (Literature [Wu et al. [See , 2001, Protein Engineering. 14(12):1025-1033]). In some embodiments, the extracellular binding domain of CD20 CAR is a heavy chain variable region of 2.1.2 (V linked by a linker, as in CAR7 described herein). H ) and light chain variable region (V LIt includes scFvs derived from monoclonal antibody 2.1.2, which includes ). Additional antibodies capable of providing an anti-CD20 binding domain include IF5, 1.5.3, rituximab, obinutuzumab, ibritumomab, ofatumumab, tocitumumab, odronextamab, veltuzumab, ublituximab, and ocrelizumab. In this paragraph, the full contents of each of the aforementioned references are incorporated by reference for everything teaching the design, structure, and activity of the anti-CD20 CAR.

[0472] In a given embodiment, CAR25 is provided herein as a CAR configuration used for anti-CD20 CAR. CAR25 mRNA encodes an amino acid sequence consisting of the following domains in order from N-terminus to C-terminus: mouse Ig-kappa signaling peptide (Igk sp), anti-CD20 scFv derived from Leu16 mAb (light chain variable domain, VL; linker, L; heavy chain variable domain (VH), IgG4 hinge, CD28 transmembrane domain (TM), 4-1BB co-stimulatory domain (co-stim), and CD3ζ signaling domain (stim). The amino acid sequence of the mature CAR25 protein (i.e., without the signaling peptide) is provided as SEQ ID NO. 19.

[0473] In some embodiments, when an anti-CD20 CAR is included, the anti-CD20 CAR includes Leu16 scFv. In some embodiments, the anti-CD20 CAR including Leu16 scFv further includes an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB co-stimulation, and a CD3ζ signaling domain. Examples of such anti-CD20 CARs include, without limitation, CAR25 (SEQ No. 19, or SEQ No. 20 having a signal peptide). In some embodiments, the anti-CD20 CAR including Leu16 scFv further includes an IgG4 hinge, a CD28 transmembrane domain and a co-stimulation domain, a 4-1BB co-stimulation, and a CD3ζ signaling domain. Examples of such anti-CD20 CARs include CAR22 (SEQ No. 21), or having a signal peptide (SEQ No. 22).

[0474] [Table 11]

[0475]

[0476] In a given embodiment comprising an anti-CD20 CAR, the anti-CD20 CAR comprises 2.1.2 scFv. In some embodiments, the anti-CD20 CAR comprising 2.1.2 scFv further comprises a CD28 hinge, a transmembrane, and a co-stimulatory domain and a CD3ζ signaling domain. Examples of such anti-CD20 CARs include, without limitation, CAR7 (SEQ No. 214), or SEQ No. 215, which has a signaling peptide.

[0477] iii) Anti-BCMA CAR

[0478] In a given embodiment, the anti-CD8 tLNP encapsulates a nucleic acid encoding an anti-BCMA chimeric antigen receptor (CAR). BCMA is a member of the tumor necrosis family receptor (TNFR) expressed in cells of the B cell lineage, exhibiting the highest expression in terminally differentiated B cells or mature B lymphocytes. BCMA is involved in mediating the survival of plasma cells for the maintenance of long-term humoral immunity. The expression of BCMA has recently been associated with numerous cancers, such as multiple myeloma, Hodgkin and non-Hodgkin lymphomas, various leukemias, and glioblastoma. Examples of anti-BCMA CARs are [Carpenter et al. [..., 2013, Clin. Cancer Res. 19(8):2048-2060] includes those comprising a BCMA binding moiety derived from C11D5.3, a mouse monoclonal antibody as described in [..., 2013, Clin. Cancer Res. 19(8):2048-2060]. Also refer to PCT International Publication WO 2010 / 104949. In some embodiments, the extracellular binding domain of the BCMA CAR is [Carpenter et al. As described in [ , 2013, Clin. Cancer Res. 19(8):2048-2060] and PCT application publication WO2010104949, it comprises a scFv derived from another mouse monoclonal antibody, C12A3.2. In some embodiments, the extracellular binding domain of the BCMA CAR is [Friedman et al. [, 2018, Hum. Gene Ther. 29(5):585-601] comprises a scFv derived from a mouse monoclonal antibody having high specificity for human BCMA, referred to as BB2121. Also refer to PCT International Publication WO 2012163805. In some embodiments, the extracellular binding domain of the BCMA CAR is [Zhao Z et al.It comprises a single variable fragment (VHH) of two heavy chains capable of binding to two epitopes of BCMA as described in [J. Hematol. Oncol. 11(1):141]. Also refer to PCT application publication number WO 2018 / 028647. In some embodiments, the extracellular binding domain of the BCMA CAR is [Lam et al. It includes a fully human heavy chain variable domain (FHVH) as described in [2020, Nat. Commun. 11(1):283]. Also refer to PCT International Publication WO 2019 / 006072. In some embodiments, the extracellular binding domain of the BCMA CAR includes a scFv derived from CT103A (or CAR0085) as described in U.S. Patent No. 11,026,975 B2. Additionally, anti-BCMA CARs are disclosed in U.S. Patent Application Publications No. 2020 / 0246381 and No. 2020 / 0339699. The full contents of each of the references mentioned in this paragraph are incorporated by reference to everything they teach regarding the design, structure, and activity of the anti-BCMA CAR.

[0479] [Table 12]

[0480]

[0481]

[0482] iv) Anti-FAP CAR

[0483] In a given embodiment comprising an anti-FAP CAR, the anti-FAP CAR comprises a scFv based on antibody 4G5 (see WO2021 / 061708 and WO2021 / 061778). In some embodiments comprising an anti-FAP CAR comprising a scFv based on antibody 4G5 further comprising a hinge and transmembrane domain from CD8, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain, examples of the anti-FAP CAR include the CAR disclosed in WO2021 / 061778.

[0484] [Table 13]

[0485]

[0486] In some embodiments, anti-CD8 tLNP encapsulates a nucleic acid encoding an anti-GPRC5D chimeric antigen receptor (CAR). GPRC5D is a G protein-coupled receptor with no known ligand and an unclear function in human tissues. However, this receptor is expressed in myeloma cell lines and in the bone marrow plasma cells of multiple myeloma patients. GPRC5D has been identified as an immunotherapeutic target in multiple myeloma and Hodgkin lymphoma. Examples of anti-GPRC5D CARs include those containing a GPRC5D binding moiety, such as MCARH109 (Mailankody et al., N Engl J Med. 387(13): 1196-1206 (2022)), BMS-986393, or OriCAR-017 (Rodriguez-Otero et al., Blood Cancer J. 14(1): 24 (2024)). Examples of anti-GPRC5D CARs include those containing a GPRC5D binding moiety derived from an antibody specific to GPRC5D, such as talquetamab (Pillarisetti et al., Blood 135:1232-43 (2020)), or Porimtamic. In some embodiments, the extracellular binding domain of the anti-GPRC5D CAR comprises a scFv derived from a 6D9 mouse antibody specific to human GPRC5D (see creative-biolabs.com / car-t / anti-gprc5d-6d9-h-41bb-cd3-car-pcdcar1-26380.htm). In some embodiments, the extracellular binding domain of the GPRC5D CAR comprises a scFv of an anti-GPRC5D antibody linked to a 4-1BB or CD28 co-stimulation domain and a CD3ζ signaling domain, which is [Mailankody et al., N Engl J Med. 387(13): 1196-1206 (2022)]; [creative-biolabs.com / car-t / anti-gprc5d-6d9-h-41bb-cd3-car-pcdcar1-26380.htm]; and as described in the literature [Rodriguez-Otero et al., Blood Cancer J. 14(1): 24 (2024)]. In this paragraph, the full content of each of the aforementioned references is incorporated by reference to all that teaches the design, structure, and activity of anti-GPRC5D CARs and anti-GPRC5D antibodies capable of providing an antigen-binding domain to a CAR or immune cell engager, each example constituting a means for binding GPRC5D. In any of the aforementioned tLNP embodiments, a given embodiment comprises a tLNP having a T cell targeting moiety, such as an anti-CD8 antibody, encapsulating a GPRC5D CAR payload encoded by RNA.

[0487] In some embodiments, the anti-CD8 tLNP encapsulates a nucleic acid encoding an anti-FCRL5 chimeric antigen receptor (CAR). FCRL5 (Fc receptor-like 5), also known as FCRH5, BXMAS1, CD307, CD307E, and IRTA2, is a protein marker expressed on the surface of plasma cells in patients with multiple myeloma. Furthermore, since contact with FCRL5 stimulates B-cell proliferation; FCRL5 has been identified as an immunotherapeutic target for this disease. Examples of anti-FCRL5 CARs include those containing an FCRL5 binding moiety, such as those described in WO2016090337, WO2017096120, WO2022263855, and WO2024047558. In some embodiments, the extracellular binding domain of the anti-FCRL5 CAR comprises scFvs specific to FCRL5, such as ET200-31, ET200-39, ET200-69, ET200-104, ET200-105, ET200-109, or ET200-117. In some embodiments, the extracellular binding domain of the anti-FCRL5 CAR comprises scFvs derived from mouse antibodies specific to human FCRL5. Such antibodies include 7D11, F25, F56, and F119, which are [Polson et al., Int. Immunol., 18(9): 1363-1373 (2006)]; [Franco et al., J. Immunol. 190(11): 5739-5746 (2013)]; As described in the literature [Ise et al., Clin. Cancer Res. 11(1): 87-96 (2005)]; and literature [Ise et al., Clin. Chem. Lab. Med. 44(5): 594-602 (2006)], all of which are incorporated herein by reference.In some embodiments, the extracellular binding domain of the anti-FCRL5 CAR comprises a binding moiety derived from an antigen-binding domain of an anti-FCRL5 antibody or nanobody, comprising sevostamab, 2A10H7, 307307, 2A10D6, 13G9, 10A8, 509f6, EPR27365-87, EPR26948-19, or EPR26948-67, or as disclosed in WO2016090337, WO2017096120, WO2022263855, or WO2024047558. In some embodiments, the extracellular binding domain of the anti-FCRL5 CAR comprises [Elkins et al., Mol. Cancer Ther. It comprises a binding moiety derived from an antibody-drug conjugate targeting FCRL5, as described in [11(10): 2222-2232 (2012)]. In some embodiments, the extracellular binding domain of the anti-FCRL5 CAR is linked to a 4-1BB or CD28 co-stimulatory domain and a signaling domain, such as a CD3ζ signaling domain. The entire contents of each of the aforementioned references in this paragraph are incorporated by reference to all that teach the design, structure, properties, and activity of anti-FCRL5 CARs and anti-FCRL5 antibodies capable of providing an antigen-binding domain to a CAR or immune cell engager. Each example constitutes a means for binding to FCRL5. In any of the aforementioned tLNP embodiments, a specific embodiment comprises a tLNP having a T cell targeting moiety, such as an anti-CD8 antibody, encapsulating an FCRL5 CAR payload encoded by RNA.

[0488] Each CAR having specificity for a specific antigen described herein constitutes a means for antigen recognition for that antigen, and collectively all CARs described herein constitute a means for antigen recognition. The action may alternatively be referred to as antigen recognition by immune cells or antigen recognition by T cells, etc.

[0489] In a given embodiment, the ORF is encoding a gene-editing nuclease, e.g., an RNA-induced nuclease, a gene or base editing protein, a prime editing protein, a gene writer protein (e.g., a modified or modularized non-long terminal repeat (LTR) retrotransposon), a retrotransposase, an RNA writer, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a transposase, a retrotransposon, a reverse transcriptase (e.g., M-MLV reverse transcriptase), a nicase or inactivated nuclease (e.g., Cas9, nCas9, dCas9), a DNA recombinase, a CRISPR nuclease (e.g., Cas9, Cas12, Cas13, Cas3, CasMINI, Cas7-11, CasX), a DNA nicase, a Cas9 nicase (e.g., D10A or It may encode H840A), or any fusion thereof or combination thereof. Genome-, gene-, and base-editing technologies are described in the literature [Anzalone et al., Nature Biotechnology 38:824-844, 2020], Literature[Sakuma, Gene and Genome Editing 3-4:100017, 2022], and literature[Zhou et al. , MedComm [3(3):e155, 2022] is reviewed, and each of these is incorporated by reference to all that they teach regarding the components and uses of such technology to the extent that it does not conflict with the present disclosure.

[0490] In a specific embodiment of any of the above embodiments, the poly(A) sequence may have at least about 80 adenosine residues to about 130 or more adenosine residues. In some embodiments, the poly(A) sequence has about 80 adenosine residues. In a specific embodiment, the poly(A) sequence has about 90 adenosine residues. In a specific embodiment, the poly(A) sequence has about 100 adenosine residues. In a specific embodiment, the poly(A) sequence has about 110 adenosine residues. In a specific embodiment, the poly(A) sequence has about 130 adenosine residues. Each CAR having specificity for a specific antigen described herein constitutes a means for antigen recognition for that antigen, and collectively, all CARs described herein constitute a means for antigen recognition. The action may alternatively be referred to as antigen recognition by immune cells or antigen recognition by T cells, etc.

[0491] Each of the various genera, subgenuses, and / or species of LNP or tLNP disclosed herein, including those based on specific lipids, specific lipid compositions, specific payloads, and / or the inclusion or exclusion of specific humanized anti-CD8 antibodies, may be used to define the category of each embodiment of the treatment method.

[0492] Unless otherwise defined, all technical terms used herein have the same meaning as generally understood by those skilled in the art to which this disclosure pertains.

[0493] For the purpose of facilitating an understanding of the principles of the present disclosure, reference will now be made to embodiments, and specific language will be used to describe them. Nevertheless, no limitation is intended to be placed on the scope of the present disclosure, and it will be understood that such modifications and further variations of the present disclosure, as exemplified herein, would normally occur to those skilled in the art to which the present disclosure relates.

[0494] Various exemplary embodiments of the composition and method according to the present invention are now described in the following non-limiting examples. The examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. In fact, in addition to those shown and described herein, various variations of the invention will become apparent to those skilled in the art from the foregoing description and the following examples and will fall within the scope of the appended claims.

[0495] [Table 14]

[0496]

[0497] Examples

[0498] Materials and Methods

[0499] Production of humanized anti-CD8α binder / antibody

[0500] The sequence of the CT8 antibody (also referred to herein as CBD1017p) is disclosed herein. The VH and VL sequences were compared with a library of known human germline sequences from the human VH gene and the human VL kappa gene ( IMGT® the international ImMunoGeneTics information system® www.imgt.org ; founder and director: Marie-Paule Lefranc, Montpellier, France); the databases used were the IMGT human VH gene (F+ORF, 273 germline sequences) and the IMGT human VL-kappa gene (F+ORF, 74 germline sequences) as used by the NCBI IgBLAST program. Recipient human germlines were selected from those whose sequences were closest to the maternal antibody.

[0501] In the case of VH, the human germline IGHV1-46*01 is used as the recipient sequence, and the human heavy chain IGHJ6 (allele 1) binding region (J gene) IMGT® the international ImMunoGeneTics information system® www.imgt.org (founder and director: Marie-Paule Lefranc, It was selected from human binding region sequences compiled in Montpellier, France (see Fig. 1a).

[0502] In the case of VL, the human germline IGKV1-39*01 is used as the recipient sequence, and the human light chain IGKJ2 (allele 1) binding region (J gene) IMGT® the international ImMunoGeneTics information system® www.imgt.org (founder and director: Marie-Paule Lefranc, It was selected from human binding region sequences compiled in Montpellier, France (see Fig. 1b).

[0503] CDRs were defined according to the AbM definition (see Dr. Andrew CR Martin's website on the Bioinformatics website for a table comparing CDR definitions). To optimize the binding of humanized antibodies to CD8-specific antigens, it is considered to change human germline framework locations (i.e., non-CDR residues within VH and VL) to corresponding maternal murine sequences. Potential changes to each humanized sequence are mentioned in Figures 1a and 1b.

[0504] Using the IMGT / BlastSearch online implementation, the CBD1017p VH and VL sequences were also compared with a library of known human germline sequences from the human VH gene and the human VL kappa gene. IGHV1-18*01 was used as the heavy chain receptor sequence, and IGKV3D-11*02 was used as the human light chain receptor sequence. BioLuminate® modeling software online ( A modified germline common sequence was obtained using a manual investigation with Inc., which was used as the starting point location for the humanization being considered (Figs. 1c and 1d).

[0505] Binding affinity (K by biolayer interferometric kinetics assay D ) measurement

[0506] Biolayer interferometric kinetics assays were performed using the GatorBio Gator Plus BLI system. Kinetic buffers containing PBS + 0.1% Tween 20 and 0.2% BSA were used for baseline calculations. To measure the binding kinetics of the anti-CD8α antibody fragment (Fab), a streptavidin-immobilized SA-XT sensor (#160029, Gator Bio) was pre-hydrated in kinetic buffer, and 100 mM biotinylated CD8α recombinant protein (CDA-H82E3, Acro Biosystems) was loaded to a reaction threshold of 10 nm at a spin rate of 400 rpm. Subsequently, baseline measurements were obtained by incubating the sensor in kinetic buffer for 300 seconds prior to each binding. Each anti-CD8α Fab fragment was diluted in kinetic buffer through 2-fold dilution at concentrations ranging from 100 nM to 3.12 nM. The antigen-loaded sensor was incubated in a diluted solution for 300 seconds to capture the Fab fragment and record the association phase. Finally, the sensor was incubated in kinetics buffer for 900 seconds to record the dissociation phase. The spin rate for all phases except the antigen loading phase was 1000 rpm. The temperature of the assay performed was 30°C or 37°C during the association and dissociation phases. Sensor data was subtracted from baseline, and overall curve fitting of kinetic data from six different analyte concentrations was performed using GatorOne software (version v2.10) in a 1:1 monovalent binding model to determine the kinetic rate constant (k) of the analyzed antibody. on and k off ) and equilibrium dissociation constant (K D Decided on ).

[0507] The binding kinetics of CBD1033 Fab and its parent CBD1017ch Fab to human CD8αα homomers, human CD8αβ heteromers, and cyanomolgus macaque CD8αα homomers were evaluated according to the same method as in the BLI evaluation of humanized Fab variants. To evaluate the binding kinetics to the cyanomolgus macaque CD8αβ heteromer protein, the antigen protein was directly coupled onto an amine-reactive biosensor tip (Gator Bio) according to the manufacturer's protocol. Briefly, the sensor was activated for 300 seconds with a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (ThermoFisher Scientific) and 10 mM N-hydroxysulfosuccinimide sodium salt (Sulfo-NHS) (Sigma-Aldrich) in water. Cynomolgus macaque CD8αβ protein diluted to 100 mM in 10 mM sodium acetate buffer, pH 6.0, was covalently captured to a threshold of approximately 5 nm for 300 seconds, and subsequently, the sensor was inactivated in 1 M ethanolamine (ETA) (Sigma-Aldrich) solution, pH 8.5, for 300 seconds. Subsequently, the sensor was rinsed in kinetic buffer for 600 seconds. An additional rinsing step of 120 seconds was repeated for baseline calculation before measuring binding kinetics. The antibody fragment was diluted in kinetic buffer through 3-fold dilution at concentrations ranging from 1000 nM to 1.37 nM. As described above, the antigen-captured sensor was incubated in the diluted solution for 1200 seconds to capture the Fab fragment and record the binding step. Subsequently, the sensor was incubated in kinetic buffer for 1200 seconds to record the dissociation phase. The shaking rate for all phases was 1000 rpm, and the temperature of the assay performed was 30℃. The kinetic parameters were determined using a 1:1 monovalent (Langmuir) coupling model with the previous method.

[0508] To measure the binding kinetics of biotin-conjugated Fab molecules, a streptavidin-immobilized SA-XT sensor (#160029, Gator Bio) was pre-hydrated in kinetic buffer, and 50 mM biotinylated Fab samples CBD1033.37 or CBD1033.24 were loaded to a reaction threshold of 10 nm. Subsequently, the sensor was briefly blocked for 60 seconds with kinetic buffer containing 50 mM biotin and 3% BSA, and further incubated in kinetic buffer for 120 seconds to achieve steady-state baseline measurements prior to binding. Custom recombinant human CD8α mouse IgG2a Fc fusion proteins were diluted in kinetic buffer through 2-fold dilution at concentrations ranging from 100 nM to 1.56 nM. The binder-loaded sensor was incubated in a diluted solution for 1200 seconds to capture CD8α protein and record the binding phase. Finally, the sensor was incubated in kinetics buffer for an additional 1200 seconds to record the dissociation phase. The spin rate for all phases was 1000 rpm. The temperature of the assay performed was 37°C during binding and dissociation. Sensor data were subtracted from baseline, and overall curve fitting of kinetic data from six different analyte concentrations was performed using GatorOne software (version v2.10) in a 1:1 monovalent binding model to determine the kinetic rate constant (k) of the analyzed binder. on and k off ) and equilibrium dissociation constant (K D Decided on ).

[0509] Binding affinity (K) by steady-state analysis of biolayer interferometry D ) measurement

[0510] Since the divalent nature of both the total antibody and CD8α complicates kinetic analysis, a steady-state analysis was used to evaluate the affinity of the total antibody. 50 nM CD8α-His (i.e., CD8α with a C-terminal oligohistidine tag; Acro) was immobilized on the Ni-NTA sensor probe. Binding was analyzed using a 3-fold antibody dilution series (60 nM, 20 nM, 6.67 nM, 2.22 nM, 0.74 nM, and 0.25 nM). The antigen-loaded sensor was incubated in the diluted solution for 300 seconds to capture the total IgG antibody and record the binding phase. The sensor was incubated in kinetic buffer for 900 seconds to record the dissociation phase. The spin rate was 1000 rpm for all steps, except for the antigen loading step, which was 400 rpm to control antigen density. The temperature of the assay performed was 30°C during the binding and dissociation phases. By subtracting the baseline from the sensor data and further analyzing it in GatorOne software (version 2.10) to fit the steady-state signal to a 1:1 binding model, the antibody's equilibrium dissociation constant (K D Decided on ).

[0511] Binding affinity (K by surface plasmon resonance assay) D ) measurement

[0512] Surface plasmon resonance (SPR) assays were performed at 25°C using a Biacore 8K SPR instrument (Cytiva) in running buffer containing 1xHBS-N (Cytiva) and 0.05% Tween-20. The CBD1033 antibody was captured by the anti-human IgG(Fc) antibody (Cytiva) immobilized on the CM5 sensor chip (Cytiva) at densities within 40 to 50 and 80 to 100 reaction units (RU), respectively, for kinetic measurements using CD8αα and CD8αβ. To measure binding kinetics, serial 2-fold dilutions of the recombinant CD8 protein were prepared in running buffer and injected into a flow cell at 30 μL / min at concentrations ranging from 200 to 6.25 nM (serial 1:2 dilutions). Binding data were collected for 180 seconds, followed by a 1200-second dissociation step. At the end of each binding cycle, the sensor surface was regenerated with 3 M MgCl2 buffer. For the cyanomolus macaque CD8αβ heteromer, binding kinetics were evaluated using the same experimental parameters, except that the concentration range was 400 nM to 12.5 nM. Sensorgrams were generated, and the background was subtracted using blank learning buffer. BIAcore Evaluation software (Cytiva) was used to analyze and determine binding kinetic parameters using a standard 1:1 monovalent binding (Langmuir) model.

[0513] Expression and Purification of Disulfide-Engineered F(ab')

[0514] Disulfide-engineered F(ab') analogs were transiently expressed using engineered CHO-K1 cell lines (Wuxi Biologics) employing a proprietary expression protocol. After 7 days, the culture supernatant was harvested by centrifugation and filtration. F(ab') analogs were first captured from the filtered supernatant by affinity chromatography using KanCap™ G resin (Kaneka) and eluted in 50 mM citrate buffer, pH 3.5. The antibody-containing eluent was buffer-exchanged by dialysis with PBS pH 6.5 containing 10 mM EDTA. Subsequently, Fab molecules were reduced by adding 5 mM 2-mercaptoethylamine (2-MEA) and incubated at room temperature for up to 90 minutes. The reduction process was monitored by taking small samples at 30-minute intervals and confirming the integrity of Fab purity by SDS-PAGE analysis. The reduced F(ab') analog was subsequently diluted to 20 mM NaAc, pH 5.0, captured by cation exchange chromatography using SP Sepharose High Performance resin (Cytiva), and eluted with a gradient of 0 to 1 M NaCl. Subsequently, the polished F(ab') protein was dialyzed against 20 mM histidine-HCl, pH 5.5, and 240 mM sucrose. The purity of the final F(ab') analog was evaluated by SDS-PAGE, analytical SEC-HPLC, and LC-MS analysis.

[0515] EC 50 Antigen CD8-specific binding measurement

[0516] EC for antigen-specific binding 50 To obtain the results shown in Fig. 3a for determining the values, CD8-overexpressing HEK293T cells were dissociated in Versine solution (pH 7.4 PBS buffer supplemented with 0.5 mM EDTA), and 2×10 5Total cells were transferred to V-bottom 96-well culture plates (Corning). Cells were washed twice with cell staining buffer (#420201, Biolegend). Each anti-CD8α antibody containing human IgG1 allotype and Fc-silencing mutants L234A, L235A, and P329A was diluted in cell staining buffer in a 4-fold antibody titration series ranging from 100 μg / mL to 6.1 ng / mL. eFluor 780 Fixable Viability dye (eBioscience) was also included in the antibody solution at a 1:4000 dilution. Cells were stained with each diluted antibody solution for 30 minutes on ice. Subsequently, primary antibody binding was detected by washing cells three times with cell staining buffer and staining them with an anti-human Fc BV421 conjugate antibody (#410704, Biolegend) at a 1:100 dilution for 30 minutes on ice. After staining with the secondary detection antibody, cells were washed three times with cell staining buffer, fixed in FluoroFix buffer (Biolegend), and stored in the dark at 4°C until analysis. Flow cytometry was performed on each sample using an Agilent NovoCyte flow cytometer. Flow cytometry data were analyzed using a Flowjo 10 (Becton, Dickinson & Company) to measure the geometric median fluorescence intensity (gMFI) of on-cell binding from each antibody. The gMFI values ​​were normalized to the gMFI measured in samples stained solely with the secondary detection antibody and plotted against the concentration of the added antibody. Binding curves were constructed using Graphpad Prism software, and EC was determined by non-linear curve fitting. 50 The value was determined.

[0517] To obtain the results shown in Figure 6, SupT1 (ATCC #CRL-1942) and HPB-ALL (DSMZ; ACC-483) were cultured in RPMI medium supplemented with 10% fetal bovine serum (#97068-085; Avantor). EC of cell binding from anti-CD8α antibody 50 To determine the value, 2×10 5 Total cells were transferred to V-bottom 96-well culture plates (Corning). Cells were washed twice with cell staining buffer (#420201, Biolegend). Anti-CD8α antibodies were diluted in cell staining buffer in a 4-fold antibody titration series ranging from 60 μg / mL to 57 pg / mL. Cells were stained with each diluted antibody solution on ice for 30 minutes. Subsequently, primary antibody binding was detected by washing cells three times with cell staining buffer and staining with a 1:200 dilution of anti-human Fc BV421 conjugate antibody (#410704, Biolegend) on ice for 30 minutes. A 1:4000 dilution of eFluor 780 Fixable Viability dye (eBioscience) was also included in the detection antibody solution. After staining with the secondary detection antibody, cells were washed three times with cell staining buffer, fixed in FluoroFix buffer (Biolegend), and stored in the dark at 4°C until analysis. Flow cytometry was performed on each sample using an Agilent NovoCyte flow cytometer. Flow cytometry data were analyzed using a Flowjo 10 (Becton, Dickinson & Company) to measure the geometric median fluorescence intensity (gMFI) of cell-phase binding from each antibody. The gMFI values ​​were normalized to the gMFI measured in samples stained solely with the secondary detection antibody and plotted against the concentration of the added antibody. Binding curves were constructed using Graphpad Prism software, and EC was determined by non-linear curve fitting. 50 The value was determined.

[0518] To obtain the results shown in Figs. 8a and 8b, 5×10 4 Canine expanded primary T cells (human, cynomolgus macaque, or rhesus monkey) were thawed and transferred to V-bottom 96-well culture plates (Corning). Cells were washed twice with cell staining buffer (#420201, Biolegend). Each anti-CD8α antibody was diluted in cell staining buffer in a 4-fold antibody titration series ranging from 60 μg / mL to 0.057 ng / mL. Cells were stained with each diluted antibody solution on ice for 30 minutes. Subsequently, primary antibody binding was detected by washing cells three times with cell staining buffer and staining with a 1:200 dilution of anti-human Fc BV421 conjugate antibody (#410704, Biolegend) on ice for 30 minutes. A 1:200 dilution of the anti-CD3 SP34-2 Alexa Fluor 700 conjugate antibody (#557917, BD Pharmagen), a 1:200 dilution of the anti-CD4 OKT4 BV650 conjugate antibody (#317436, Biolegend), and a 1:5000 dilution of the eFluor 780 Fixable Viability dye (eBioscience) were also included in the detection antibody solution. After staining with the secondary detection antibodies, cells were washed three times with cell staining buffer, fixed in eBioscience™ IC fixation buffer, and stored in the dark at 4°C until analysis. Flow cytometry was performed on each sample using an Agilent NovoCyte flow cytometer. Flow cytometry data were analyzed using a Flowjo 10 (Becton, Dickinson & Company) to measure the geometric median fluorescence intensity (gMFI) of cell-phase binding from each antibody. The gMFI values ​​were normalized to the gMFI measured in samples stained solely with the secondary detection antibody and plotted against the concentration of the added antibody. Binding curves were constructed using Graphpad Prism software, and EC was determined by non-linear curve fitting.50 The value was determined.

[0519] tLNP formation

[0520] An initial LNP was formed by mixing an aqueous solution of mCherry mRNA and an ethanol solution of lipids in the ratio of CICL1:DSPC:CHOL:DSG-PEG(2k):DSPE-PEG(2k)-MAL[58:10:30.5:1.4:0.1] (composition F9 mentioned in Table 14). Subsequently, stepwise phosphate and Tris buffer dilution and tangential flow filtration (TFF) purification followed.

[0521] The total antibody was coupled to LNP via N-succinimidyl S-acetylthioacetate (SATA)-maleimide conjugation chemistry. The antibody was modified using SATA (Sigma-Aldrich) to introduce sulfhydryl groups to accessible lysine residues, enabling conjugation to maleimide. The SATA was deprotected using 0.5 M hydroxylamine, followed by the removal of unreacted components using a G-25 Sephadex Quick Spin Protein column (Roche Applied Science, Indianapolis, Indiana, USA). Subsequently, reactive sulfhydryl groups on the antibody were conjugated to the maleimide moiety on the LNP using thioether conjugation chemistry. Purification of the conjugated tLNP (LNP conjugated with the targeting antibody) was performed using a Sepharose CL-4B gel filtration column (Sigma-Aldrich) or TFF (tangential flow filtration). The tLNP was frozen at -80°C until use.

[0522] As described in the previous paragraph, the diabody and F(ab')2 were conjugated by first partially reducing the cysteine ​​bonds within the antibody using tris(2-carboxy)phosphine (TCEP) to generate thiol groups for conjugation through the maleimide ...

Claims

Claim 1 An isolated antibody or antigen-binding fragment thereof comprising a humanized immunoglobulin antigen-binding domain that specifically binds to human CD8, comprising: (a) a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of the framework region of SEQ ID NO. 9 or 31 (wherein VH comprises a heavy chain CDR1 (VH-CDR1) having the amino acid sequence RYTFTDYX1LH (SEQ ID NO. 45), a VH-CDR2 having the amino acid sequence FIYPYX1GGTG (SEQ ID NO. 46) or FIYPYX2GGTG (SEQ ID NO. 47), and a VH-CDR3 having the amino acid sequence DHRYX1EGVSFDY (SEQ ID NO. 48); and (b) a light chain variable region (VL) comprising an amino acid sequence having at least 90% identity with the amino acid sequence of the framework region of SEQ ID NO. 15 or 37 (wherein VL comprises a CDR1 (VL-CDR1) comprising the amino acid sequence RASESVX3GFGX1SFMN (SEQ ID NO. 49), a VL-CDR2 comprising the amino acid sequence LASX2LES (SEQ ID NO. 50), and a VL-CDR3 comprising the amino acid sequence QQX2X2EX3PYT (SEQ ID NO. 51), wherein each X1 is independently N, S, Q, or A; each X2 is independently N, Q, D, S, or A; and each X3 is independently D, E, S, or A, an isolated antibody or its antigen-binding fragment. Claim 2 In paragraph 1, X1 of VH-CDR2 is an isolated antibody or its antigen-binding fragment that is S, Q, or A. Claim 3 In paragraph 1 or 2, X2 of VL-CDR1 is an isolated antibody or its antigen-binding fragment, which is S or A. Claim 4 In claim 1 or 2, VH is an isolated antibody or an antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO. 27, SEQ ID NO. 66, SEQ ID NO. 28, SEQ ID NO. 67, SEQ ID NO. 29, or SEQ ID NO.

68. Claim 5 In paragraphs 1 and 3, VL is an isolated antibody or an antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO. 64 or SEQ ID NO.

65. Claim 6 The isolated antibody or its antigen-binding fragment, comprising: (a) a human heavy chain variable region (VH) comprising a heavy chain CDR1 (VH-CDR1) having the amino acid sequence of SEQ ID NO. 2, a VH-CDR2 having the amino acid sequence of SEQ ID NO. 3, 58, 59, or 60, and a VH-CDR3 having the amino acid sequence of SEQ ID NO. 4; and (b) a light chain variable region (VL) comprising a CDR1 (VL-CDR1) having the amino acid sequence of SEQ ID NO. 6, 227, or 228, a VL-CDR2 having the amino acid sequence of SEQ ID NO. 7, and a VL-CDR3 having the amino acid sequence of SEQ ID NO.

8. Claim 7 An isolated antibody or an antigen-binding fragment thereof, wherein (a) VH comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, or SEQ ID NO. 14, wherein VH-CDR1 comprises the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprises the amino acid sequence of SEQ ID NO. 3, and VH-CDR3 comprises the amino acid sequence of SEQ ID NO. 4; and (b) VL comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO. 16, SEQ ID NO. 17, or SEQ ID NO. 18, wherein VL-CDR1 comprises the amino acid sequence of SEQ ID NO. 6, VL-CDR2 comprises the amino acid sequence of SEQ ID NO. 7, and VL-CDR3 comprises the amino acid sequence of SEQ ID NO.

8. Claim 8 An isolated antibody or its antigen-binding fragment, wherein (a) VH comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, or SEQ ID NO. 36, wherein VH-CDR1 comprises the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprises the amino acid sequence of SEQ ID NO. 3, and VH-CDR3 comprises the amino acid sequence of SEQ ID NO. 4; and (b) VL comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO. 38, SEQ ID NO. 39, or SEQ ID NO. 40, wherein VL-CDR1 comprises the amino acid sequence of SEQ ID NO. 6, VL-CDR2 comprises the amino acid sequence of SEQ ID NO. 7, and VL-CDR3 comprises the amino acid sequence of SEQ ID NO.

8. Claim 9 An isolated antibody or its antigen-binding fragment, wherein VH comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 66, SEQ ID NO. 67, or SEQ ID NO. 68, and VL comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 64, or SEQ ID NO.

65. Claim 10 In claim 1, 6, or 9, (a) VH comprises the amino acid sequence of SEQ NO 10, SEQ NO 11, SEQ NO 12, SEQ NO 13, SEQ NO 14, SEQ NO 27, SEQ NO 28, SEQ NO 29, SEQ NO 35, or SEQ NO 36, and VL comprises the amino acid sequence of SEQ NO 16; (b) VH comprises the amino acid sequence of SEQ NO 10, SEQ NO 11, SEQ NO 12, SEQ NO 13, SEQ NO 14, SEQ NO 27, SEQ NO 28, SEQ NO 29, SEQ NO 35, or SEQ NO 36, and VL comprises the amino acid sequence of SEQ NO 17; or (c) VH comprises SEQ NO 10, SEQ NO 11, SEQ NO 12, SEQ NO 13, SEQ NO 14, SEQ NO 27, SEQ NO 28, SEQ NO 29, SEQ NO (d) an isolated antibody or its antigen-binding fragment comprising the amino acid sequence of SEQ ID NO. 35, or SEQ ID NO. 36, and VL comprises the amino acid sequence of SEQ ID NO. 18; and VH comprises the amino acid sequence of SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 35, or SEQ ID NO. 36, and VL comprises the amino acid sequence of SEQ ID NO.

39. Claim 11 An isolated antibody or an antigen-binding fragment thereof, wherein (a) VH comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO. 11, wherein VH-CDR1 comprises the amino acid sequence of SEQ ID NO. 2, VH-CDR2 comprises the amino acid sequence of SEQ ID NO. 3, and VH-CDR3 comprises the amino acid sequence of SEQ ID NO. 4; and (b) VL comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO. 17, wherein VL-CDR1 comprises the amino acid sequence of SEQ ID NO. 6, VL-CDR2 comprises the amino acid sequence of SEQ ID NO. 7, and VL-CDR3 comprises the amino acid sequence of SEQ ID NO.

8. Claim 12 In any one of claims 1 to 11, VH and VL are isolated antibodies or antigen-binding fragments thereof linked in scFv or diabody. Claim 13 An isolated antibody or its antigen-binding fragment comprising a kappa, lambda, human IgG1, human IgG2, human IgG3, or human IgG4 constant region, in any one of claims 1 to 12. Claim 14 In paragraph 13, an isolated antibody or its antigen-binding fragment comprising a silenced Fc region. Claim 15 In claim 14, the isolated antibody in which the silenced Fc region comprises sequence number 43 or 44. Claim 16 In claim 15, an isolated antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO.

61. Claim 17 In paragraph 13 or 14, the isolated antibody, which is the whole antibody. Claim 18 In claim 13, the kappa constant region is an isolated antibody or its antigen-binding fragment having the amino acid sequence of SEQ ID NO.

41. Claim 19 In claim 13, the human IgG1 constant region is an isolated antibody having the amino acid sequence of SEQ ID NO. 42, SEQ ID NO. 43, or SEQ ID NO.

44. Claim 20 In paragraph 13, an antigen-binding fragment that is an analog of F(ab), F(ab'), or F(ab'). Claim 21 In claim 20, an antigen-binding fragment comprising a human IgG1 F(ab') constant region having the amino acid sequence of SEQ ID NO.

76. Claim 22 In claim 21, the F(ab') heavy chain is an antigen-binding fragment having the amino acid sequence of SEQ ID NO.

78. Claim 23 In claim 20, an antigen-binding fragment comprising a human IgG4 F(ab') constant region having the amino acid sequence of SEQ ID NO.

79. Claim 24 In paragraph 23, the F(ab') heavy chain is an antigen-binding fragment having the amino acid sequence of SEQ ID NO.

80. Claim 25 An antigen-binding fragment comprising a kappa invariant region having the amino acid sequence of SEQ ID NO. 41, in any one of claims 20 to 24. Claim 26 In paragraph 20, the F(ab') analog is an antigen-binding fragment comprising IgG1 or IgG4 CH1 F174C substitution and Cκ S162C substitution. Claim 27 In claim 26, the F(ab') analog is an antigen-binding fragment further comprising a Cκ C214S substitution, a Cκ C214S substitution and an IgG1 hinge C233S substitution, or a Cκ C214S substitution and an IgG1 hinge cleavage at T238. Claim 28 In paragraph 26, the F(ab') analog is an antigen-binding fragment further comprising IgG4 CH1 C127S substitution, or IgG4 CH1 C127S substitution and Cκ C214S substitution. Claim 29 In claim 20, the IgG1 constant region is an antigen-binding fragment having the amino acid sequence of SEQ ID NO. 81, SEQ ID NO. 85, SEQ ID NO. 90, SEQ ID NO. 95, SEQ ID NO. 97, or SEQ ID NO.

99. Claim 30 In claim 27, an antigen-binding fragment comprising a heavy chain having the amino acid sequence of SEQ ID NO. 78, SEQ ID NO. 82, SEQ ID NO. 86, SEQ ID NO. 92, SEQ ID NO. 96, SEQ ID NO. 98, SEQ ID NO. 102, SEQ ID NO. 105, SEQ ID NO. 106, SEQ ID NO. 108, SEQ ID NO. 109, SEQ ID NO. 110, SEQ ID NO. 111, SEQ ID NO. 113, or SEQ ID NO.

114. Claim 31 In claim 27, an antigen-binding fragment comprising a heavy chain having the amino acid sequence of SEQ ID NO. 92, SEQ ID NO. 98, or SEQ ID NO.

102. Claim 32 In claim 27, an antigen-binding fragment comprising a heavy chain having the amino acid sequence of SEQ ID NO. 98, SEQ ID NO. 102, SEQ ID NO. 110, SEQ ID NO. 111, SEQ ID NO. 113, or SEQ ID NO.

114. Claim 33 In claim 20, the IgG4 constant region is an antigen-binding fragment having the amino acid sequence of SEQ ID NO. 83, SEQ ID NO. 87, SEQ ID NO. 93, or SEQ ID NO.

103. Claim 34 In claim 33, an antigen-binding fragment comprising a heavy chain having the amino acid sequence of SEQ ID NO. 84, SEQ ID NO. 88, SEQ ID NO. 94, or SEQ ID NO.

104. Claim 35 In claim 26, Cκ is an antigen-binding fragment having the amino acid sequence of SEQ ID NO. 89 or SEQ ID NO.

100. Claim 36 In claim 35, an antigen-binding fragment comprising a light chain having the amino acid sequence of SEQ ID NO. 91, SEQ ID NO. 101, SEQ ID NO. 107, or SEQ ID NO.

112. Claim 37 An antigen-binding fragment according to claim 31 or 36, comprising: (a) a heavy chain having the amino acid sequence of SEQ ID NO. 92 and a light chain having the amino acid sequence of SEQ ID NO. 91; (b) a heavy chain having the amino acid sequence of SEQ ID NO. 98 and a light chain having the amino acid sequence of SEQ ID NO. 91; or (c) a heavy chain having the amino acid sequence of SEQ ID NO. 102 and a light chain having the amino acid sequence of SEQ ID NO.

101. Claim 38 An antigen-binding fragment according to claim 32 or 36, comprising: (a) a heavy chain having the amino acid sequence of SEQ ID NO. 98 and a light chain having the amino acid sequence of SEQ ID NO. 91; (b) a heavy chain having the amino acid sequence of SEQ ID NO. 102 and a light chain having the amino acid sequence of SEQ ID NO. 101; (c) a heavy chain having the amino acid sequence of SEQ ID NO. 110 and a light chain having the amino acid sequence of SEQ ID NO. 107; (d) a heavy chain having the amino acid sequence of SEQ ID NO. 113 and a light chain having the amino acid sequence of SEQ ID NO. 112; (e) a heavy chain having the amino acid sequence of SEQ ID NO. 111 and a light chain having the amino acid sequence of SEQ ID NO. 107; or (f) a heavy chain having the amino acid sequence of SEQ ID NO. 114 and a light chain having the amino acid sequence of SEQ ID NO.

112. Claim 39 In any one of paragraphs 1 to 38, the humanized antibody or its antigen-binding fragment is at an agglutination temperature of 60°C or higher (T agg ) and melting temperature of 65°C or higher (T M An isolated antibody or its antigen-binding fragment having ). Claim 40 In any one of claims 1 to 39, the humanized antibody or its antigen-binding fragment is an isolated antibody or its antigen-binding fragment having a low propensity for self-interaction. Claim 41 In any one of claims 1 to 40, the humanized antibody or its antigen-binding fragment is (a) double-stranded DNA and insulin; (b) baculovirus particles; (c) human cell surface proteins and secreted proteins; or (d) an isolated antibody or its antigen-binding fragment lacking multiple reactivity to any combination of (a) to (c). Claim 42 In any one of claims 1 to 41, the humanized antibody or its antigen-binding fragment is an isolated antibody or its antigen-binding fragment having minimal to undetectable off-target binding. Claim 43 An isolated antibody or its antigen-binding fragment comprising a thiolized lysine residue in Lys248 or Lys288 of any one of claims 1 to 42. Claim 44 An isolated antibody or its antigen-binding fragment, which is F(ab), F(ab'), F(ab')2, scFv, diabody, or minibody, in any one of claims 1 to 14. Claim 45 An F(ab') analog comprising VH and VL of the isolated antibody or its antigen-binding fragment according to any one of claims 1 to 11. Claim 46 F(ab') analog, comprising a rearranged interchain disulfide bond and an antigen-binding domain that binds to the CT8 epitope of CD8. Claim 47 F(ab') analog, comprising an antigen-binding domain that competes for binding to an epitope bound by the anti-CD8 antibody CT8, TRX2, or YTC182.

20. Claim 48 An F(ab') analogue comprising, in claim 46 or 47, means for binding to a CT8 epitope or means for binding to the same epitope as that bound by CT8, TRX2, and / or YTC182.

20. Claim 49 An F(ab') analog comprising IgG1 or IgG4 CH1 F174C substitution and Cκ S162C substitution in any one of claims 46 to 48. Claim 50 An F(ab') analog of claim 49, further comprising a Cκ C214S substitution, or a Cκ C214S substitution and an IgG1 hinge C233S substitution, or a Cκ C214S substitution and an IgG1 hinge cleavage at T238. Claim 51 In paragraph 49, an F(ab') analog further comprising IgG4 CH1 C127S substitution, or IgG4 CH1 C127S substitution and Cκ C214S substitution. Claim 52 An F(ab') analogue according to any one of claims 46 to 51, comprising VH and VL, wherein VH comprises a heavy chain CDR1 (VH-CDR1) having the amino acid sequence of SEQ ID NO. 220, a VH-CDR2 having the amino acid sequence of SEQ ID NO. 221, and a VH-CDR3 having the amino acid sequence of SEQ ID NO. 222; wherein VL comprises a CDR1 (VL-CDR1) having the amino acid sequence of SEQ ID NO. 223, a VL-CDR2 having the amino acid sequence of SEQ ID NO. 224, and a VL-CDR3 having the amino acid sequence of SEQ ID NO.

225. Claim 53 An F(ab') analogue comprising VH and VL of YTC182.20 in any one of claims 46 to 51. Claim 54 An F(ab') analog comprising VH and VL of CT8 in any one of claims 46 to 51. Claim 55 In any one of claims 46 to 51, (a) a heavy chain variable region (VH) comprising an amino acid sequence having at least 90% identity with the amino acid sequence of the framework region of SEQ ID NO. 9 or 31 (wherein VH comprises a heavy chain CDR1 (VH-CDR1) comprising the amino acid sequence RYTFTDYX1LH (SEQ ID NO. 45), a VH-CDR2 comprising the amino acid sequence FIYPYX1GGTG (SEQ ID NO. 46) or FIYPYX2GGTG (SEQ ID NO. 47), and a VH-CDR3 having the amino acid sequence DHRYX1EGVSFDY (SEQ ID NO. 48); and (b) a light chain variable region (VL) comprising an amino acid sequence having at least 90% identity with the amino acid sequence of the framework region of SEQ ID NO. 15 or 37 (wherein VL comprises a CDR1 (VL-CDR1) comprising the amino acid sequence RASESVX3GFGX1SFMN (SEQ ID NO. 49), a VL-CDR2 comprising the amino acid sequence LASX2LES (SEQ ID NO. 50), and a VL-CDR3 comprising the amino acid sequence QQX2X2EX3PYT (SEQ ID NO. 51), wherein each X1 is independently N, S, Q, or A; each X2 is independently N, Q, D, S, or A; and each X3 is independently D, E, S, or A, an F(ab') analog. Claim 56 LNP comprising an isolated antibody of any one of claims 1 to 55 or an antigen-binding fragment thereof conjugated to a lipid nanoparticle (LNP). Claim 57 In paragraph 56, a structure of about 35 to about 65 mol% Ionizable cationic lipids of (CICL) (wherein R is , or LNP, comprising a lipid formulation comprising about 0.5 to about 3 mol% of PEG-lipids (wherein the PEG-lipids include functioned PEG-lipids and non-functioned PEG-lipids), about 7 to about 13 mol% of phospholipids, and about 27 to about 50 mol% of sterols, wherein the antibody or its antigen-binding fragment is conjugated to the functioned PEG-lipids. Claim 58 In paragraph 56 or 57, a) about 40 mol% to about 62 mol% ionizable cationic lipids, about 7 mol% to about 13 mol% phospholipids, about 30 mol% to about 50 mol% sterols, about 0.5 mol% to about 3 mol% total functionalized PEG-lipids and non-functionalized PEG-lipids, and about 0.1 mol% to 0.3 mol% functionalized PEG-lipids; b) about 50 mol% CLCL, about 10 mol% phospholipids, about 38.5 mol% sterols, about 1.4 mol% non-functionalized PEG-lipids, and about 0.1 mol% functionalized PEG-lipids; c) about 58 mol% CLCL, about 10 mol% phospholipids, about 30.5 mol% sterols, about 1.4 mol% non-functionalized PEG-lipid, and about 0.1 mol% of functionalized PEG-lipid; or d) a lipid composition comprising about 62 mol% CLCL, about 10 mol% phospholipid, about 26.5 mol% sterol, about 1.4 mol% non-functionalized PEG-lipid, and about 0.1 mol% functionalized PEG-lipid, LNP. Claim 59 In Article 57 or 58, R of CICL is LNP, wherein the phospholipid is distearoylphosphatidylcholine (DSPC), the sterol is cholesterol, the non-functionalized PEG-lipid is 1,2-distearoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000 (DSPE-PEG(2k)), and the functionalized PEG-lipid is DSPE-PE(2k)-maleimide (DSPE-PE(2k)-MAL). Claim 60 In any one of claims 57 to 59, the isolated humanized monoclonal antibody or its antigen-binding fragment is covalently attached to a PEG-lipid functioned through a modified lysine residue or cysteine ​​residue of the antibody or its binding fragment, LNP. Claim 61 LNP comprising an isolated antibody of any one of claims 1 to 44 or an antigen-binding fragment thereof conjugated to a lipid nanoparticle (LNP). Claim 62 LNP comprising an F(ab') analog of any one of claims 45 to 55 conjugated to a lipid nanoparticle (LNP). Claim 63 Lipid nanoparticles (LNPs) conjugated with F(ab') analogs containing rearranged interchain disulfide bonds. Claim 64 A composition comprising an isolated antibody or its antigen-binding fragment of any one of claims 1 to 44, an F(ab') analog of any one of claims 45 to 55, or an LNP of any one of claims 56 to 63, and a pharmaceutically acceptable carrier or excipient. Claim 65 A method for delivering a payload into a CD8-positive cell, comprising the step of contacting the CD8-positive cell with the LNP of any one of claims 56 to 63 or the composition of claim 64. Claim 66 In paragraph 65, the method comprises the step of delivering the payload, which includes the step of transfecting CD8-positive cells. Claim 67 In paragraph 66, the method comprises a payload including mRNA, circular RNA, self-amplifying RNA, or guide RNA. Claim 68 In paragraph 65, the step of contacting is a method that occurs in vivo, extracorporeally, or in vitro. Claim 69 In paragraph 65, the payload is a method for mediating the reprogramming of CD8-positive cells. Claim 70 In claim 69, the method comprises a payload containing a nucleic acid encoding an immune receptor or an immune cell engager. Claim 71 In claim 69, the method comprises a payload comprising a nucleic acid encoding a gene / genome editing enzyme and / or a guide RNA or other components of a gene / genome editing system.

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