Humanized anti-cd8 antibodies and uses thereof

Abstract

The present disclosure provides humanized antibodies and antigen-binding domains thereof that bind CD8a, as well as other antibody formats comprising these antigen-binding domains, their use as targeted moieties on lipid nanoparticles (tLNPs) to deliver therapeutic payloads (such as nucleic acid molecules) or other types of payloads. The present disclosure also relates to pharmaceutical compositions comprising humanized anti-CD8a antibodies and CD8-targeted tLNPs that encapsulate a payload.

Description

Reference to electronic sequence list

[0001] This application contains a sequence list, which has been electronically submitted and is incorporated herein by reference in its entirety. The sequence list was created on December 15, 2024, named “23-1742-US _SequenceListing.xml”, and has a size of 250,755 bytes. Cross-reference to related applications

[0002] This application claims priority to the following U.S. provisional applications: 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 of which are expressly incorporated herein by reference. Background Technology

[0003] The differentiation cluster 8 (CD8) antigen is a cell surface glycoprotein found on most cytotoxic T lymphocytes, which helps mediate efficient cell-cell interactions. The CD8 antigen binds to class I major histocompatibility complex (MHC) molecules and acts as a co-receptor for the T cell receptor (TCR) on T lymphocytes to recognize antigens presented by antigen-presenting cells (APCs) in the context of class I MHC molecules. The co-receptor functions as a homodimer composed of two α chains (CD8 subunit α-CD8α) or as a heterodimer composed of one CD8α and one CD8β chain (CD8β).

[0004] CD8-positive T cells are mediators of adaptive immunity. They 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).

[0005] Several anti-CD8 antibodies have been developed. However, many of these antibodies have already been produced in mice, where they possess the ability to elicit an immune response if introduced into humans. Therefore, there remains a need for humanized (or human) anti-CD8 antibodies with reduced immunogenicity potential while retaining biological activity, particularly binding to CD8-expressing cells. Furthermore, such humanized anti-CD8 antibodies must possess biophysical properties suitable for clinical development and manufacturing. Therefore, there is a need to develop modified anti-CD8 antibodies for therapeutic purposes. Attached Figure Description

[0006] This disclosure will be better understood when considered in the following detailed description, and features, aspects, and advantages in addition to those set forth above will become apparent. Such a detailed description is illustrated in the following figures.

[0007] Figure 1A 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 is shown. Bold text indicates the CDR region based on AbM definition (see bioinf.org.uk / abs / ). Underlined text indicates that frame residues will be mutated to amino acids found in the parental mouse antibody. Other possible variations are shown below the sequence. Seq: Simple consecutive amino acid numbering. AbM: Chothia amino acid numbering.

[0008] Figure 1B The humanized sequence of the variable light chain (VL) of CBD1017vl (mouse anti-CD8 antibody clone CT8) in the IGKV1-39*01 (VK1-39) line is shown. Bold text indicates the CDR region based on AbM definition. Underlined text indicates that frame residues will be mutated to amino acids found in the parental mouse antibody. Other possible variations are shown below the sequence. Seq: Simple consecutive amino acid numbering. AbM: Chothia amino acid numbering.

[0009] Figure 1C 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 is shown. Dark bars indicate CDR regions. Amino acid residues in CBD1017vh that differ from the VH1-18 sequence are shaded, as are residues in the humanized sequence using mouse residues. Simple consecutive amino acid numbering is used.

[0010] Figure 1D 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 is shown. Dark bars indicate CDR regions. Amino acid residues in CBD1017vh that differ from the VH1-18 sequence are shaded, as are residues in the humanized sequence using mouse residues. Simple consecutive amino acid numbering is used.

[0011] Figure 2A This is a summary of the binding constants of different humanized anti-CD8 antibody binding fragments (Fab) based on the VH1-46 and VK1-39 lineages, measured at 30°C and 37°C using biolayer interference (BLI) kinetic assays.

[0012] Figure 2B The binding kinetics of CBD1033Fab to CD8αα antigen at specified concentrations are shown in a biolayer interferometric kinetic assay at 30°C and 37°C. Global fitting was performed using a 1:1 fitting model, as described below, with GatorOne software.

[0013] Figure 2C This is a summary of the steady-state binding constants of different humanized anti-CD8 antibodies with hIgG1-LALAPA (human IgG1 (hIgG1) isotypes with humanized Fc silencing mutations L234A, L235A and P329A (LALAPA) (SEQ ID NO:43) humanized using the biolayer interference kinetic assay) as measured by the biolayer interference kinetic assay.

[0014] Figure 2D This paper presents a binding kinetics sensor plot of the interaction between CBD1033 IgG1 and a specified concentration of human CD8αα antigen in a biolayer interferometric kinetic assay. A global fit was performed using the GatorOne software described below with a 1:1 fitting model.

[0015] Figure 2E It shows the basis Figure 2D The data in the study analyzed the steady-state of the humanized antibody CBD1033, in which the human IgG1 constant region with Fc silencing mutations L234A, L235A and P329A (LALAPA) (CBD1033-hIgG1-LALAPA) (SEQ ID NO: 43) interacts with human CD8αα.

[0016] Figure 2F This is a summary of the steady-state binding constants of different humanized anti-CD8 antibodies based on human CD8αα-interacting VH1-18 and VK3D-11 lineages with hIgG1-LALAPA (human IgG1 (hIgG1) isotypes with Fc silencing mutations L234A, L235A and P329A (LALAPA) (SEQ ID NO: 43)) as measured by biolayer interference kinetics.

[0017] Figure 2G It shows the method for determining Figure 2F K in the report D Biological layer interference binding kinetics of humanized CBD1043 anti-CD8 antibody at a specified concentration of human CD8αα. Global fitting was performed using the GatorOne software described below with a 1:1 fitting model.

[0018] Figure 2H It shows the basis Figure 2G The data in the middle is used to determine Figure 2F K in the report D Steady-state analysis of humanized CBD1043 anti-CD8 antibody.

[0019] Figure 2I This is a summary of the binding constants of CBD1033 and CBD1017ch Fab for human and cynomolgus monkey CD8αα homodimers and CD8αβ heterodimers, measured at 37°C or 30°C using the biolayer interferodynamics method.

[0020] Figure 2J This paper presents a binding kinetic sensor plot showing the interaction between humanized CBD1033 and CBD1017chFab and a specified concentration of cynomolgus CD8αα homodimeric antigen at 30 °C using a biolayer interferometric kinetic assay. A global fit was performed using the GatorOne software described below with a 1:1 fitting model.

[0021] Figure 2K This is a summary of the binding constants of the intact CBD1033 antibody against human or cynomolgus CD8αα homodimer or CD8αβ heterodimer, as measured by surface plasmon resonance (SPR).

[0022] Figure 2L The binding kinetics of the intact humanized CBD1033 antibody to human or cynomolgus CD8αα homodimer or CD8αβ heterodimer in the SPR assay are shown in the sensory map. Binding was analyzed serially with two-fold dilutions from 200 nM to 6.25 nM for the cynomolgus CD8αβ heterodimer, except for the cynomolgus CD8αβ heterodimer. For the cynomolgus CD8αβ heterodimer, binding was analyzed serially with two-fold dilutions from 400 nM to 12.5 nM. Global fitting was performed using the GatorOne software described below with a 1:1 fitting model.

[0023] Figure 3A The binding of different concentrations of intact anti-CD8 antibodies containing human IgG1 (hIgG1) isotypes with Fc silencing mutations L234A, L235A, and P329A (LALAPA) (SEQ ID NO: 43) to CD8-overexpressing HEK293T cells is illustrated using a normalized geometric median fluorescence intensity (gMFI) assay. Binding was measured by fluorescence from anti-human Fc secondary antibodies conjugated to the BV421 fluorophore that recognizes the binding agent. gMFI was normalized relative to the gMFI of the secondary antibody in the absence of an anti-CD8α binding agent.

[0024] Figure 3B Through Figure 3AEC50 of anti-CD8α antibody as determined by normalized gMFI in the experiment shown 50 Summary.

[0025] Figures 4A to 4D The transfection rates of a group of targeted lipid nanoparticles (tLNPs) derived from different donors and encapsulated with mRNA encoding mCherry, conjugated with a specified anti-CD8 antibody, are shown. Figures 4A to 4B ) and level of expression ( Figures 4C to 4D The tLNP added to the cells provided 0.6 µg, 0.3 µg, 0.15 µg, 0.075 µg, and 0 µg of mRNA. Transfection rate was measured as the percentage of CD4-CD8+ T cells expressing mCherry.

[0026] Figures 5A to 5C This study compares mCherry expression after transfection with mCherry-encoding mRNA encapsulated in CD8-targeting tLNPs using CBD1017ch, humanized antiCD8 binding agents derived from antiCD8 antibody clones CT8 and OKT8, and cetuximab (negative control) as the targeting moiety.

[0027] Figure 6 The normalized fold increase in gMFI, measured by flow cytometry, is shown after different concentrations of the anti-CD8α binding agent CBD1033 bind to CD8-expressing SupT1 and HPB-ALL lymphoma T cells. The fluorescence measurement is based on the fluorescence of the anti-human Fc secondary antibody conjugated to the BV421 fluorophore that recognizes the binding agent. gMFI is normalized relative to the gMFI of the secondary antibody in the absence of the anti-CD8α binding agent.

[0028] Figure 7A and Figure 7B Transfection rate (% mCherry positive) and % mCherry positive relative to equivalent soluble fluorescent dye (MESF) molecules are shown, as measured by flow cytometry of cynomolgus CD8+ T cells transfected with mRNA encoding mCherry encapsulated in anti-CD8α binding agent-targeting lipid nanoparticles (tLNP).

[0029] Figure 8A and Figure 8B Normalized to show a fold increase in gMFI relative to background as measured by flow cytometry, demonstrating the binding of chimeric anti-CD8α binders and humanized anti-CD8α binders to CD4-CD8+ rhesus monkey, cynomolgus monkey, or human T cells at different concentrations. Binding was measured by fluorescence measurements of anti-human Fc secondary antibodies conjugated to the BV421 fluorophore recognizing the binder. gMFI was normalized relative to the gMFI of the secondary antibody in the absence of the anti-CD8α binder.

[0030] Figure 9A and Figure 9B EC values ​​in µg / mL and nM are listed separately. 50 The values ​​showed that, compared to humanized 5D7 antibodies (anti-CD5 antibodies), anti-CD8 binding agents from different species (cross-species) had comparable CD8 specificity among T cells, and they lacked binding to CD4+ T cells.

[0031] Figure 10 This study demonstrates the efficient and cell-specific in vivo delivery of mCherry mRNA to CD8+ T cells using an intact IgG1 antibody silenced by the anti-CD8 binding agent CBD1033 Fc as the targeting moiety on tLNP. The antibody-to-mRNA (w / w) ratios for tLNP were 0.37, 0.72, and 1.09, as indicated.

[0032] Figure 11A The affinity-capture self-interaction nanoparticle spectrometry (AC-SINS) scores of six anti-CD8α hIgG1-LALAPA antibodies are shown. All six binders showed a low tendency for self-association. Bercocilizumab was used as a positive control, while aliximumab and bercocilizumab NEI were used as negative controls.

[0033] Figure 11B yes Figure 11A The results are summarized graphically. The results for positive and negative controls are plotted as horizontal lines, while the results for the six anti-CD8α antibodies are plotted as circles.

[0034] Figure 12A The melting temperature (T) of the anti-CD8α hIgG1-LALAPA antibody is shown. m The dot plot distribution is shown. Shaded areas (above 65°C) indicate a range with good exploitability. All results are within the range sufficient to meet chemical, manufacturing, and control (CMC) exploitability.

[0035] Figure 12B T is measured by differential scanning fluorometry (DSF). m And the aggregation temperature (T) as measured by static light scattering (SLS) of anti-CD8 antibodies. agg (Summary)

[0036] Figure 13A The dot plot distribution of the multireactive ELISA scores for six anti-CD8 binding agents against double-stranded DNA (dsDNA) and insulin is shown.

[0037] Figure 13B yes Figure 13A The table shows the experimental values.

[0038] Figure 14 Data from a multiple reactive ELISA of baculovirus particles (BVP) are described. PC: positive control. NC: negative control. This assay measures nonspecific binding to an array of membrane proteins on baculovirus particles carrying multiple proteins from the host cells that produce the virus.

[0039] Figure 15 The binding interactions of anti-CD8α binders to human cell membrane proteins are shown in a membrane proteome array assay. No other significant specific interactions were observed besides the CD8A gene product.

[0040] Figure 16A The percentage of deamidation of CBD1017ch at the N55 position in the VH-CDR1 region is shown after 7 days at high pH (8.5) and high temperature (40°C).

[0041] Figure 16B The percentage of deamidation of anti-CD8α binders CBD1033, CBD1035 and CBD1039 at the N55 position in the VH-CDR2 region is shown after 7 days at high pH (8.5) and high temperature (40°C).

[0042] Figure 16C The percentage of deamidation of CBD1033 at the N55 position in the VH-CDR2 region over time is shown at high pH (8.5) and high temperature (40°C).

[0043] Figure 16D This demonstrates the minimal loss of binding affinity against CD8α binders due to high pH stress.

[0044] Figure 16E The effect of the mutation at the N55 position in the VH-CDR2 region on binding affinity is shown.

[0045] Figure 16F The effect of mutations at the N55 position of the VH-CDR2 region or the D30 position of the VL-CDR1 region, or both, on the binding affinity of the Fab fragment is shown.

[0046] Figure 17A and Figure 17B The study demonstrated that the deamidation process, mimicked by the N55 mutation to aspartic acid (D), was reduced in two separate donors via mCherry transfection with anti-CD8α tLNP. Mutations at the N55 position in the VH-CDR2 region encoding glutamine (Q), serine (S), or alanine (A) did not result in loss of this function.

[0047] Figure 17C Is Figure 17A and Figure 17B A summary of mutations tested and evaluated.

[0048] Figure 18A and Figure 18B The transfection efficiency and expression level of anti-CD8α-targeting tLNPs in T cells are shown. Various antibody forms of the anti-CD8α antigen-binding domain CBD1033 (biantibody synthetic hinge [groups (GRP) 1-3], biantibody IgG3 hinge [groups 4-7], recombinant IgG1 F(ab')2 [groups 8-10], recombinant IgG4 F(ab')2 [groups 11-13], or enzymatically digested IgG1 F(ab')2 [groups 14-16]) were conjugated to tLNPs. Antibody densities and reduction conditions also varied (see Example 8 and Table 15 for details). tLNPs with intact IgG1 antibodies silencing CBD1033 Fc were used as a positive control (group 17), and untransfected cells (NTD) were used as a negative control.

[0049] Figure 19A The design strategies for disulfide bond engineered F(ab') constructs are summarized.

[0050] Figure 19B It is shown that F(ab') dimers are not present because the engineered interchain disulfide bonds of F(ab') are purified under reducing conditions without breaking them.

[0051] Figure 20A Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of anti-CD8 F(ab') analogs CBD1033.37 or CBD1033.24 under complete reduction (R) or non-reduction (NR) conditions is shown.

[0052] Figure 20B The 280 nm absorption peaks corresponding to the anti-CD8 Fab' binders CBD1033.37 and CBD1033.24 are shown in the size exclusion high performance liquid chromatography (SEC-HPLC) chromatogram.

[0053] Figure 20C Results are shown from non-reducing liquid chromatography-mass spectrometry (LC-MS) analysis of anti-CD8 F(ab') fragments with high abundance peaks corresponding to the F(ab') fraction of CBD1033.37 or CBD1033.24.

[0054] Figure 21A This is a summary of the binding constants of various engineered anti-CD8 binding F(ab') fragments measured by the biolayer interferodynamics method.

[0055] Figure 21B The binding kinetics of the interaction between CBD1033.37 F(ab') and a specified concentration of CD8αα homodimer is shown in a biolayer interferometric kinetics assay. A global fit was performed using the GatorOne software described below with a 1:1 fitting model.

[0056] Figure 22A The conjugation reaction of humanized anti-CD8 F(ab') with maleimide-PEG-biotin was demonstrated.

[0057] Figure 22B An immunoblot of a biotin-conjugated F(ab') analogue, as detected by streptavidin-horseradish peroxidase (HRP), is shown.

[0058] Figure 22C The 280 nm absorbance peak corresponding to the biotin-conjugated F(ab') fraction is shown in the SEC-HPLC chromatogram.

[0059] Figure 22D The results of LC-MS analysis of maleimide-PEG-biotin conjugated CBD1033.24 and CBD1033.37 are shown, which have high abundance peaks corresponding to the F(ab') fraction, respectively.

[0060] Figure 22E The abundance of modifications detected in conjugated CBD1033.24 and CBD1033.37 is shown 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 are numbered sequentially. C227 and C230 correspond to C239 in the Kabat numbers for IgG4 and IgG1, respectively.

[0061] Figure 23A This is a summary of the binding constants of maleimide-PEG-biotin conjugated CBD1033.24 and CBD1033.37 F(ab') against human CD8α mouse Fc fusion protein, as measured at 37°C by biolayer interferokinetic assay.

[0062] Figure 23B The binding kinetics of maleimide-PEG-biotin-conjugated CBD1033.37 to a specified concentration of human CD8α mouse Fc fusion protein at 37 °C is shown in a biolayer interferometric kinetics assay. Global fitting was performed using a 1:1 fitting model with GatorOne software as described below.

[0063] Figure 24AThe expression level of mCherry in primary human activated T cells transfected with anti-CD8α F(ab')-conjugated lipid nanoparticles (tLNPs) encapsulating mCherry mRNA is shown, expressed as gMFI. The tLNPs added to the cells were formulated with a binder-to-mRNA ratio (w / w) of 0.1, 0.3, 0.5, 0.75, or 0.35. Transfection rate was measured as the percentage of CD4-CD8+ T cells expressing mCherry. tLNPs conjugated to CBD1033.29 (containing the LALAPA Fc silencing mutation (CBD1033.3) and thiolated intact IgG1 via the AJICAP process) were used as a positive control.

[0064] Figure 24B This study demonstrates efficient and cell-specific in vivo delivery of mCherry mRNA to human CD8+ T cells in the blood and spleen of NCG mice implanted with human PBMCs, using natural and engineered disulfide-bonded anti-CD8 F(ab') as the targeting moiety on tLNP. The antibody-to-mRNA (w / w) ratio on tLNP was 0.35 for the intact antibody (CBD1033.29) and 0.3 for F(ab')s. CBD1033.29 is a CBD1033 antigen-binding domain linked to SEQ ID NO: 43 (an IgG1 constant region with an Fc-silenced LALAPA mutation (CBD1033.3) thiolated via the AJICAP process).

[0065] Figure 25A and Figure 25B Expression levels in expanded human CD8+ T cells from two donors are shown. These cells were transfected in vitro with modified tLNP-98219, in which various engineered anti-CD8 F(ab') analogues were replaced with intact antibodies as the targeting moiety. Transfection was performed in duplicate with 0.6 µg doses of mRNA. CAR expression in CD8+ T cells transfected with duplicate tLNPs from both donors is shown as transfection efficiency (percentage CAR+) at 24 hours post-transfection. Figure 25A ) and CAR expression level (median fluorescence intensity) Figure 25B tLNP-98219 is an anti-CD8 targeting composition F9 tLNP encapsulating RM_61461 mRNA (SEQ ID NO: 195) encoding anti-CD19 CAR2. The CBD1033.29 positive control is an intact antibody IgG1 (LALAPA) that is thiolated and conjugated to LNP via the AJICAP process and silenced.

[0066] Figure 26A and Figure 26BThe CAR transfection rates of CD4+ and CD8+ T cells from two donors, expanded human T cells, are shown. These T cells were transfected with anti-CD8-targeting tLNPs encapsulating anti-CD19 CAR-encoding mRNA. Two mRNA constructs encoding anti-CD19 were used: the modified RM_61461 construct (SEQ ID NO: 195) or the basic RM_61512 construct (SEQ ID NO: 196). The modified construct is known to have higher expression than the basic construct, and both were used as assay controls. Groups 1-16 are tLNPs conjugated with various anti-CD8 F(ab') antibodies with different antibody design and a tendency for engineered mutations in the variable domain. Groups 1-16 express the modified mRNA RM_61461 (SEQ ID NO: 195). The improved (RM_61461) and basic (RM_61512) control mRNAs were encapsulated in tLNPs, with the targeting portion containing CBD1033.29 conjugated to tLNPs (the improved control and the basic control, respectively).

[0067] Figure 26C and Figure 26D It shows that respectively in and Figure 26A and Figure 26B In a similar experiment, the CAR expression levels of CD4+ and CD8+ T cells were measured using phycoerythrin fluorescence.

[0068] Figure 26E It shows Figures 26A to 26D A summary of the CBD number for each group, along with the design and tendency for engineered mutations.

[0069] Figure 27A This is the workflow for cross-linking mass spectrometry studies to identify interaction sites between antibodies and their antigens. This applies to the identification of epitopes in CBD1033.

[0070] Figure 27B The identification and mapping of cross-linked amino acid positions on an existing structural model of human CD8αα homodimer are shown.

[0071] Figure 27C The structure of human CD8αα homodimer with identified epitopes is shown.

[0072] Figure 28AThe binding kinetics of a competitive binding experiment between CBD1033.3 and either OKT8 or TRX2 is shown in the sensory plot. After capturing CD8αα, CBD1033.3 was loaded to form a complex with CD8αα. While the subsequent addition of OKT8 resulted in a reaction shift, indicating binding to the CD8αα-CBD1033.3 complex, TRX2 did not induce a reaction shift, indicating that TRX2 could not bind to the CD8αα-CBD1033.3 complex.

[0073] Figure 28B A summary of competitive binding with human CD8αα homodimer is shown, where a spectral shift value below 0.7 indicates competitive binding.

[0074] Figure 28C This represents two epitope bins, each containing the same or overlapping epitopes bound by the antibody shown.

[0075] Figure 29A The in vitro mCherry expression levels in primary human T cells are shown using antibodies against CBD1033, TRX2, SK1, OKT8, humanized OKT8 variant 1 (VL and VH are shown in Table 16 as SEQ ID NO: 229 and 230, respectively, taken from US11254744B2) or humanized OKT8 variant 2 (LC and HC are shown in Table 16 as SEQ ID NO: 231 and 232, respectively, taken from US11739150B2) as binding sites for tLNPs of mCherry encoded by mRNA.

[0076] Figure 29B The use of CBD1033 or TRX2 antibodies as binding moieties of tLNPs encapsulating mCherry encoded by mRNA to equivalent in vivo mCherry delivery to CD8+ T cells in blood or spleen tissue is demonstrated. Detailed Implementation

[0077] This document provides humanized antibody antigen-binding domains that specifically bind to CD8α (also known as CD8a and CD8 alpha, and encoding a gene called CD8A), complete antibodies and other antibody forms containing these antigen-binding domains, their use as targeting portions (tLNPs) in lipid nanoparticles to deliver payloads (e.g., nucleic acid molecules), and compositions of anti-CD8α tLNPs. This document also provides compositions comprising humanized anti-CD8α antibodies, anti-CD8 tLNPs encapsulating payloads, and methods of using them. In a particular embodiment, the payload is mRNA. In a further embodiment, the mRNA encodes an antigen-specific protein that reprograms 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 adaptor.

[0078] This article specifically provides a CD8α binding moiety containing an immunoglobulin antigen-binding domain, which specifically binds to human CD8α in the CD8αα homodimer and CD8αβ heterodimer containing the frame region of genes derived from human germline heavy and light chain variable domains.

[0079] In some embodiments, the humanized anti-CD8α antibody and its antigen-binding fragment of this disclosure specifically bind to human and non-human primate (NHP) CD8. In some embodiments, the isolated humanized anti-CD8α monoclonal antibody or its antigen-binding fragment of this disclosure has an aggregation temperature ≥60°C (Tg). agg It has a melting temperature of ≥65°C, low self-interaction tendency (i.e., tendency to aggregate), lacks CT8 cross-reactivity, and lacks multi-reactivity with: (a) double-stranded DNA and insulin; (b) baculovirus particles; or (c) human cell surface and secretory proteins.

[0080] On the other hand, there are tLNPs containing an anti-CD8 targeting moiety, which binds to a proximal membrane epitope near the CD8 dimerization interface. Antibodies CT8, TRX2, and YTC182.20 compete for binding to this epitope. An epitope is a structural (i.e., non-linear) epitope containing or adjacent to amino acid 40-47, 86-95, and 103-106 of CD8α. This epitope (whether defined by cross-competition for antibody binding, antibody-antigen cross-linking, or localization within the secondary or tertiary structure of CD8) will be referred to herein as the CT8 epitope. Therefore, the antigen-binding domains of CT8, TRX2, and YTC182.20 constitute a means for binding to this CT8 epitope or for targeting tLNPs to CD8+ cells or CD8+ T cells. tLNPs whose targeting moiety contains an antigen-binding domain that binds to the CT8 epitope transfect CD8+ cells more efficiently than tLNPs whose targeting moiety contains an antigen-binding domain that recognizes some other CD8α epitopes. Therefore, tLNPs, whose targeting portion includes an antigen-binding domain that binds to the CT8 epitope, constitute a means for efficient transfection of CD8+ cells or CD8+ T cells.

[0081] It should be understood that the specific aspects described herein are not limited to the proposed specific embodiments and can vary. It should also be understood that the terminology used herein is for descriptive purposes only and is not intended to be limiting unless expressly defined herein. Furthermore, as those skilled in the art will recognize, the specific embodiments disclosed herein can be combined with other embodiments disclosed herein without limitation.

[0082] definition

[0083] Before elaborating on this disclosure in more detail, providing definitions for certain terms used throughout the disclosure may aid in understanding it. Other definitions are set forth throughout the disclosure.

[0084] Throughout this specification, unless the context clearly indicates otherwise, the terms “comprise” and “include”, and their variations (e.g., “comprises”, “comprising”, “includes”, and “including”) should be understood to mean including the said component, feature, element, or step, or a group of said components, features, elements, or steps, but not excluding any other component, feature, element, or step, or a group of said components, features, elements, or steps. Any of the terms “comprise”, “substantially constitutes”, and “consisting of” can be replaced by any of the other two terms while retaining their ordinary meaning.

[0085] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural referents. It should be understood that, as used herein, the terms “a” and “an” refer to “one or more” of the listed components.

[0086] Unless otherwise stated or apparent from the context and from the understanding of one of ordinary skill in the art, values ​​expressed herein as ranges may be assumed to be any particular value or subrange within the range in different embodiments of this disclosure, accurate to one-tenth of the unit of the lower limit of the range, unless the context expressly specifies otherwise.

[0087] As used herein and in the accompanying figures, ranges and quantities may be expressed as “about” a specific value or range. “About” also includes the exact quantity. For example, “about 5%” means “about 5%” as well as “5%”. The term “about” can also refer to ±10% of a given value or range of values. For example, “about 5%” means 4.5%–5.5%.

[0088] As used herein, the terms “or” and “and / or” are used to describe multiple components that are combined or mutually exclusive. For example, “x, y and / or z” can refer to a single “x”, a single “y”, a single “z”, “x, y and z”, “(x and y) or z”, “x or (y and z)”, or “x or y or z”.

[0089] Throughout this disclosure, unless otherwise stated, any concentration range, percentage range, ratio range, or integer range shall be construed as including any integer value within the range, and, where appropriate, fractions of that integer (such as tenths and hundredths of an integer). Furthermore, unless otherwise stated, any numerical range of this disclosure relating to any physical characteristic (such as polymer subunits, size, or thickness) shall be construed as including any integer within the range. Throughout this disclosure, unless otherwise specifically stated, numerical ranges include their enumerated endpoints.

[0090] The phrase “at least one of…” when followed by a list of items or elements refers to an open set of one or more elements in the list, which may, but does not necessarily, include more than one element.

[0091] As used herein, the term “subject” refers to a warm-blooded animal, such as a mammal, preferably a human or human child, that has or may have one or more diseases and conditions.

[0092] As used herein, a “derivative” refers to a chemically or biologically modified form of a compound that is structurally similar to the parent compound and (in practice or theoretically) derived from it. Generally, a “derivative” differs from an “analogue” in that the parent compound can be the starting material for producing the “derivative,” but the parent compound is not necessarily used as the starting material for producing the “analogue.” For example, a derivative may be more hydrophilic or hydrophobic than the parent compound, or it may have altered reactivity. Derivatives can be obtained through physical (e.g., biological or chemical) modifications of the parent compound, but derivatives can also be conceptually derived, for example, when a protein sequence is designed based on one or more known sequences, a nucleic acid encoding it is constructed, and a derived protein is obtained through the expression of the nucleic acid.

[0093] As used herein, “lipid nanoparticles” (LNPs) refer to solid particles, which are distinct from liposomes having aqueous chambers. The core of an LNP, like the chamber of a liposome, is surrounded by a lipid layer, which may be, but is not necessarily, a continuous lipid monolayer, bilayer, or multilayer with three or more lipid layers.

[0094] As used herein, "artificial sequence" or "synthetic sequence" refers to an amino acid or nucleotide sequence that is designed for a specific purpose and is not derived from a specific sequence existing in nature. Uses of such sequences can include linkers, spacers, restriction sites, and untranslated regions.

[0095] As used herein, “transfection” or “transfecting” refers to the introduction of nucleic acids into cells by non-viral methods. Transfection can be mediated by calcium phosphate, cationic polymers, magnetic beads, electroporation, and lipid-based reagents. In the preferred embodiments disclosed herein, transfection is mediated by solid lipid nanoparticles (LNPs) (including targeted LNPs (tLNPs)). The term transfection is used to distinguish it from transduction (the transfer of genetic material from cells to cells or from viruses to cells) and transformation (the uptake of extracellular genetic material by the cell’s natural processes). As used herein, phrases such as “delivering nucleic acids into cells” are synonymous with transfection.

[0096] As used in this article, “reprogramming” of immune cells refers to altering the antigen-specific function of immune cells by inducing the expression of exogenous T-cell receptors (TCRs), chimeric antigen receptors (CARs), or immune cell connectors (collectively referred to as “reprogramming agents”). Typically, T lymphocytes and natural killer (NK) cells can be reprogrammed using TCRs, CARs, or immune cell connectors, but only CARs or immune cell connectors can be used to reprogram monocytes. In the case of immune cell connectors, immune cells that connect to the connector and redirect their tracking of antigens are reprogrammed cells, regardless of whether these immune cells express the reprogramming agent. Reprogramming can be transient or persistent, depending on the nature of the engineered agent.

[0097] As used herein, “engineering agent” refers to a reagent that enables immune cells (particularly non-B lymphocytes or monocytes) to express a reprogramming agent. Engineering agents may include nucleic acids encoding the reprogramming agent, including mRNA. Engineering agents may also include nucleic acids that are components of or encode gene editing systems, such as RNA-directed nucleases, guide RNA, and nucleic acid templates for knocking in reprogramming agents or knocking out endogenous antigen receptors. Gene editing systems include base editors, leader editors, or gene writers. RNA-directed nucleases include CRISPR nucleases such as Cas9, Cas12, Cas13, Cas3, CasMINI, Cas7-11, and CasX. For transient expression of reprogramming agents (such as CARs), mRNA encoding the reprogramming agent can be used as an engineering agent. For persistent expression of reprogramming agents, such as exogenous, modified, or corrected genes (and their gene products), engineering agents may contain mRNA-encoded RNA-directed nucleases, guide RNA, nucleic acid templates, and other components of the gene / genome editing system.

[0098] Examples of gene-editing components encoded by nucleic acid molecules include mRNAs encoding the following: RNA-directed nucleases, gene or base-editing proteins, leader editing proteins, gene-writing 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), large-scale nucleases, transposases, retrotransposons, reverse transcriptases (e.g., M-MLV reverse transcriptases), nicking enzymes or inactive nucleases (e.g., Cas9, nCas9, dCas9), DNA recombinases, CRISPR nucleases (e.g., Cas9, Cas12, Cas13, Cas3, CasMINI, Cas7-11, CasX), DNA nicking enzymes, Cas9 nicking enzymes (e.g., D10A or H840A), or any fusions or combinations thereof. Other components include guide RNA (gRNA), single guide RNA (sgRNA), leader editing guide RNA (pegRNA), clustered regularly spaced short palindromic repeat (CRISPR) RNA (crRNA), trans-activating clustered regularly spaced short palindromic repeat (CRISPR) RNA (tracrRNA), or DNA molecules to be inserted or to serve as templates for double-strand break (DSB) repair at specific genomic loci. Genome, gene, and base editing technologies have been reviewed in 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. To the extent that they do not conflict with this disclosure, all content taught in each of these documents regarding the components and uses of this technology is incorporated herein by reference.

[0099] As used in this article, "target antigen" or "targeting antigen" refers to the surface antigen of immune cells that can be specifically bound by the targeting portion of tLNP.

[0100] As used herein, “tracking antigen” refers to an antigen recognized by a reprogramming agent, such as a TCR, CAR, or immune cell connector. The term target (or targeted) antigen is commonly used in the art to refer to any antigen that binds to an antigen (or other) receptor. This can be confusing when it involves antigens of two different functional classes. To avoid this confusion, as used herein, target (or targeted) antigen refers to an antigen that binds to the targeted portion of a nanoparticle, and tracking antigen (or cell or tissue or indication, etc.) refers to an antigen that binds to a reprogramming agent. (The terms “effectant to target ratio,” “target cell,” “off-target,” and “on-target” are replaced, as these tend to increase potential confusion rather than reduce it.) In the treatment of a disease, the tracking antigen will be expressed by pathogenic cells, but it can also be expressed by normal cells.

[0101] As used herein, "opsonizer" refers to a biological response modifier (BRM) that enhances the efficiency of engineered immune cells, expands the number of engineered immune cells available for use or the number of engineered cells in target tissues (e.g., tumors, fibrotic tissues, or tissues subjected to autoimmune attack), promotes the activity of engineered cells in target tissues, or broadens the scope of operational mechanisms that contribute to therapeutic immune responses. Opsonizers can be provided by delivering a nucleic acid encoding a tLNP. Exemplary BRMs include cytokines such as IL-7, IL-15, or IL-18.

[0102] As used herein, the term “immune cell” can refer to any cell of the immune system. However, certain aspects may exclude polymorphonuclear leukocytes and / or B cells, or limit to non-B lymphocytes such as T cells and / or NK cells, or limit to monocytes such as various forms of dendritic cells and / or macrophages.

[0103] As used herein, unless otherwise specified in the context, the term "nucleic acid" or "nucleic acid molecule" refers to RNA or DNA molecules, particularly those encoding expressible polypeptides. The description of the disclosed (t)LNP payload focuses on mRNA molecules having a typical mRNA structure. However, polypeptides can also be encoded and expressed by circular and self-amplifying (also known as self-replicating) RNA molecules. Therefore, the sequence of any linear mRNA molecule disclosed herein can be incorporated into circular or self-amplifying / self-replicating RNA molecules. Similarly, each of these RNA molecules can be encoded as a DNA molecule. Each of the disclosed nucleic acid sequences in RNA or DNA should be understood as disclosing the corresponding DNA or RNA sequence, respectively.

[0104] As used herein, “antibody” refers to a protein containing an immunoglobulin domain with a hypervariable region that determines the specificity of antibody binding to an antigen, called the complementarity-determining region (CDR). The term antibody can refer to a complete antibody (also called a whole antibody or full-length antibody) as well as antibody fragments and constructs containing the antigen-binding portion of the complete antibody. While typical natural antibodies have a pair of heavy and light chains, camelids (from camels, alpacas, llamas, etc.) produce antibodies with typical structures and antibodies containing only the heavy chain. The variable region of camelid-only heavy-chain antibodies has a unique structure with an elongated CDR3, called a VHH, or, when produced as a fragment, a nanobody. Antigen-binding fragments and constructs of antibodies include F(ab')2, F(ab'), F(ab), (sometimes equivalently referred to as Fab'2, Fab', and Fab) microbodies, Fv, single-chain Fv (scFv), biantibodies, and VH. These elements can be combined to produce bispecific and multispecific agents, such as BiTE (bispecific T-cell adaptor). The term “monoclonal antibody” originated from hybridoma technology but is now used to refer to any single molecular type of antibody, regardless of its origin or production. Similarly, the terms F(ab), F(ab'), and Fc are derived from the proteolytic analysis of antibodies but now refer to such fragments obtained in any way, whether or not they have precise ends produced by historical proteolysis. Antibodies can be obtained through immunization, selection from natural or immune libraries (e.g., by phage display), alteration of the isolated antibody coding sequence, or any combination thereof. Many antibodies that can be used as binding moieties are known in the art. Wilkinson & Hale, 2022, MAbs 14(1):2123299 (including its supplementary tables) is an excellent source of information (including sequence information) on antibodies for which International Nonproprietary Medicine Names (INNs) have been proposed or recommended, and all that this literature teaches about single antibodies and the various antibody forms that can be constructed is incorporated herein by reference. U.S. Patent No. 11,326,182 (especially its Table 9, entitled "Cancer, Inflammation and Immune System Antibodies") is a source of sequences and other information for a wide range of antibodies, including many antibodies that do not have INNs, and all that this document teaches about individual antibodies is incorporated herein by reference.

[0105] If the antibody or its binding fragment or other binding part (or its fusion protein) is equal to or greater than 10 5 M −1 Affinity or K a(That is, the equilibrium association constant of a specific binding interaction in units of 1 / M) If an antibody or its binding fragment or other binding part (or its fusion protein) binds to the target without significantly binding other components present in the test sample, then the antibody "specifically binds" to the target. Binding domains (or their fusion proteins) can be classified as "high-affinity" binding domains (or their fusion proteins) and "low-affinity" binding domains (or their fusion proteins). "High-affinity" binding domains refer to K... a For 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 −1 Or at least 10 9 M −1 Those binding domains. "Low affinity" binding domains refer to those with a Ka value as high as 10. 8 M −1 Up to 10 7 M −1 Up to 10 6 M −1 Up to 10 5 M −1 Those binding structural domains. Alternatively, affinity can be defined as the equilibrium dissociation constant (K0) of a particular binding interaction. D ), its unit is molar concentration (M) (e.g., 10 -5 M to 10 -13 M). The affinity of the binding domain peptide and the fusion protein according to this disclosure can be readily determined using conventional techniques (see, for example, Scatchard et al., 1949, Ann.NY Acad. Sci. 51:660; and U.S. Patent Nos. 5,283,173, 5,468,614, or equivalents).

[0106] As used herein, "binding agent," "binding moiety," or "targeting moiety" refers to a protein, polypeptide, oligopeptide, peptide, carbohydrate, nucleic acid, or combination thereof capable of specifically binding to one or more targets. A binding agent includes any naturally occurring, synthetic, semi-synthetic, or recombinant binding conjugate of a biomolecule or another target of interest. Exemplary binding moieties of this disclosure include antibodies or their antigen-binding domains, Fab', F(ab')2, Fab, Fv, rIgG, scFv, hcAb (heavy chain antibody), single-domain antibody (sdAb), VHH, variable neoantigen receptor (VNAR), nanobodies, receptor extracellular domains or their ligand-binding moieties, or ligands (e.g., cytokines, chemokines). A "Fab" (antigen-binding fragment) is a portion of an antibody that binds an antigen and includes a variable region and a first heavy chain constant (CH1) domain linked to a light chain via an interchain disulfide bond. In other embodiments, the binding moiety comprises a receptor or a ligand-binding domain of a receptor ligand. In some embodiments, the binding moiety may have more than one specificity, including, for example, bispecific or multispecific binding agents. Various assays are known for identifying the binding moieties of this disclosure that specifically bind to a particular target, including Western blotting, ELISA, biolayer interferometry, and surface plasmon resonance. Binding moieties (such as those containing variable domains of immunoglobulin light and heavy chains (e.g., scFv)) can be incorporated into a variety of protein scaffolds or structures as described herein, such as antibodies or their antigen-binding fragments, scFv-Fc fusion proteins, or fusion proteins containing two or more such immunoglobulin binding domains.

[0107] "Frame" or "FW" refers to the variable domain residues other than the CDR residues. The variable domain FW typically consists of four FW regions: FW1, FW2, FW3, and FW4. Therefore, the CDR and FR sequences usually appear in VH or VL with the following sequence: FW1-CDR1-FW2-CDR2-FW3-CDR3-FW4.

[0108] Several schemes exist for identifying hypervariable regions, using a simple sequential numbering of the antibody sequence throughout the application. In some cases, a Chothia number is used and specifically indicated. Several commonly used CDR numbering systems exist. The differences between Chothia numbers and Kabat numbers (as well as the definitions of Kabat, Chothia, AbM, and Contact CDRs) are described on the antibody information page of bioinf.org.uk, which belongs to Professor Andrew C.R. Martin's team at UCL.

[0109] A "humanized antibody" is a chimera, a genetically engineered antibody in which a CDR from an antibody (e.g., a mouse antibody (donor antibody)) is grafted to the CDR position of the receptor sequence of a human antibody (recipient antibody). Therefore, a humanized antibody is an antibody having a CDR from a donor, a non-human antibody, and a variable framework and constant region (when present) from a human antibody. In some embodiments, the human framework sequence in the humanized antibody may be modified at certain positions to contain residues present at those positions in the donor antibody in an attempt to better maintain (or improve) the affinity, specificity, stability, and / or other properties of the donor antibody.

[0110] Although humanized antibodies are chimeric, the term "chimeric antibody" is generally reserved to refer to an antibody containing the variable region of a donor antibody and the constant region of a recipient antibody (e.g., the constant region of a human antibody), as opposed to CDR transplanted antibodies in which the variable region itself is chimeric. This convention was observed in this paper. While chimeric antibodies exhibit lower immunogenicity when administered to the species containing the recipient antibody, they most commonly induce clinically useful immune responses that are limited or eliminated by repeated or prolonged exposure, whereas humanized antibodies avoid or reduce the occurrence of such harmful immune responses.

[0111] As used herein, the terms “monovalent” or “bivalent” refer to one or two antigen-binding sites on an intact antibody or antibody fragment.

[0112] As used in this article, the mouse anti-CD8α antibody clone RPA-T8 is referred to as the "CT8" antibody and used as a donor for humanization. As expressed on human cells, CD8 is a dimer, typically consisting of two α chains or one α and one β chain. Most CD8+ T cells express the αβ heterodimer. CT8 recognizes epitopes on the α chain. CT8 and its humanized derivatives can bind to both the α2 and αβ dimers.

[0113] The humanized anti-CD8α antigen-binding domain of this disclosure can be incorporated into various antibody forms, such as antigen-binding fragments (F(ab), F(ab'), or F(ab')2), single-chain variable fragments (scFv), biantibodies, microantibodies, and other antibody forms described elsewhere (Wilkinson and Hale, 2022, Mabs 14(1): e2123299). The term “F(ab)” refers to an antigen-binding monovalent fragment having a molecular weight of approximately 50,000 Daltons and antigen-binding activity, and consisting of VH and VL, a light chain constant domain (CL), and a first heavy chain constant domain (CH1). The term “(Fab')2” refers to an antibody bivalent fragment having a molecular weight of approximately 100,000 Daltons and antigen-binding activity, which comprises two antigen-binding fragments (F(ab)) connected by a disulfide bridge in 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 by recombinant DNA methods involving truncation or substitution of relevant hinge cysteine ​​residues. While various Fab fragments are classically generated by proteolysis, generation via recombinant DNA methods has become standard, especially for monoclonal reagents. This allows for variations and modifications to their amino acid sequence and ends, but the Fab terminology still applies to such similar molecules. The term "scFv" refers to the N-terminal portion of a Fab fragment and consists of variable portions (VH and VL) of a light and heavy chain linked in any order by short linker peptides of 10–25 amino acids. The term "biantibody" refers to a bivalent fragment consisting of two chains, each containing VH and VL domains from the same or different antibodies. In the biantibody form, the two variable domains (VH and VL) are linked by a short linker, typically 5 residues. Compared to scFv, the linker in a biantibody is often too short for the two domains in the same polypeptide chain to associate with each other. The term "microantibody" refers to a bivalent fragment derived from scFv having two scFvs, each fused to a constant heavy chain domain 3 (CH3), and in some embodiments, is bispecific.

[0114] As used herein, the term "monoclonal antibody" or "mAb" refers to an antibody molecule consisting of a single amino acid that targets a specific antigen and can be produced by a single clone of B cells or hybridomas or by recombinant methods. Using antibody components derived from humanized monoclonal antibodies avoids potential problems associated with immunogenicity in mouse constant regions and / or frame regions. Rodent monoclonal antibodies against specific antigens can be obtained by methods known to those skilled in the art (see, for example, Kohler and Milstein, Nature 256: 495 (1975) and Coligan et al. (eds.), Current Protocols In Immunology, VOL.1, pp. 2.5.1–2.6.7 (John Wiley & Sons 1991)).

[0115] The various anti-human CD8α antigen-binding domains described herein are generally designated by the initials CBD followed by four digits. In various experiments, these anti-CD8α antigen-binding domains were constructed as complete antibodies (e.g., as human IgG1 with a silencing LALAPAFc mutation; see SEQ ID NO: 43 or 44), F(ab), and other antigen-binding forms. The initials CBD may also be followed by numbers in the form xxxx.y or xxxx.yy, where four digits again indicate the antigen-binding domain, and one or two digits after the decimal point indicate F(ab') or other antibody forms (see Table 17).

[0116] As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain of two or more amino acids, not a product of a specific length. Therefore, 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 in place of or interchangeably with any of these terms.

[0117] As used throughout this disclosure, "identical" or "identical" means the similarity between a DNA, RNA, nucleotide, amino acid, or protein sequence and another DNA, RNA, nucleotide, amino acid, or protein sequence, respectively. Identity can be expressed as a percentage of sequence identity between a first sequence and a second sequence. The percentage of sequence identity (%) relative to a reference DNA sequence can be the percentage of DNA nucleotides in the candidate sequence that are identical to DNA nucleotides in the reference DNA sequence after sequence alignment. The percentage of sequence identity (%) relative to a reference amino acid sequence can 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 sequence alignment and the introduction of vacancies (if necessary) to achieve the maximum percentage of sequence identity, and without considering any conserved substitutions as part of sequence identity. As used throughout this disclosure, the NCBI BLAST 2.0 software, as defined by Altschul et al., “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs,” Nucleic Acids Res. 2007, 25, 3389-3402, generates the percentage of sequence identity values, with the parameters set to default values.

[0118] Humanized anti-CD8 binding site

[0119] This disclosure provides anti-CD8α antibodies (e.g., isolated monoclonal antibodies), also known as anti-CD8α antibodies or antigen-binding fragments thereof. In some embodiments of this disclosure, the anti-CD8α antibody or antigen-binding fragment thereof comprises two light-chain polypeptides (light chains) and two heavy-chain polypeptides (heavy chains) covalently held together by disulfide bonds.

[0120] In specific embodiments, the VH and VL of this disclosure can be expressed as separate polypeptides that associate with each other to form an antigen-binding fragment specific to CD8α, as they do in natural antibodies or various F(ab') fragments known in the art. In other embodiments, the VH and VL of this disclosure can be contained in a single polypeptide chain linked by a linker peptide. If the linker is of sufficient length, VH and VL of the same polypeptide chain can associate to form a single-chain Fv (scFv) that specifically binds to CD8α. Shorter linkers can be used such that VH and VL in one polypeptide chain associate with VL and VH of a second polypeptide chain, respectively, to form a biantibody. Generally, antigen-binding domains can be used in a modular manner and combined with other protein domains.

[0121] 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 grafted onto 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 this disclosure comprises an IgG1 constant region. Exemplary heavy chain constant regions include the human IgG1 heavy chain constant region (SEQ ID NO: 42) and the human IgG1 null heavy chain constant region (SEQ ID NO: 43 or 44).

[0122] In some embodiments, the light chain comprises a light chain variable region (VL) and a light chain constant region. The humanized anti-CD8α variant of this disclosure is grafted onto all or a portion of the κ light chain constant region or the λ light chain constant region or a portion thereof. Non-limiting exemplary light chain constant regions include both κ and λ constant regions. A non-limiting exemplary human κ constant region is shown in SEQ ID NO: 41.

[0123] Constant domains provide the general framework of an antibody and may not be directly involved in antibody-antigen binding, but can participate in various effector functions, such as antibody-dependent cytotoxicity (ADCC), ADCP (antibody-dependent phagocytosis), CDC (complement-dependent cytotoxicity), and complement fixation, and binding to Fc receptors (e.g., CD16, CD32, FcRn). As used herein, “Fc” or “Fc region” refers to a heavy chain constant segment (“crystallizable fragment” region or Fc region) from the Fc fragment of an antibody, which may contain one or more constant domains, such as CH2, CH3, CH4, or any combination thereof. In some embodiments, the Fc region includes 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.

[0124] The Fc region can interact with different types of Fc receptors (FcRs). These different types of FcRs can include, for example, FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB, FcαRI, FcµR, FcεRI, FcεRII, and FcRn. FcRs can 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 an FcR is bound to an Fc region, it can initiate the various effector functions described above. When FcRs are aggregated with antibodies on the cell surface, they can deliver signals. The aggregation of FcRs with immune receptor tyrosine-based activation motifs (ITAMs) can sequentially activate SRC family tyrosine kinases and SYK family tyrosine kinases. SRC and SYK kinases can link transduced signals to a common activation pathway. In the treatment of certain indications (such as autoimmune diseases), such signals may be undesirable.

[0125] 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 silencing region. As used herein, an "Fc null" or "Fc silencing" region refers to a domain that exhibits weak to no binding with any Fcγ receptor.

[0126] 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 affinity of the Fc region to the FcR. In some embodiments, the Fc region exhibits reduced binding affinity with FcγRI (CD64), FcγRIIA (CD32), FcγRIIIA (CD16a), FcγRIIIB (CD16b), or any combination thereof. To reduce the binding affinity of the Fc region to the FcR, the Fc region may contain one or more amino acid substitutions that have the effect of reducing the affinity of the Fc region to the FcR. In some implementations, the Fc region is IgG1, and one or more substitutions in the Fc region comprise any one or more of the IgG1 heavy chain mutations corresponding to E233P, L234V, L234A, L235A, L235E, ∆G236, G237A, E318A, K320A, K322A, A327G, P329A, A330S, or P331S according to the EU index of the Kabat number.

[0127] In some embodiments, the Fc region may contain a sequence of an IgG1 isotype modified from the wild-type IgG1 sequence. Modification may include substitutions at more than one amino acid residue, such as substitutions at two different amino acid residues, including S239D / I332E (IgG1 SDIE) according to the EU index of Kabat numbering. Modification may include substitutions at more than one amino acid residue, such as substitutions at 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. Modification may include substitutions at more than one amino acid residue, such as substitutions 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 SEQ ID NO: 43 and 44.

[0128] The Fc portion of an antibody can also mediate functional interactions with other agents besides the Fc receptor, including the mannose receptor, complement component C1q, and TRIM21. To prevent these interactions and their functional effects, antibody forms without the Fc region can be used, including scFv, F(ab), F(ab'), F(ab')2, and their variants. Table 17 presents exemplary sequences of wild-type and engineered Cκ and F(ab') recurrent constant domains (in some cases, truncated to remove some or all of the hinge regions found in classic F(ab').

[0129] Binding affinity (usually reported as the dissociation constant K) D The binding rate can be determined by kinetic or steady-state (equilibrium) analysis, i.e., by the ratio of dissociation rate to binding rate or by the binding concentration profile, respectively. In some embodiments, the anti-CD8α antigen binding fragment having a framework region containing variable domains of the human heavy and light chains from this 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 silent IgG1 antibody of SEQ ID NO: 43). To determine the K of these different anti-CD8α binders... DKinetic analyses were performed on F(ab) and F(ab'); and steady-state analyses were performed on 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 biolayer interferometry (BLI) kinetics and surface plasmon resonance (SPR) binding assays. This assay measured the binding affinity to the CD8αα homodimer. In these embodiments, the humanized anti-CD8α F(ab), F(ab'), or Fc-ineffective full-length antibody had a Kc of approximately 10 nM or less. D In some implementations, CBD1032, CBD1033, CBD1035, CBD1036, CBD1037, CBD1038, CBD1039, or CBD1040 F(ab) has a Kc value of less than 8 nM. D In some implementations, CBD1032, CBD1033, CBD1037, or CBD1039 F(ab) has a Kc value of less than 5 nM. D In some implementations, the CBD1032, CBD1033, CBD1034, CBD1035, CBD1036, CBD1037, CBD1038, CBD1039, CBD1040, CBD1042, CBD1043, CBD1045, CBD1047, CBD1048, CBD1049, or CBD1050 Fc ineffective full-length antibodies have a Kc of less than 7 nM. D In some implementations, the CBD1032, CBD1033, CBD1034, CBD1035, CBD1037, CBD1038, CBD1039, CBD1040, CBD1042, CBD1043, CBD1045, CBD1047, or CBD1049 Fc ineffective full-length antibodies have a K+ of less than 5 nM. D In some implementations, CBD1033.24 or CBD1033.37 F(ab') has a K0 value of less than 7 nM. D The most widely used affinity substitution indicator is EC. 50 This refers to the concentration at which the half-maximal effect is achieved. In some embodiments, the anti-CD8α Fc ineffective antibody of this disclosure has an EC50 of about 6 nM, about 3 nM, about 2 nM, about 1 nM, or about 0.5 nM. 50Studies have found that the humanized anti-CD8 antigen-binding fragment disclosed and tested in this application exhibits binding affinity for both the CD8αα homodimer and the CD8αβ heterodimer, and the binding strength to the CT8 epitope is sufficient to deliver tLNP to CD8+ cells. Specifically, CBD1033 F(ab) binds to both human and cynomolgus monkey CD8αα homodimers and CD8αβ heterodimers. However, the binding affinity of CBD1033 F(ab) is 1 / 5 that of the parental CBD1017ch F(ab). Furthermore, compared to the CD8αα homodimer, the binding affinity of the CBD1033 F(ab) or Fc ineffective full-length antibody to the CD8αβ heterodimer is 1 / 10 to 1 / 6. However, in CD8+ T cells known to primarily express CD8αβ heterodimers, tLNPs targeting CBD1033 have shown comparable to, and in some cases better than, CBD1017ch transfection efficiencies in vitro, and in vivo transfection efficiencies up to 80%. Further studies on the CD8 binding site revealed that CBD1033 binds to a specific epitope on CD8 (called the CT8 epitope), which exhibits higher transfection efficiency compared to other epitopes bound by other antibodies such as SK1 and OKT8. Therefore, the humanized anti-CD8 binding fragment disclosed herein possesses sufficient binding affinity to the CT8 epitope of either the CD8αα homodimer or the CD8αβ dimer to maintain tLNP transfection function in vivo.

[0130] In some embodiments, the anti-CD8α antibody of this disclosure or its antigen-binding fragment specifically binds to non-human primate and human CD8αα homodimers and CDαβ heterodimers. In some cases, the humanized anti-CD8α antibody of this disclosure or its antigen-binding fragment specifically binds to CD8 in cynomolgus monkeys or rhesus monkeys. In some embodiments, the humanized anti-CD8α antibody of this disclosure or its antigen-binding fragment competitively binds to the same epitope on CD8α that is bound by TRX2, and vice versa, as shown in Figure 5 of US20060002921. Figure 6The contents of the teachings concerning TRX2 described herein are incorporated herein by reference. The sequence of the TRX2 antibody is shown in Table 19. In some embodiments, the humanized anti-CD8 antibody of this disclosure or its antigen-binding fragment competitively binds to the same epitope on CD8α bound by YTC182.20 (described in Jonker, M. et al. (1989) Reactivity of mAb specific for human CD markers with Rhesus monkeyleucocytes. Leucocyte Typing IV. Oxford University Press, pp. 1058-1063, the contents of which are incorporated herein by reference), and vice versa. The CT8 epitope bound by these antibodies is located in the proximal membrane portion of the extracellular domain of CD8α above the stem (or hinge) emerging from the cell membrane and near the dimer interface. Crosslinking analysis revealed that CBD1033 binds to or near amino acid residues 40, 45, 47, 86, 91, 95, 103, 105, and 106 of CD8α, indicating that CBD1033 interacts with the CC' loop, C' chain, F chain turn, F chain, and G chain of CD8α (as predicted by AlphaFold2), and thus identifies the location of the CT8 epitope. (See Srinivasan et al., 2024 Front. Immunol. 15:1412513, whose teachings on the structure of CD8 and its interaction with mAbs are incorporated herein by reference). Residues 40 and 45 are in the CC' loop, residue 47 is in the C' chain, residue 86 is in the F chain turn, residues 91 and 95 are in the F chain, and residues 103, 105, and 106 are in the G chain. Competitive binding analysis showed that the anti-CD8 antibody OKT8 does not compete for binding to the same epitope. Anti-CD8 antibody SK1 does not competitively bind to the CT8 epitope, but it blocks T cell activation and competitively binds to the same epitope as anti-CD8 antibody HIT8α. In various embodiments, the antigen-binding domain that binds to the CT8 epitope is a designated means for binding the CT8 epitope or a means for competitively binding to the same epitope as that bound by CT8, TRX2, and / or YTC182.20.

[0131] The variable region of an antibody contains the antigen-binding site of the molecule. The variable heavy chain (VH) and variable light chain (VL) are the domains of the larger and smaller polypeptide subunits of the antibody, respectively, and form the antigen-binding site. The VH and VL domains of an antibody typically have similar structures, with each domain including four conserved frame regions (FWs) and three hypervariable regions. Most of the sequence variability of the antibody occurs in six hypervariable regions, each called a "complementarity-determining region" (CDR), three for each VH and VL chain (VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, VL-CDR3). In some embodiments, the antigen recognition region of the variable domain of the anti-CD8 antibody of this disclosure comprises six CDRs or hypervariable regions located within the frame of the heavy chain variable regions and light chain variable regions at the N-termini of the two heavy chains and two light chains. For example, the CD8 binding domain includes heavy chain complementarity determination region 1 (VH-CDR1), heavy chain complementarity determination region 2 (VH-CDR2), heavy chain complementarity determination region 3 (VH-CDR3), light chain complementarity determination region 1 (VL-CDR1), light chain complementarity determination region 2 (VL-CDR2), and light chain complementarity determination region 3 (VL-CDR3).

[0132] In some respects, an anti-CD8α antibody or its antigen-binding fragment having a framework region of variable domains from human heavy and light chains comprises: (a) a VH comprising: a VH-CDR1 having the amino acid sequence RYTFTDYX1LH (SEQ ID NO: 45), wherein X1 is N, S, Q, or A; a VH-CDR2 having the amino acid sequence FIYPYX1GGTG (SEQ ID NO: 46) or FIYPYX2GGTG (SEQ ID NO: 47), wherein X2 is N, Q, D, S, or A; a VH-CDR3 having the amino acid sequence DHRYX1EGVSFDY (SEQ ID NO: 48); and a VL comprising: a VL-CDR1 having the amino acid sequence RASESVX3GFGX2SFMN (SEQ ID NO: 49), wherein X3 is an amino acid identified by the symbols D, E, S, or A; and a VL having the amino acid sequence LASX2LES (SEQ ID NO: 49). VL-CDR2 having the amino acid sequence QQX2X2EX3PYT (SEQ ID NO: 51); and VL-CDR3 having the amino acid sequence RYTFTDYNLH (SEQ ID NO: 2). In some embodiments, VH-CDR1 has the amino acid sequence FIYPYNGGTG (SEQ ID NO: 3), FIYPYSGGTG (SEQ ID NO: 58), FIYPYQGGTG (SEQ ID NO: 59), or FIYPYAGGTG (SEQ ID NO: 60). In some embodiments, VH-CDR3 has the amino acid sequence DHRYNEGVSFDY (SEQ ID NO: 4). In some embodiments, VL-CDR1 has the amino acid sequence RASESVDGFGNSFMN (SEQ ID NO: 6), SEQ ID NO: 227, or SEQ ID NO: 228. In some embodiments, VL-CDR2 has the amino acid sequence LASNLES (SEQ ID NO: 7). In some embodiments, VL-CDR3 has the amino acid sequence QQNNEDPYT (SEQ ID NO: 8).

[0133] 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: 60. (a) VH-CDR3 containing the amino acid sequence SEQ ID NO: 6; and (b) VL-CDR1 containing the amino acid sequence SEQ ID NO: 7, VL-CDR2 containing the amino acid sequence SEQ ID NO: 8, and VL-CDR3 containing the amino acid sequence SEQ ID NO: 8.

[0134] In any of the above embodiments, (a) the receptor sequence of the derived heavy chain framework region is from IGHV1-46*01 / IGHJ6*01, as shown in SEQ ID NO: 9; (b) the receptor sequence of the derived light chain framework region is from IGKV1-39*01 / IGKJ2*01, as shown in SEQ ID NO: 15; (c) the receptor sequence of the derived heavy chain framework region is from a modified IGHV1-18*01, as shown in SEQ ID NO: 31; and (d) the receptor sequence of the derived light chain framework region is from a modified form of IGKV3D-11*01, as shown in SEQ ID NO: 9. As shown in Figure 37; (e) the receptor sequence of the derived heavy chain framework region is from IGHV1-46*01 / IGHJ6*01 and the receptor sequence of the derived light chain framework region is from IGKV1-39*01 / IGKJ2*01; (f) the receptor sequence of the derived heavy chain framework region is from a modified form of IGHV1-18*01 and the receptor sequence of the derived light chain framework region is from a modified form of IGKV3D-11*01; (g) the receptor sequence of the derived heavy chain framework region is from IGHV1-46*01 / IGHJ6*01 and the receptor sequence of the derived light chain framework region is from a modified form of IGKV3D-11*01; (h) the receptor sequence of the derived heavy chain framework region is from a modified form of IGHV1-18*01 and the receptor sequence of the derived light chain framework region is from IGKV1-39*01 / IGKJ2*01.

[0135] In some embodiments, the anti-CD8α antibody or its antigen-binding fragment comprises: a heavy chain variable region (VH) containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to that 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 a light chain variable region (VL) containing the amino acid sequence identical to that of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19. 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 64 or SEQ ID NO: 65 have amino acid sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical.

[0136] In some embodiments, the anti-CD8α antibody or its antigen-binding fragment comprises: a heavy chain variable region (VH) containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to that 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, and wherein VH-CDR1 contains the amino acid sequence of SEQ ID NO: 2, VH-CDR2 contains the amino acid sequence of SEQ ID NO: 3, 58, 59, or 60, and VH-CDR3 contains the amino acid sequence of SEQ ID NO: 4; and a light chain variable region (VL) containing the amino acid sequence identical to that of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19. The amino acid sequences of 39 are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, and wherein VL-CDR1 contains the amino acid sequence of SEQ ID NO: 6, 227, or 228, VL-CDR2 contains the amino acid sequence of SEQ ID NO: 7, and VL-CDR3 contains the amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-CD8α antibody or its antigen-binding fragment comprises: VH, which contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 10, 11, 12, 13, or 14; and VL, which contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of 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) remain unchanged.In a further embodiment, the anti-CD8α antibody or its antigen-binding fragment comprises: VH, which contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 10, 11, 12, 13, or 14; and VL, which contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 17, provided that the amino acid sequences of VH-CDR and VL-CDR remain unchanged. In a further embodiment, the anti-CD8α antibody or its antigen-binding fragment comprises: VH, which contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 10, 11, 12, 13, or 14; and VL, which contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 18, provided that the amino acid sequences of VH-CDR and VL-CDR remain unchanged. In a further embodiment, the anti-CD8α antibody or its antigen-binding fragment comprises: VH, which contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO:35 or 36; and VL, which contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO:39, provided that the amino acid sequences of VH-CDR and VL-CDR remain unchanged.

[0137] In some embodiments, the anti-CD8α antibody or its antigen-binding fragment comprises: a heavy chain variable region (VH) containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34, wherein VH-CDR1 contains the amino acid sequence of SEQ ID NO: 2, VH-CDR2 contains the amino acid sequence of SEQ ID NO: 3, and VH-CDR3 contains the amino acid sequence of SEQ ID NO: 4; and a light chain variable region (VL) containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 40, wherein VL-CDR1 contains the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 40. The amino acid sequence of SEQ ID NO: 6, VL-CDR2 contains the amino acid sequence of SEQ ID NO: 7, and VL-CDR3 contains the amino acid sequence of SEQ ID NO: 8.

[0138] In some embodiments, the anti-CD8α antibody or its antigen-binding fragment comprises a VL region having an amino acid sequence having one of SEQ ID NO: 16-18, 38-40, 64, or 65. In some embodiments, the anti-CD8α antibody or its antigen-binding fragment comprises a VH region having an amino acid sequence having one of SEQ ID NO: 10-14, 27-30, 32-36, or 66-68. In a further embodiment, the anti-CD8α antibody or its antigen-binding fragment comprises a VL region having an amino acid sequence having one of SEQ ID NO: 17 and a VH region having an amino acid sequence having one of SEQ ID NO: 11 or 27-30. In a further embodiment, the anti-CD8α antibody or its antigen-binding fragment comprises: (a) a VH region comprising the amino acid sequence of SEQ ID NO: 10 and a VL region comprising the amino acid sequence of SEQ ID NO: 16; (b) a VH region comprising the amino acid sequence of one of SEQ ID NO: 11-14 and a VL region comprising the amino acid sequence of SEQ ID NO: 17; or (c) a VH region comprising the amino acid sequence of one of SEQ ID NO: 11-14 and a VL region comprising the amino acid sequence of SEQ ID NO: 18. In a further embodiment, the anti-CD8α antibody or its antigen-binding fragment comprises a VL region having the amino acid sequence of SEQ ID NO: 64 and a VH region having the amino acid sequence of one of SEQ ID NO: 11, 13, 28, 29, 67, or 68. In a further embodiment, the anti-CD8α antibody or its antigen-binding fragment comprises a VL region having the amino acid sequence of SEQ ID NO: 65 and a VH region having the amino acid sequence of one of SEQ ID NO: 11 and 13.

[0139] In a further embodiment, the anti-CD8α antibody or its antigen-binding fragment comprises: (a) a 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, and 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) a 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, and 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) a 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: 28, 19, 10, 11, 12, 13, 14, 35, or 36, wherein VH-CDR1 comprises the amino acid sequence of SEQ ID NO: 28, 19, 10, 11, 12, 13, 14, 35, or 36, and VL comprises the amino acid sequence of SEQ ID NO: 28, 19, 10, 11, 12, 13, 14, 35, or 36, and VH-CDR1 ... (d) A VH containing the amino acid sequence of SEQ ID NO: 29 or 68, wherein VH-CDR1 contains the amino acid sequence of SEQ ID NO: 2, VH-CDR2 contains the amino acid sequence of SEQ ID NO: 4, and VL contains the amino acid sequence of SEQ ID NO: 16; (e) A VH containing the amino acid sequence of SEQ ID NO: 10, 11, 12, 13, 14, 35, or 36, wherein VH-CDR1 contains the amino acid sequence of SEQ ID NO: 2, VH-CDR2 contains the amino acid sequence of SEQ ID NO: 3, VH-CDR3 contains the amino acid sequence of SEQ ID NO: 4, and VL contains the amino acid sequence of SEQ ID NO: 16; (f) A 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, and 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) A 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) A 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: 17; (i) A 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: 29, VH-CDR2 comprises the amino acid sequence of SEQ ID NO: 39, VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 3 ... (j) A VH containing the amino acid sequence of SEQ ID NO: 27 or 66, wherein VH-CDR1 contains the amino acid sequence of SEQ ID NO: 2, VH-CDR2 contains the amino acid sequence of SEQ ID NO: 58, VH-CDR3 contains the amino acid sequence of SEQ ID NO: 4, and VL contains the amino acid sequence of SEQ ID NO: 18; (k) A VH containing the amino acid sequence of SEQ ID NO: 28 or 67, wherein VH-CDR1 contains the amino acid sequence of SEQ ID NO: 2, VH-CDR2 contains the amino acid sequence of SEQ ID NO: 59, VH-CDR3 contains the amino acid sequence of SEQ ID NO: 4, and VL contains the amino acid sequence of SEQ ID NO: 18; (l) A VH containing the amino acid sequence of SEQ ID NO: 28 or 67. VH is a sequence of amino acids of 29 or 68, wherein VH-CDR1 contains the amino acid sequence of SEQ ID NO: 2, VH-CDR2 contains the amino acid sequence of SEQ ID NO: 60, VH-CDR3 contains the amino acid sequence of SEQ ID NO: 4, and VL contains the amino acid sequence of SEQ ID NO: 18;(m) A 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, and VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 4, and VL comprises the amino acid sequence of SEQ ID NO: 39; (n) A 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: 39; (o) A 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: 38, VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 39, VH-CDR2 comprises the amino acid sequence of SEQ ID NO: 39 ... (a) A VH containing the amino acid sequence of SEQ ID NO: 59, wherein VH-CDR1 contains the amino acid sequence of SEQ ID NO: 4, and VL contains the amino acid sequence of SEQ ID NO: 39; (b) A VH containing the amino acid sequence of SEQ ID NO: 29 or 68, wherein VH-CDR1 contains the amino acid sequence of SEQ ID NO: 2, VH-CDR2 contains the amino acid sequence of SEQ ID NO: 60, VH-CDR3 contains the amino acid sequence of SEQ ID NO: 4, and VL contains the amino acid sequence of SEQ ID NO: 39; (c) A VH containing the amino acid sequence of SEQ ID NO: 10, 11, 12, 13, 14, 33, 34, 35, or 36, wherein VH-CDR1 contains the amino acid sequence of SEQ ID NO: 2, VH-CDR2 contains the amino acid sequence of SEQ ID NO: 3, VH-CDR3 contains the amino acid sequence of SEQ ID NO: 4, and VL contains the amino acid sequence of SEQ ID NO: 64; (d) A VH containing the amino acid sequence of SEQ ID NO: 59, wherein VH-CDR1 contains the amino acid sequence of SEQ ID NO: 2, VH-CDR2 contains the amino acid sequence of SEQ ID NO: 3, VH-CDR3 contains the amino acid sequence of SEQ ID NO: 4, and VL contains the amino acid sequence of SEQ ID NO: 64; (e) A VH containing the amino acid sequence of SEQ ID NO: 69; VH of amino acid sequence NO: 27 or 66, wherein VH-CDR1 contains the amino acid sequence of SEQ ID NO: 2, VH-CDR2 contains the amino acid sequence of SEQ ID NO: 58, and VH-CDR3 contains the amino acid sequence of SEQ ID NO: 4, and VL contains the amino acid sequence of SEQ ID NO: 64;(s) A 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: 64; (t) A 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: 64; (u) A VH 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: 29, VH-CDR2 comprises the amino acid sequence of SEQ ID NO: 29, VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 29, VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 29, VH-CDR2 ... (v) A VH containing the amino acid sequence of SEQ ID NO: 27 or 66, wherein VH-CDR1 contains the amino acid sequence of SEQ ID NO: 2, VH-CDR2 contains the amino acid sequence of SEQ ID NO: 58, VH-CDR3 contains the amino acid sequence of SEQ ID NO: 4, and VL contains the amino acid sequence of SEQ ID NO: 65; (w) A VH containing the amino acid sequence of SEQ ID NO: 28 or 67, wherein VH-CDR1 contains the amino acid sequence of SEQ ID NO: 2, VH-CDR2 contains the amino acid sequence of SEQ ID NO: 59, VH-CDR3 contains the amino acid sequence of SEQ ID NO: 4, and VL contains the amino acid sequence of SEQ ID NO: 65; (x) A VH containing the amino acid sequence of SEQ ID NO: 28 or 67. VH of amino acid sequences of 29 or 68, wherein VH-CDR1 contains the amino acid sequence of SEQ ID NO: 2, VH-CDR2 contains the amino acid sequence of SEQ ID NO: 60, and VH-CDR3 contains the amino acid sequence of SEQ ID NO: 4, and VL contains the amino acid sequence of SEQ ID NO: 65;

[0140] Examples of humanized anti-CD8α variants containing the variable domains described herein are shown in Tables 3-5. The VH or VL domains described herein can be grafted onto classical or engineered heavy or light chain constant regions, respectively. The constant regions can be full-length, F(ab), F(ab'), F(ab')2, single-chain fragment variable regions (scFv), biantibodies, microantibodies, or other antibody forms. 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 κ and λ constant regions.

[0141] In the context of F(ab'), the term "engineering" refers to the modification or mutation of amino acid residues in the constant region. In some embodiments, the modification is a truncation of the constant region; for example, a truncation after proline at position 245 (P245), P240, or P241 in the hinge region of IgG1 F(ab') or IgG4 F(ab'); or a truncation after T238 in the hinge region of IgG1 F(ab') (Table 17). In some embodiments, the mutation is a mutation of a cysteine ​​residue to serine or other non-cysteine ​​amino acid to remove a disulfide bond; for example, C214S in the constant region of the κ chain, C233S in the CH1 domain of IgG1, or C127S in the CH1 domain of IgG4 F(ab') (Table 17). In some implementations, the mutation is a non-cysteine ​​amino acid mutation to cysteine ​​to support the formation of a new disulfide bond; for example, the F174C of the CH1 domain and the S162C of the κ constant region of IgG1 F(ab') or IgG4 F(ab') form a CH1174-Cκ162 disulfide bond (Table 17).

[0142] The VH domain described herein is transplantable into the heavy chain constant region containing the amino acid sequences SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 90, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, or SEQ ID NO: 103. The VL domain described herein is transplantable into the light chain constant region containing the amino acid sequences SEQ ID NO: 41, SEQ ID NO: 89, or SEQ ID NO: 100. Combinations of the disclosed VH and VL domains with the heavy and light constant regions, respectively, are shown in Table 17.

[0143] Biophysical characteristics of humanized anti-CD8 binding sites

[0144] In addition to binding affinity and specificity to the desired target molecule, therapeutic antibodies advantageously meet a set of criteria regarding the feasibility of their manufacture, storage stability, and absence of off-target binding (“stickiness”). This set of characteristics is often referred to as “developability.” The biophysical properties of an antibody greatly influence developability. For example, the protein’s “melting temperature” (T0) m The aggregation temperature (T0) is the temperature at which half of a protein group is in a folded state, and therefore an indicator of thermal stability, which helps determine the stability of antibodies during storage and manufacturing. Similarly, the aggregation temperature (T0) is... agg The detection of the initiation of aggregation occurs at a temperature at which molecules tend to aggregate and are associated with protein unfolding. Furthermore, multiple studies have shown that monoclonal antibodies can interact nonspecifically with themselves (self-aggregation) and other serum proteins; therefore, low self-aggregation properties can avoid antibody aggregation, off-target effects, and rapid antibody clearance in vivo.

[0145] The “off-target” assessment of the humanized anti-CD8α antibodies and their antigen-binding fragments disclosed herein (including multireactivity and cross-reactivity assessments, such as DNA and insulin multireactivity ELISA assays, baculovirus particle (BVP) multireactivity assays, and analysis of human cell membrane proteome arrays containing cell surface and secreted proteins) is used to measure binding to targets other than the CD8 antigen. As used herein, the term “multireactivity” refers to the ability of an antibody to bind to multiple self-antigens 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 assess the ability of these antibodies to bind to antigens other than CD8, which have structures that are similar to and different from CD8, respectively. Multireactivity and cross-reactivity (or off-target) effects affect a variety of factors, including in vivo pharmacokinetics, bioavailability, clearance, and toxicity, all of which contribute to successful drug / antibody development.

[0146] In some embodiments, the anti-CD8α binding agents of this disclosure exhibit similar binding activity and affinity for CD8 in non-human primates as in humans. The term "cross-species binding" refers to the ability of an antibody to bind to the same or related antigens in different species ("target specificity") while retaining its antigen specificity for CD8 molecules in such different species. This property is particularly useful in research and clinical development because experimental data in non-human species can be obtained and it can be reliably translated into humans.

[0147] Those skilled in the art will understand that the properties of the anti-CD8α antibodies and their antigen-binding fragments (including any of the embodiments described above) disclosed herein are generally unrelated to the binding affinity and specificity of the antibody. Furthermore, those skilled in the art will recognize that variations in the amino acid sequence can affect the biophysical properties of the antibody. Therefore, merely preserving affinity will not ensure the developability of humanized antibodies, nor can these biophysical properties be reliably predicted solely from the sequence. However, testing on at least some variants, in addition to revealing their properties, can provide some indication of the robustness of specific combinations of the framework and CDR sequences, and what variations in the sequence might be problematic or not.

[0148] Antibody thermal stability serves as an indicator of antibody developability, reflecting its stability during storage and during various purification steps that may require harsh or stressful conditions. A sensitive measure of antibody thermal stability is the aggregation initiation temperature (Tagg), which indicates the onset of protein denaturation. Typically, a Tagg is preferred. agg >60℃. By this standard, intact antibodies incorporating the anti-CD8α antigen-binding fragments CBD1033, CBD1034, CBD1035, CBD1039, and CBD1040 all exhibit acceptable T... agg And CBD1032's T agg Not ideal. Melting temperature (Tm) (another measure of thermal stability) indicates the midpoint of protein denaturation. For potentially good exploitability, antibodies with a Tm >65°C are preferred. By this standard, intact antibodies incorporating the anti-CD8α antigen-binding fragments CBD1033, CBD1035, CBD1039, and CBD1040 all have acceptable Tm. Therefore, in some embodiments, the humanized CT8 antibody and its antigen-binding fragments have ideal thermal stability, and CBD1033, CBD1035, CBD1039, and CBD1040 constitute a humanization means for binding CD8α with ideal thermal stability.

[0149] One potential problem encountered in CDR transplantation is that engineered antibodies may become multireactive or have an increased tendency for self-interaction or self-association. These properties can contribute to antibody aggregation, off-target effects, and rapid clearance (reducing their effectiveness or potency).

[0150] Self-interactions can be assessed using affinity-captured self-interaction nanoparticle spectroscopy (AC-SINS) (Phan et al., 2022, MAbs 14(1): 2094750). Full-length antibodies against CD8α antigen-binding fragments containing intact CBD1032, CBD1033, CBD1034, CBD1035, CBD1039, or CBD1040 were evaluated using AC-SINS and found to all exhibit low self-interaction tendencies. Therefore, CBD1032, CBD1033, CBD1034, CBD1035, CBD1039, and CBD1040 constitute a humanized approach for binding CD8α with low self-interaction tendencies.

[0151] Several tests are available for assessing multireactivity. One test assesses reactivity with double-stranded DNA (dsDNA) and insulin. By this standard, intact antibodies incorporating anti-CD8α antigen-binding fragments of CBD1032, CBD1033, CBD1034, CBD1035, CBD1039, or CBD1040 are not multireactive and constitute humanized means for binding CD8α, which lack multireactivity with dsDNA and insulin.

[0152] Another multireactivity test assesses the ability to bind baculovirus particles (BVP). By this standard, the intact antibody incorporating the anti-CD8α antigen-binding fragment of CBD1033 is not multireactive, while CBD1032 is weakly multireactive. Therefore, CBD1033, constituting a humanized means for binding CD8α, lacks multireactivity against BVP.

[0153] A more comprehensive test of multireactivity and cross-reactivity evaluated binding to a group of >6000 human cell surface proteins and secreted proteins in the form of integrated 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 intended for use in humans (or similar bodies), this is relevant to the developability and success of such products. Through this evaluation, the intact CBD1017ch antibody, containing the parent antigen-binding domain, showed no cross-reactivity with any antigen in this group. The intact CBD1033, CBD1035, and CBD1039 antibodies maintained this lack of cross-reactivity and did not introduce any multireactivity. Therefore, CBD1033, CBD1035, and CBD1039 constitute a humanized means for binding CD8α that lacks multireactivity and cross-reactivity with human cell surface and soluble proteins.

[0154] In some aspects of this disclosure, certain amino acid modifications can be made to VH-CDRs (including amino acid sequence changes N33Q, N33S, N33A, N55Q, N55S, N55A, N103Q, N103S, N103A, or combinations thereof) and VL-CDRs (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) to remove unstable amides and prevent potential deamidation. In some embodiments, these substitutions do not affect or only minimally affect binding affinity, but may be advantageous for antibody purification, storage, and other processing, regardless of whether such binding moieties are under high-stress conditions such as high pH and high temperature. As used herein, the term "prone to engineered mutation" refers to a mutation that removes unstable amino acid residues (such as asparagine and aspartic acid) through deamidation and isoflavone formation, respectively, which are at risk of post-translational modification, including during product manufacturing. In some embodiments, unstable engineered mutations include one or more of the VH-CDR and VL-CDR mutations mentioned above.

[0155] The term "high stress conditions" encompasses extreme environmental conditions that can affect molecular stability, such as high pH (pH ≥ 8), low pH (pH ≤ 6), high temperature (≥ 40°C), or combinations thereof.

[0156] Humanized antiCD8α as the targeting component on LNP

[0157] Because CD8-positive T cells play a crucial role in adaptive immunity, the ability of humanized anti-CD8α antibodies to target CD8-positive T cells could provide therapeutic or diagnostic benefits in the treatment of cancer, infections, immune disorders, inflammatory diseases, and autoimmune diseases. CD8 is also expressed on natural killer (NK) cells, which are potent and therapeutically attractive mediators of cytotoxic activity.

[0158] In some aspects, any of the aforementioned humanized anti-CD8α antibodies or their antigen-binding fragments can be used as a targeting moiety on nanoparticles. A variety of nanoparticles suitable for delivering payload molecules to or into cells are known in the art, including nanoparticles containing polymers and / or lipids, and the disclosed anti-CD8 antibody or other peptides containing their antigen-binding domains can be attached to the nanoparticles as a targeting moiety. In some embodiments, the nanoparticles are lipid nanoparticles (LNPs). In the specific embodiments disclosed herein, the term "tLNP" refers to an LNP containing 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 another target molecule; in particular, the targeting moiety of an LNP is an anti-CD8 antibody capable of binding the CD8 antigen on cells expressing CD8.

[0159] CD8-targeting tLNPs can be used to deliver payloads, particularly negatively charged payloads such as nucleic acids for incorporating cationic lipids, into CD8+ cells. This can be done in vitro or in vivo (e.g., as described in PCT / US2024 / 035902, which is incorporated herein by reference for all information on the use of tLNPs for in vitro or in vitro cell transfection) 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 in vitro or in vivo use than in vivo use. Therefore, humanized antigen-binding domains of CT8 with a larger number of mouse residues or those not yet characterized in other measures of multireactivity or potential cross-reactivity with non-CD8 antigens, or even unhumanized antigen-binding domains, can be used to provide binding specificity for the targeting moiety of tLNPs for in vitro or in vivo use.

[0160] Within the range of affinities exhibited by the various antigen-binding domains disclosed herein, affinity is not a valid variable for either transfection rate resulting from incorporating those antigen-binding domains into the target portion of the tLNP, nor is it a valid variable for the expression level of transfected mRNA. However, emerging 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 incorporating antigen-binding domains from the various anti-CD8 antibodies disclosed herein. When the anti-CD8 monoclonal antibodies SK1 and OKT8 provide the antigen-binding domain for the target portion of the tLNP, the in vitro transfection efficiency and payload expression levels are significantly lower than when the antigen-binding domain is provided by CBD1033 (a humanized form of the anti-CD8 monoclonal antibody CT8) or TRX2 (another humanized monoclonal anti-CD8 antibody). The in vivo payload expression levels are also similar for tLNPs with target portions incorporating the antigen-binding domains of CBD1033 or TRX2. CBD1033 and TRX2 compete with each other for epitope binding, as does the anti-CD8 antibody YTC182.20; OKT8 and SK1 do not compete with these antibodies. Unbound by any particular theory, these data suggest that binding to the CT8 epitope (as defined above) allows for significantly higher engineerability compared to binding elsewhere, and demonstrate that certain molecular conformational changes induced by binding to a specific site can determine the cellular signaling that leads to the uptake of active particles. Therefore, incorporating the antigen-binding domain of CT8, TRX2, or YTC182.20 into its targeting moiety constitutes a means for efficient particle internalization or for efficient transfection of effective payload nucleic acids (such as DNA, RNA, or mRNA). In various embodiments, such means specifically include or exclude any antibody, antibody form, or antigen-binding domain disclosed herein as part of its targeting moiety.

[0161] The humanized anti-CD8-targeting tLNPs incorporating cationic lipids (such as ionizable cationic lipids) disclosed herein can deliver negatively charged cargo / payloads (such as nucleic acids, peptides, and small molecules) to cells expressing CD8. The nucleic acids thus introduced can encode the expression of proteins beneficial to a subject treating a disease. In some aspects, methods for delivering nucleic acids (or other negatively charged payloads) into cells are disclosed herein, including contacting the cells with tLNPs encapsulating nucleic acids or other payloads. In some embodiments, contact occurs ex vivo. In some embodiments, contact occurs in vivo. In some cases, in vivo contact includes intravenous, intramuscular, subcutaneous, intranodal, or intralymphatic administration. Therefore, each of the genera, subgenera, and / or species of LNPs or tLNPs disclosed herein (including those based on the inclusion or exclusion of specific lipids, specific lipid compositions, specific payloads, and / or specific CD8-targeting portions) can be used to define the delivery of payloads to CD8. + The scope of cell-based methods.

[0162] Nucleic acids may include messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASO), self-replicating RNA, or circular RNA. In some embodiments, the payload is mRNA encoding a detectable biomarker (e.g., mCherry fluorescent protein). In some embodiments, the delivery method is a transfection method.

[0163] In some implementations, the encapsulated nucleic acid is mRNA encoding a chimeric antigen receptor (CAR), a T-cell receptor (TCR) or an immune cell connector (such as BiTE (bispecific T-cell connector)), cytokines, chemokines, chemokine receptors, dominant or negative cytokine receptors, cell recognition protein tags, fluorescent proteins, or molecular switches.

[0164] The encapsulated nucleic acid can also be mRNA encoding gene / genome editing enzymes and / or guide RNA or other components of the gene / genome editing system. Gene / genome editing components can be RNA-guided nucleases or guide RNA for other nucleic acid editing enzymes. Examples of gene-editing components encoded by nucleic acid molecules include mRNAs encoding the following: RNA-directed nucleases, gene or base-editing proteins, leader editing proteins, gene-writing 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), large-scale nucleases, transposases, retrotransposons, reverse transcriptases (e.g., M-MLV reverse transcriptases), nicking enzymes or inactive nucleases (e.g., Cas9, nCas9, dCas9), DNA recombinases, CRISPR nucleases (e.g., Cas9, Cas12, Cas13, Cas3, CasMINI, Cas7-11, CasX), DNA nicking enzymes, Cas9 nicking enzymes (e.g., D10A or H840A), or any fusions or combinations thereof. Other components include guide RNA (gRNA), single guide RNA (sgRNA), leader editing guide RNA (pegRNA), clustered regularly spaced short palindromic repeat (CRISPR) RNA (crRNA), trans-activating clustered regularly spaced short palindromic repeat (CRISPR) RNA (tracrRNA), or DNA molecules to be inserted or to serve as templates for double-strand break (DSB) repair at specific genomic loci. Genome, gene, and base editing technologies have been reviewed in 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. To the extent that they do not conflict with this disclosure, all content taught in each of these documents regarding the components and uses of this technology is incorporated herein by reference.

[0165] Delivering mRNA into cells provides transient expression of the encoded protein (which could be, for example, a CAR, TCR, or an immune cell adjuvant) lasting for several days. This may be sufficient, and even desirable, for therapeutic effects, and can be repeated if slightly longer expression is required. Delivery of components of gene / genome editing systems enables more permanent changes, while the editing system itself will only exist for a short time, yet the changes to cellular DNA will be persistent. Gene / genome editing systems may also allow for broader changes. In addition to conferring the expression of specific proteins, gene / genome editing systems also allow for the regulation of the expression of individual proteins to be altered or the knockout of protein expression.

[0166] In some embodiments comprising multiple reagents, the nucleic acid may be polycistronic. In other embodiments comprising multiple reagents or components, each reagent or component is encoded or contained as a separate nucleic acid substance. In some embodiments involving multiple payload nucleic acid substances, two or more nucleic acid substances are encapsulated together in a single LNP substance. In other embodiments, a subset of the payload nucleic acid substances to be delivered (e.g., a single nucleic acid substance) is encapsulated in one LNP substance, while another subset of the nucleic acid substances is encapsulated in another LNP substance. Different tLNP substances may differ only in the payload they contain. Different tLNP substances may be combined in a single formulation for administration.

[0167] F(ab') and F(ab') analogues

[0168] F(ab') and F(ab')-like forms offer certain advantages as targeting moieties of tLNPs. While any antibody fragment having a structure similar to or derived from the classic proteolytically produced F(ab') is generally referred to as F(ab'), the term "F(ab') analogue" is used herein to refer to engineered sequences containing amino acid substitutions and / or being truncated, distinguishing them from the polymerized native sequences. F(ab') are smaller than intact antibodies, which can be advantageous in manufacturing. When used as targeting moieties on tLNPs, their antigen-binding domains are further away from the LNP than, for example, scFv, which can facilitate interaction with the target cell surface. F(ab') molecules have cysteine ​​residues in a partially hinged region that can readily conjugate to functionalized PEG-lipids (e.g., maleimide-functionalized PEG-lipids). Furthermore, F(ab') can be engineered to have uniquely accessible cysteine ​​residues, enabling site-specific conjugation, which is desirable for product consistency. This can be achieved using recombinant DNA technology by truncating the hinge region of F(ab') or by changing cysteine ​​residues to another amino acid (such as serine) or both.

[0169] The cysteine ​​residue in the hinge region can form a cystine residue with another F(ab') molecule, resulting in F(ab')2. This would prevent the cysteine ​​from being used for conjugation with LNPs (more specifically, their functionalized lipids). This can be prevented by treating F(ab') under mild reducing conditions; however, this carries the risk of breaking the interchain disulfide bond between CL and CH1. This risk can be avoided by repositioning the interchain bond to a less accessible region within the molecule.

[0170] Some aspects combine the constant region of F(ab') or F(ab') analogues with the humanized immunoglobulin antigen-binding domain derived from the anti-CD8α antibody CT8 disclosed herein.

[0171] Some aspects combine the constant region of an F(ab') analog with the antigen-binding domain of an anti-CD8 antibody. 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 includes a humanized immunoglobulin antigen-binding domain derived from the anti-CD8α antibody CT8 disclosed herein.

[0172] In some respects, such as the engineered F(ab') analogues disclosed herein, they are conjugated with LNPs, but are general in terms of the variable structural domains of F(ab') analogues and their specificity.

[0173] In some aspects, the F(ab') or F(ab') analog constant region is combined with the antigen-binding domain of the anti-CD8 antibody conjugated to the 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.

[0174] Regarding these foregoing aspects, in some embodiments, the F(ab') analogue suitably includes a repositioned interchain disulfide bond, such as a Cκ S162C substitution paired with an IgG1 or IgG4 CH1 F174C substitution. In further embodiments, one, another, or both cysteine ​​residues involved in forming the native interchain disulfide bond are mutated, for example, 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 a cysteine ​​residue for conjugation with an LNP, such as those containing SEQ ID NO: 100. Such Cκ domains, such as those containing SEQ ID NO: 100, are particularly suitable for pairing with heavy chains that do not retain readily accessible cysteine ​​residues for conjugation with LNPs, such as SEQ ID NO: 99 and the .45 design exemplified by CBD1033.45 (see Table 17). In some embodiments, the Cκ domain does not retain C214, such as those containing SEQ ID NO: 89. Such Cκ domains, such as those containing SEQ ID NO: 89, are particularly suitable for pairing with heavy chains that retain easily accessible cysteine ​​residues for LNP conjugation, such as those containing SEQ ID NO: 85, 87, 90, 93, 95, 97, or 103. In some embodiments, the F(ab') analogue has a truncated CH region at P245, such as SEQ ID NO: 81 or 83. In some embodiments, the F(ab') analog has a truncated CH region at P241 and has substitutions for P240A and P241A, such as SEQ ID NO: 85, 87, 90, 93. In some embodiments, the F(ab') analog has a truncated IgG1 CH region at P240, such as SEQ ID NO: 95. In some embodiments, the F(ab') analog has a truncated IgG1 CH region at T238, such as SEQ ID NO: 97. In some embodiments, the F(ab') analog has a truncated IgG4 CH region at C239, such as SEQ ID NO: 103. In some embodiments, the cysteine ​​residue of the F(ab') analog for conjugation with the LNP is C239, such as SEQ ID NO: 85, 87, 90, 93, 95, or 103.In some embodiments, the cysteine ​​residue of the F(ab') analog used for conjugation with LNP is C233, for example, 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 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 comprises 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 containing 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 containing 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') analogue comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 84, SEQ ID NO: 88, SEQ ID NO: 94 or SEQ ID NO: 104.

[0175] The CBD1033 variable domain has been linked to the aforementioned constant region in various F(ab') and F(ab') analog designs, as shown in Table 17 (below). In various embodiments, the humanized CT8 variable domain or variable domains of other antibodies may be incorporated into these designs. Therefore, in various embodiments, the targeting portion of the LNP can be any of the F(ab') and F(ab') analog designs listed 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 (LNPs) and targeting LNPs (tLNPs)

[0176] Various LNP compositions are known in the art and can be used as the basis for targeting LNPs (tLNPs). For in vivo use, LNPs composed of cationic lipids (particularly ionizable cationic lipids), neutral lipids (such as phospholipids), sterols (such as cholesterol), and polymer-conjugated lipids (such as polyethylene glycol (PEG)-lipids) have shown advantageous properties. In specific embodiments of this 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 shown to form mRNA encapsulating LNPs, and peptides containing antibodies or their antigen-binding domains can be conjugated to them as targeting moieties. In some embodiments, the targeting moieties are engineered F(ab') as disclosed herein. In some embodiments, the targeting moieties comprise an antigen-binding domain specific to CD8 (such as specific to human CD8), regardless of whether the targeting moieties are intact antibodies, 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 in Table 14. In some embodiments, composition F9 in Table 14 is used to generate a tLNP containing the anti-CD8 binding moiety as its targeting moiety.

[0177] In any of the above-described tLNP embodiments, some embodiments include tLNPs in which the targeting portion comprises one of the humanized antigen-binding domains of CT8 disclosed herein, such as tLNPs comprising a VL region having the amino acid sequence of SEQ ID NO: 17 and a VH region having the amino acid sequence of one of SEQ ID NO: 11 or 27-29. In some such embodiments, the targeting portion is a fully humanized anti-CD8 antibody comprising a heavy chain having a silenced Fc region, such as a silenced Fc region having the amino acid sequence of SEQ ID NO: 43 or 44. In some cases, a fully humanized anti-CD8 antibody comprising a heavy chain having a silenced Fc region comprises a light chain comprising the sequence of CBD1033HC (SEQ ID NO: 61) and / or a sequence comprising CBD1033LC (SEQ ID NO: 62). In some embodiments, the targeting portion is anti-CD8 F(ab') of classical F(ab'). In some embodiments, the targeting portion is anti-CD8 F(ab') of engineered F(ab'). Examples of such anti-CD8 F(ab') of classic 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 κ constant region, wherein the κ constant region has the amino acid sequence SEQ ID NO: 41. In some embodiments, the anti-CD8 F(ab') comprises a light chain having an engineered κ constant region, wherein the κ constant region has the amino acid sequence SEQ ID NO: 89 or SEQ ID NO: 100. In some embodiments, the anti-CD8 F(ab') comprises a heavy chain having wild-type IgG1 F(ab'), wherein IgG1F(ab') has the amino acid sequence SEQ ID NO: 76. In some embodiments, anti-CD8 F(ab') comprises a heavy chain having engineered IgG1 F(ab'), wherein IgG1 F(ab') has the amino acid sequence 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, anti-CD8 F(ab') comprises a heavy chain having wild-type IgG4 F(ab'), wherein IgG4 F(ab') has the amino acid sequence SEQ ID NO: 79. In some embodiments, anti-CD8 F(ab') comprises a heavy chain having engineered IgG4 F(ab'), wherein 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, anti-CD8 F(ab') comprises a light chain having the following amino acid sequence: SEQ ID NO: 77, SEQ ID NO: 91, SEQ ID NO: 101, SEQ ID NO: 107, or SEQ ID NO: 112. In some embodiments, anti-CD8 F(ab') comprises a heavy chain having the following amino acid sequence: 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, anti-CD8 F(ab') comprises a heavy chain having the following amino acid sequence: SEQ ID NO: 80, SEQ ID NO: 84, SEQ ID NO: 88, SEQ ID NO: 94, or SEQ ID NO: 104. In any of the above-described tLNP embodiments encapsulating the mRNA disclosed herein, in some embodiments, the tLNP comprises, as its targeting portion, an antibody or its antigen-binding portion comprising one of the humanized antigen-binding domains of CT8 disclosed herein.

[0178] In some embodiments, the targeting portion of the tLNP is a classic or engineered F(ab'). In some embodiments, the targeting portion of the tLNP comprises engineered F(ab'). Examples of such F(ab') containing wild-type or engineered constant regions are listed in Table 17. In some such embodiments, the F(ab') comprises a light chain having a wild-type κ constant region, wherein the κ constant region has the amino acid sequence SEQ ID NO: 41. In some such embodiments, the F(ab') comprises a light chain having an engineered κ constant region, wherein the κ constant region has the amino acid sequence SEQ ID NO: 89 or SEQ ID NO: 100. In some such embodiments, the F(ab') comprises 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 an engineered IgG1 F(ab') constant region, wherein the IgG1 F(ab') constant region has the amino acid sequence 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 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 SEQ ID NO: 79. In some such embodiments, F(ab') comprises a heavy chain having an engineered IgG4 F(ab') constant region, wherein the IgG4 F(ab') constant region has the amino acid sequence SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 93, or SEQ ID NO: 103.

[0179] In some embodiments, tLNP comprises F(ab') or anti-CD8 F(ab') with an S162C κ chain substitution and an IgG1 or IgG4 CH1 F174C substitution. In some embodiments, the F(ab') or anti-CD8 F(ab') heavy chain further contains an IgG1CH1 C233S or IgG4 CH1 C127S substitution. In some embodiments, the F(ab') or anti-CD8 F(ab') light chain further contains a C214S κ chain substitution.

[0180] In some embodiments of the tLNP comprising an F(ab') targeting portion containing a constant region disclosed herein, the tLNP comprises one or more ionizable cationic lipids disclosed herein. In some embodiments of the tLNP comprising an F(ab') targeting portion containing a constant region disclosed herein, the tLNP comprises LNP compositions as disclosed in Table 14, such as F9.

[0181] In some implementations, the LNP (or tLNP) includes a binding moiety specific to immune cell antigens selected from: 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 receptor ‡ In a further embodiment, tLNP comprises a binding moiety specifically for HSC surface molecules selected from CD117. † CD34* ‡ CD44* ‡ CD45 †‡ CD90 (Thy1) ‡ CD105 ‡ CD133 ‡ BMPR2 ‡ and Sca-1; or specific binding sites to MSC surface molecules selected from the following: 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 ‡ In various embodiments, the binding portion is an antibody or its antigen-binding portion. (* indicates an exemplary antibody having the indicated specificity, from which the binding portion can be derived, and can be found in Table 9 or Table 10 of U.S. Patent No. 11,326,182B2.) † Exemplary antibodies with the indicated specificity are shown, from which the binding moiety can be derived; these can be found in Wilkinson & Hale, 2022. Both references cited above are incorporated herein by reference. ‡ Exemplary antibodies with the specificity shown are described, from which the binding moiety can be derived, and can be found in the Therapeutic Antibody Database (TABS) at tabs.craic.com. Other suitable antibodies can be found in Appendix A.

[0182] The LNP disclosed herein is a multi-component composition comprising a payload and multiple lipid components, including ionizable cationic lipids, unfunctionalized and / or functionalized PEG-lipids, phospholipids, and sterols. The tLNP disclosed herein is a multi-component composition comprising an LNP and a binding moiety such as a humanized anti-CD8 binding agent / antibody. As used herein, the term "tLNP composition" refers to the same characteristics as an LNP composition with an anti-CD8 binding moiety added as a targeting moiety, and the density of the binding moiety on the tLNP can be expressed as a w / w ratio to the payload. Table 14 provides a list of lipid compositions that have been shown to form LNPs. 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 typically surrounded by or located within the LNP or tLNP. As disclosed herein, dosage always refers to the amount of payload provided.

[0183] As used herein, the term "LNP composition" refers to the lipid components present in an LNP, their molar ratios relative to each other (e.g., mol%), and the ratio of payload to total lipids. In some aspects, the payload comprises one or more nucleic acid molecules or other negatively charged molecules. That is, in some embodiments, the payload comprises only a single nucleic acid substance or other negatively charged molecule (or is composed of such a substance or molecule), while in other embodiments, the payload comprises multiple nucleic acid substances or other negatively charged molecules, such as 2, 3, or 4 such substances or molecules. In some embodiments in which the payload comprises multiple nucleic acid substances or other negatively charged molecules, up to and including more than one substance of all substances that reacts with the same target or encodes a polypeptide that reacts with the same target.

[0184] LNP and tLNP composition

[0185] LNP compositions facilitate the formation of stable LNPs and tLNPs, efficiently encapsulate payloads, protect payloads from degradation until they are delivered to the cell, and promote payload escape from endosomes into the cytoplasm. These functions are largely independent of the specificity of the binding moiety (or moieties) used to direct or deflect tLNPs to specific cell types. Additional LNP and tLNP compositions are substantially disclosed in PCT / US2024 / 032141 (titled LipidNanoparticle Formulations and Compositions), filed May 31, 2024, all of which teaches the design, formation, characterization, properties, and uses of LNPs and tLNPs and is incorporated herein by reference.

[0186] LNPs and / or tLNPs may contain sufficient amounts of various components to provide nanoparticles with the desired shape, flowability, and bioacceptability as described herein. Regarding the LNPs or tLNPs of this 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 mol% to about 65 mol%, or any integer bounded sub-range thereof, for example, about 40 mol% to about 65 mol%, or about 40 mol% 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 an ionizable cationic lipid. In some embodiments, the LNP (or tLNP) comprises phospholipids in an amount ranging from about 7 mol% to about 30 mol%, or any integer bounded sub-range thereof, for example, about 13 mol% to about 30 mol%. In some embodiments, the LNP or tLNP comprises about 10 mol% phospholipids. In some embodiments, the LNP (or tLNP) comprises sterols in an amount ranging from about 20 mol% to about 50 mol%, or any integer bounded thereto, such as from about 20 mol% to about 45 mol%, or from about 30 mol% to about 50 mol%, or from about 30 mol% to about 45 mol%. In some embodiments, the LNP or tLNP comprises about 30.5 mol%, 26.5 mol%, or 23.5 mol% sterols. In some embodiments, the LNP (or tLNP) comprises at least one accessory lipid in an amount ranging from about 1 mol% to about 30 mol%. In some embodiments, the LNP or tLNP comprises total PEG-lipids in an amount ranging from about 1 mol% to about 5 mol%, or any integer × 10⁻⁶. -1 Within the bounded subscale, for example, in amounts ranging from about 1 mol% to about 2 mol% of total PEG-lipids. In some embodiments, LNP (or tLNP) comprises at least one nonfunctionalized PEG-lipid in an amount of 0 mol% to about 5 mol%, or any integer thereof × 10⁻⁶. -1 Within the bounded range, for example, in amounts ranging from 0 mol% to about 3 mol%, or from about 0.1 mol% to about 5 mol%, or from about 0.5 mol% to about 5 mol%, or from about 0.5 mol% to about 3 mol%. In some embodiments, the LNP or tLNP comprises about 1.4 mol% of unfunctionalized PEG-lipids. In some embodiments, the LNP or tLNP comprises at least one functionalized PEG-lipid in an amount ranging from about 0.1 mol% to about 5 mol%, or any integer × 10⁻⁶ of the same. -1Within the bounded subdomain range, for example, in the range of about 0.1 mol% to 0.3 mol%. In some embodiments, the LNP or tLNP comprises about 0.1 mol%, about 0.2 mol%, or about 0.3 mol% of functionalized PEG-lipids. In some embodiments, the LNP or tLNP comprises about 0.1 mol% of functionalized PEG-lipids. In some embodiments, the functionalized PEG-lipids are conjugated to a binding moiety. In some embodiments, the binding moiety is an engineered F(ab') as disclosed herein. In some embodiments, the binding moiety comprises an anti-CD8 antigen binding domain, such as the humanized anti-CD8α antigen binding domain disclosed herein. In some cases, the tLNP comprises an intact anti-CD8α antibody as the binding moiety, which is present at an antibody:mRNA ratio (w / w) of about 0.3 to about 1.0.

[0187] In some aspects, this disclosure provides an LNP or tLNP comprising about 35 mol% to about 65 mol% of an ionizable cationic lipid, about 0.5 mol% to about 3 mol% of a PEG-lipid (including unfunctionalized PEG-lipids and optionally functionalized PEG-lipids), about 7 mol% to about 13 mol% of a phospholipid, and about 30 mol% to about 50 mol% of a sterol. In some embodiments, the LNP or tLNP comprises a payload having a net negative charge, such as a peptide, polypeptide, protein, small molecule, or nucleic acid molecule, or a combination thereof. The payload is typically surrounded by or located within the LNP or tLNP. As disclosed herein, a dose always refers to the amount of payload provided. In some embodiments, the payload comprises one or more types of nucleic acid molecules. For tLNP-encapsulated mRNA, the dose is typically in the range of 0.05 mg / kg to 5 mg / kg, regardless of the recipient species. In some embodiments, the dose is in the range of 0.1 mg / kg to 1 mg / kg.

[0188] The ratio of payload to total lipids can be expressed on a w / w basis, or for nucleic acid molecules, as an N / P ratio. Regarding the LNP or tLNP of this disclosure, in some embodiments, the ratio of total lipids to nucleic acids is from about 10:1 to about 50:1 by weight. In some embodiments, the ratio of total lipids to nucleic acids is about 10:1, about 20:1, about 30:1, or about 40:1 to about 50:1, or 10:1 to 20:1, 30:1, 40:1, or 50:1, or any range defined by a pair of these ratios. The ratio of lipids to nucleic acids can also be reported as an N / P ratio, which is the ratio of positively charged lipid amine (N = nitrogen) groups to negatively charged nucleic acid phosphate (P) groups. In some embodiments, the N / P ratio is from about 3 to about 9, from about 3 to about 7, from about 3 to about 6, from about 4 to about 6, from about 5 to about 6, or about 6. In some implementations, the N / P ratio is 3 to 9, 3 to 7, 3 to 6, 4 to 6, 5 to 6, or 6.

[0189] Due to physiological and manufacturing limitations, a hydrodynamic diameter of LNP or tLNP particles for in vivo use is ideally between about 50 nm and 150 nm. Therefore, in some embodiments, LNPs or tLNPs have a hydrodynamic diameter of 50 nm to 150 nm, and in some embodiments, the hydrodynamic diameter is ≤120 nm, ≤110 nm, ≤100 nm, or ≤90 nm. Particle size uniformity is also desirable, where a polydispersity index (PDI) of ≤0.2 (on a scale of 0 to 1) is acceptable. Both the hydrodynamic diameter and the polydispersity index are determined by dynamic light scattering (DLS). Particle size assessed by cryo-transmission electron microscopy (Cryo-TEM) can be smaller than the value determined by DLS.

[0190] The density of the binding moiety on the tLNP can be defined based on the amount of antibody input based on the conjugation reaction or the ratio (w / w) of antibody (binding agent) to mRNA as measured in the tLNP. For intact antibodies (e.g., whole IgG), in some embodiments, the preferred ratios for the input or final measured binding agent ratio 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. In some 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 for the input or final measured binding moiety density ratio. In some embodiments, if the binding agent differs in size from the intact antibody (e.g., scFv, biantibody, or microantibody, etc.), the w / w ratio is adjusted for the different sizes of the binding moiety.

[0191] Ionizable cationic lipids

[0192] In specific embodiments, ionizable cationic lipids are described in US20230320995A1, international applications PCT / US2024 / 049627 and PCT / US2024 / 049649, and US Provisional Applications 63 / 632,944 and 63 / 632,940, the disclosures of which are incorporated herein by reference in their entirety. Ionizable cationic lipids are useful components for compounding with negatively charged payloads and for facilitating the delivery of payloads to the cytoplasm of cells following endocytosis. Therefore, each genus and species of ionizable cationic lipids disclosed herein can be used in the LNP and tLNP formulations and compositions of this disclosure, as well as methods of using them. In some embodiments, ionizable cationic lipids of LNPs having a measured pKa of 6 to 7 can remain substantially neutral in the bloodstream and interstitial space, but ionize upon uptake into cells as endosomal acidification occurs. Upon acidification in the endosome space, lipids become protonated and associate more tightly with the phosphate backbone of nucleic acids. This destabilizes the structure of LNPs and promotes the release of nucleic acids from LNPs into the cytoplasm (also known as endosome escape). Therefore, the ionizable cationic lipids disclosed herein constitute a means for destabilizing the structure of LNPs (when ionized) or for promoting nucleic acid release or endosome escape.

[0193] For simplicity, the chemical part is primarily defined and referred to throughout as the monovalent chemical part (e.g., alkyl, aryl, etc.). However, such terms may also be used to convey the corresponding polyvalent part where appropriate structures are clear to those skilled in the art. For example, while the "alkyl" part generally refers to a monovalent group (e.g., CH3-CH2-), in some cases the divalent linking part can be "alkyl," in which case those skilled in the art will understand that alkyl is a divalent group (e.g., -CH2-CH2-), which is equivalent to the term "alkylene". (Similarly, where a divalent part is required and is stated as "aryl," those skilled in the art will understand that the term "aryl" refers to the corresponding divalent part, arylene.) All atoms are understood to have their normal valence 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).

[0194] As used herein, the term "alkyl" refers to a saturated straight-chain and branched aliphatic group having 1 to 12 carbon atoms. Therefore, "alkyl" includes C1, C2, C3, C4, C5, C6, C7, C8, C9, C1 ...1, C1, C1, C1, C1, C1, C1 10 C 11 and C 12 Group.

[0195] As used herein, the term "alkenyl" refers to an unsaturated straight-chain or branched aliphatic group having one or more carbon-carbon double bonds and containing 2 to 12 carbon atoms. Therefore, "alkenyl" includes C2, C3, C4, C5, C6, C7, C8, C9, C16, C17, C18, C19, C19, C10, C11, C12, C13, C14, C15, C16, C17, C18 ... 10 C 11 and C 12 Group.

[0196] In some embodiments, the hydrocarbon chain is unsubstituted. In other embodiments, one or more hydrogen atoms of the alkyl or alkenyl group may be substituted with the same or different substituents.

[0197] An aryl group is an aromatic or heteroaromatic ring that lacks a hydrogen atom, leaving a bond to attach to another part of an organic molecule. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, pyridine, pyrimidine, pyrazine, pyrrole, furan, thiophene, imidazole, thiazole, oxazole, etc.

[0198] Aryl-alkyl refers to a moiety comprising one or more aryl rings and one or more alkyl moieties. The position of the one or more aryl rings can vary within the alkyl moieties of the moiety. For example, the one or more aryl rings may be located at the end of the one or more alkyl moieties, fused to the carbon chain of the one or more alkyl moieties, or replace one or more hydrogens of the one or more alkyl moieties; and the one or more alkyl moieties may replace one or more hydrogens of the one or more aryl rings. In some embodiments, a single ring is present; in other embodiments, multiple rings are present.

[0199] Branched alkyl groups are saturated alkyl moieties in which the alkyl groups are not straight chains. Alkyl moieties such as methyl, ethyl, propyl, and butyl can be attached to variable positions on the main alkyl chain. In some embodiments, a single branch exists; in other embodiments, multiple branches exist.

[0200] A branched alkenyl group refers to an alkenyl group whose main chain contains at least one branch, which can be formed by replacing one or more hydrogen atoms in the main chain with the same or different alkyl groups (e.g., but not limited to methyl, ethyl, propyl, butyl, etc.). In some embodiments, the branched alkenyl group is a single-branched structure, while in other embodiments, the branched alkenyl group can have multiple branches.

[0201] Straight-chain alkyl groups are the unbranched, non-cyclic forms of the aforementioned alkyl moiety.

[0202] Straight-chain alkenyl groups are the unbranched, non-cyclic forms of the alkenyl groups mentioned above.

[0203] In some respects, the ionizable cationic lipids of this disclosure have the structure of formula M5:

[0204]

[0205] in:

[0206] Each R 1 Independently selected from C7-C 11 Alkyl or C7-C 11 alkenyl,

[0207] A 1 (CH2) 1-2 ,

[0208] A 2 For O,

[0209] A 3 (CH2) 1-5 Where X is N, then A 3 Not CH2

[0210] X is N, CH, or C-CH3.

[0211] A 4 It can be CH2, C=O, NH, NCH3 or O.

[0212] If A 4 If C=O, then A 5 For non-existent, O, S, NH or NCH3, or if A 4 If C=O, then A 5 For C=O,

[0213] A 6 It can be O, S, NH, NCH3, or (CH2). 0-2 ,

[0214] A 7 (CH2) 0-6 If A 6 If the components are O, S, NH, and NCH3, then A 7 (CH2) 2-4 ,

[0215] Y is , , , , , , , , , , , , , , , , , , , , or ,

[0216] Where Z is a bond; and

[0217] R 2 For O, R 3 For C=O and W is CH or N, or R 2 For C=O, R 3 It is O and W is CH;

[0218] Where A 6 and A 7 Not simultaneously (CH2)0, unless A 5 For C=O;

[0219] in

[0220] a)A 1 For CH2, A 3 (CH2) 2-5 X is N, A 4 For C=O, A 5 For O, S, NH, NCH3, A 6 (CH2) 1-2 A 7 (CH2) 1-4 ,or

[0221] b)A 1 For CH2, A 3 (CH2) 1-4 X is CH, A 4 For CH2, NH, NCH3, O, A 5 For C=O, A 6 For O, NH, NCH3, A 7 For (CH) 2-6 ,or

[0222] c)A 1 For (CH2)2, A 3 (CH2) 1-4 X is C-CH3, A 4 For C=O, A 5 For O, NH, NCH3, A 6 (CH2) 1-2 A 7 (CH2) 1-4 ,or

[0223] d)A 1 For CH2, A 3 (CH2) 2-5X is N, A 4 For C=O, A 5 Does not exist, A 6 For (CH2)0, A 7 It is (CH2)0, and Y is ,or

[0224] e)A 1 For CH2, A 3 (CH2) 1-5 X is CH, A 4 For CH2, NH, NCH3 or O, A 5 For C=O, A 6 For (CH2)0, A 7 It is (CH2)0, and Y is ,or

[0225] f)A 1 For (CH2)2, A 3 (CH2) 1-5 X is CCH3, A 4 For C=O, A 5 Does not exist, A 6 For (CH2)0, A 7 It is (CH2)0, and Y is ;

[0226] in

[0227] The number of consecutive atoms present in a span:

[0228] It is in the range of 7 to 17.

[0229] As used in this article, when the subscript has a value of "0", the group does not exist. For example, when A 6 When it is (CH2)0, A 6 It does not exist.

[0230] In some implementations of formula M5, R 2 For O, R 3 C=O and W is CH or N. For example, in some embodiments of formula M5, R 2 For O, R 3 C=O and W is CH.

[0231] In some implementations of formula M5, R 2 For C=O, R 3 It is O and W is CH.

[0232] In some implementations of M5, A 1 For CH2, A 3(CH2) 2-5 X is N, A 4 For C=O, A 5 For O, S, NH, NCH3, A 6 (CH2) 1-2 And A 7 (CH2) 1-4 For example, in some implementations, A 1 For CH2, A 3 (CH2) 2-5 X is N, A 4 For C=O, A 5 for O,A 6 (CH2) 1-2 And A 7 (CH2) 1-4 .

[0233] In some implementations of M5, A 1 For CH2, A 3 (CH2) 1-4 X is CH, A 4 For CH2, NH, NCH3, O, A 5 For C=O, A 6 It is O, NH, NCH3 or CH2, and A 7 For (CH) 2-6 In some implementations as described herein, A 1 For CH2, A 3 (CH2) 1-4 X is CH, A 4 For NH, A 5 For C=O, A 6 It is O, NH, NCH3 or CH2, and A 7 For (CH) 2-6 For example, in some embodiments of formula M5, A 1 For CH2, A 3 (CH2) 1-4 X is CH, A 4 For NH, A 5 For C=O, A 6 It is O, and A 7 For (CH) 2-6 For example, in some embodiments of formula M5, A 1 For CH2, A 3 (CH2) 1-4 X is CH, A 4 For NH, A 5 For C=O, A 6 It is CH2, and A7 For (CH) 2-6 In some implementations as described herein, A 1 For CH2, A 3 (CH2) 1-4 X is CH, A 4 For CH2, A 5 For C=O, A 6 It can be O, NH, NCH3 or CH2, or A 7 For (CH) 2-6 For example, in some embodiments of formula M5, A 1 For CH2, A 3 (CH2) 1-4 X is CH, A 4 For CH2, A 5 For C=O, A 6 for O,A 7 For (CH) 2-6 In some implementations as described herein, A 1 For CH2, A 3 (CH2) 1-4 X is CH, A 4 for O,A 5 For C=O, A 6 It is O, NH, NCH3 or CH2, and A 7 For (CH) 2-6 For example, in some embodiments of formula M5, A 1 For CH2, A 3 (CH2) 1-4 X is CH, A 4 for O,A 5 For C=O, A 6 It is CH2, and A 7 For (CH) 2-6 In some implementations as described herein, A 1 For CH2, A 3 (CH2) 1-4 X is CH, A 4 For NCH3, A 5 For C=O, A 6 It is O, NH, NCH3 or CH2, and A 7 For (CH) 2-6 For example, in some embodiments of formula M5, A 1 For CH2, A 3 (CH2) 1-4 X is CH, A 4 For NCH3, A 5 For C=O, A6 It is CH2, and A 7 For (CH) 2-6 .

[0234] In some implementations of M5, A 1 For (CH2)2, A 3 (CH2) 1-4 X is C-CH3, A 4 For C=O, A 5 For O, NH, NCH3, A 6 (CH2) 1-2 , or A 7 (CH2) 1-4 For example, in some implementations, A 1 For (CH2)2, A 3 (CH2) 1-4 X is C-CH3, A 4 For C=O, A 5 for O,A 6 (CH2) 1-2 , or A 7 (CH2) 1-4 .

[0235] In some implementations of formula M5, the number of consecutively connected atoms present in a span is: The range is 7 to 17. For example, in some implementations, the number of consecutively connected atoms present in a span is: Within the range of 7 to 11 or 7 to 10. In some implementations, the number of consecutively connected atoms present in a span: Within the range of 10 to 17 (e.g., within the range of 10 to 16, 10 to 14, or 10 to 12). For example, in some embodiments, the number of consecutively connected atoms present in a span: The value is 10. For example, in some implementations, the number of consecutively connected atoms present in a span is: The value is 7. The inventors have discovered that changing the number of consecutively connected atoms present in each span allows for modulation of the pKa of cationic lipids.

[0236] In some implementations of formula M5, Y is... And Z is the key. In some implementations of formula M5, Y is... And Z is the key. For example, in some implementations of formula M5, Y is... And Z is the key.

[0237] In some implementations of formula M5, Y is... And Z is the key. For example, in some implementations of formula M5, Y is... And Z is the key.

[0238] In some implementations of formula M5, Y is... And Z is the key. For example, in some implementations of formula M5, Y is... And Z is the key.

[0239] In some implementations of formula M5, Y is... And Z is the key.

[0240] In some implementations of formula M5, Y is... And Z is the key.

[0241] In some implementations of formula M5, Y is... And Z is the key.

[0242] In some implementations of formula M5, Y is... And Z is the key.

[0243] In some implementations of formula M5, Y is... And Z is the key.

[0244] In some implementations of formula M5, Y is... And Z is the key.

[0245] In some implementations of formula M5, Y is... And Z is the key.

[0246] In some implementations of formula M5, Y is... And Z is the key.

[0247] In some implementations of formula M5, Y is... And Z is the key.

[0248] In some implementations of formula M5, Y is... And Z is the key.

[0249] In some implementations of formula M5, Y is... And Z is the key.

[0250] In some implementations of formula M5, Y is... And Z is the key.

[0251] In some implementations of formula M5, Y is... And Z is the key.

[0252] In some implementations of formula M5, Y is... And Z is the key.

[0253] In some implementations of formula M5, Y is... And Z is the key.

[0254] In some implementations of formula M5, Y is... And Z is the key.

[0255] In some implementations of formula M5, Y is... And Z is the key.

[0256] In some implementations of formula M5, Y is... And Z is the key.

[0257] In some implementations of formula M5, Y is... And Z is the key.

[0258] In some embodiments as described herein, the ionizable cationic lipid has the structure of Formula 1:

[0259]

[0260] in:

[0261] Y can be O, NH, N-CH3, or CH2.

[0262] n is an integer from 0 to 4.

[0263] X is , , , , , , , or ,

[0264] m is an integer from 1 to 3.

[0265] o is an integer from 1 to 4.

[0266] p is an integer from 1 to 4.

[0267] When p=1:

[0268] Each R is independently C6 to C 16 Straight-chain alkyl; C6 to C 16 Branched alkyl groups; C6 to C 16 Straight-chain alkenyl; C6 to C 16 Branched alkenyl groups; C9 to C 16 Cycloalkyl-alkyl, wherein the cycloalkyl group is a C3 to C8 cycloalkyl group located at any end or within the alkyl chain; or a C8 to C8 cycloalkyl group. 18Aryl-alkyl, wherein the aryl group is phenyl or naphthyl and is located at either end or inside the alkyl chain;

[0269] When p=2:

[0270] Each R is independently C6 to C 14 Straight-chain alkyl; C6 to C 14 Straight-chain alkenyl; C6 to C 14 Branched alkyl groups; C6 to C 14 Branched alkenyl groups; C9 to C 14 Cycloalkyl-alkyl, wherein the cycloalkyl group is a C3 to C8 cycloalkyl group located at any end or within the alkyl chain; or a C8 to C8 cycloalkyl group. 16 Aryl-alkyl, wherein the aryl group is phenyl or naphthyl and is located at either end or inside the alkyl chain;

[0271] When p=3:

[0272] Each R is independently C6 to C 12 Straight-chain alkyl; C6 to C 12 Straight-chain alkenyl; C6 to C 12 Branched alkyl groups; C6 to C 12 Branched alkenyl groups; C9 to C 12 Cycloalkyl-alkyl, wherein the cycloalkyl group is a C3 to C8 cycloalkyl group located at any end or within the alkyl chain; or a C8 to C8 cycloalkyl group. 14 Aryl-alkyl, wherein the aryl group is phenyl or naphthyl and is located at either end or inside the alkyl chain; and

[0273] When p=4:

[0274] Each R is independently C6 to C 10 Straight-chain alkyl; C6 to C 10 Straight-chain alkenyl; C6 to C 10 Branched alkyl groups; C6 to C 10 Branched alkenyl groups; C9 to C 10 Cycloalkyl-alkyl, wherein the cycloalkyl group is a C3 to C8 cycloalkyl group located at either end or within the alkyl group; or a C8 to C8 cycloalkyl group. 12 Aryl-alkyl, wherein the aryl group is phenyl or naphthyl and is located at either end or inside the alkyl chain.

[0275] In some implementations, the ionizable cationic lipid has a CICL structure:

[0276]

[0277] Where R is , , or .

[0278] In some implementations, when R is (Right now When CICL is ionized cationic lipid, it is called CICL1.

[0279] In some implementations, when R is (Right now When CICL is ionized cationic lipid, it is called CICL2.

[0280] In some implementations, when R is (Right now When CICL is ionized cationic lipid, it is called CICL3.

[0281] In some implementations, when R is (Right now When CICL is ionized cationic lipid, it is called CICL4.

[0282] In some implementations, the ionizable cationic lipid has the structure CICL-IE:

[0283]

[0284] Where R is , , or .

[0285] In some implementations, when R is (Right now When CICL-IE is used, the ionizable cationic lipid is called CICL250.

[0286] In some implementations, when R is (Right now When CICL-IE is used, the ionizable cationic lipid is called CICL250.2.

[0287] In some implementations, when R is (Right now When CICL-IE is used, the ionizable cationic lipid is called CICL250.3.

[0288] In some implementations, when R is (Right now When CICL-IE is used, the ionizable cationic lipid is called CICL250.4.

[0289] In some respects, the constrained ionizable cationic lipids of this disclosure have a structure of formula M6:

[0290]

[0291] Where X is , , , , , , , , , , , , , , , , , , , , , or ;and

[0292] Y is O, S, NH, or NCH3;

[0293] Z is O, NH, or NCH3;

[0294] R 2 For O, R 3 For C=O and W is CH or N, or R 2 For C=O, R 3 O is O and W is CH; and

[0295] Each R 1 Independently selected from C7-C 11 Alkyl or C7-C 11 alkenyl;

[0296] Each A 1 A 2 A 3 and A 4 Independently selected from (CH2)0 and (CH2)1,

[0297] A 5 Selected from (CH2) 0-4 CH=CH and CH2-CH=CH-CH2; and

[0298] The wavy bond indicates that any relative or absolute stereoconfiguration or mixture of stereoconfigurations of the corresponding ring atom can be assumed.

[0299] As used in this article, when the subscript has a value of "0", the group does not exist. For example, when A 1 When it is (CH2)0, A 1 It does not exist.

[0300] In some implementations of formula M6, R 2 For O, R 3 C=O and W is CH or N. For example, in some embodiments of formula M6, R 2 For O, R 3 C=O and W is CH.

[0301] In some implementations of formula M6, R 2 For C=O, R 3 It is O and W is CH.

[0302] In the various implementation schemes of M6, option A is selected. 1 To A 4 This results in only two main chain atoms between each nearest ester oxygen in the cyclic nitrogen and the nearest tail group.

[0303] In some implementations of M6, A 1 For (CH2)0, A 2 For (CH2)0, A 3 For (CH2)1, A 4 It is (CH2)1, and A 5 (CH2) 1-4 Or CH2-CH=CH-CH2.

[0304] In some implementations of M6, A 1 For (CH2)0, A 2 For (CH2)1, A 3 For (CH2)1, A 4 It is (CH2)0, and A 5 It is (CH2)1.

[0305] In some implementations of M6, A 1 For (CH2)1, A 2 For (CH2)1, A 3 For (CH2)0, A 4 It is (CH2)0, and A 5 It is (CH2)0.

[0306] In some implementations of M6, A 1 For (CH2)1, A 2 For (CH2)1, A 3 For (CH2)0, A 4 It is (CH2)0, and A 5 It is (CH2)1.

[0307] In some implementations of M6, A 1 For (CH2)1, A2 For (CH2)1, A 3 For (CH2)0, A 4 It is (CH2)0, and A 5 It is (CH2)2 or CH=CH.

[0308] In some implementations of formula M6 as described herein, X is... For example, in some implementations of formula M6, X is... .

[0309] In some implementations of formula M6 as described herein, X is... .

[0310] In some implementations of formula M6 as described herein, X is... .

[0311] In some implementations of formula M6 as described herein, X is... In some implementations, X is... .

[0312] In some implementations of formula M6 as described herein, X is... .

[0313] In some implementations of formula M6 as described herein, X is... .

[0314] In some implementations of formula M6 as described herein, X is... .

[0315] In some implementations of formula M6 as described herein, X is... .

[0316] In some implementations of formula M6 as described herein, X is... .

[0317] In some implementations of formula M6 as described herein, X is... .

[0318] In some implementations of formula M6 as described herein, X is... .

[0319] In some implementations of formula M6 as described herein, X is... .

[0320] In some implementations of formula M6 as described herein, X is... .

[0321] In some implementations of formula M6, X is... In some implementations of formula M6, X is... In some implementations of formula M6, X is... .

[0322] In some implementations of formula M6, X is... In some implementations of formula M6, X is... .

[0323] In some implementations of formula M6, X is... In some implementations of formula M6, X is... In some implementations of formula M6, X is... .

[0324] In some implementations of formula M6, X is... In some implementations of formula M6, X is... In some implementations of formula M6, X is... In some implementations of formula M6, X is... .

[0325] In some implementations of formula M6, X is... In some implementations of formula M6, X is... In some implementations of formula M6, X is... .

[0326] In some implementations of formula M6, X is... In some implementations of formula M6, X is... In some implementations of formula M6, X is... .

[0327] In some implementations of formula M6, X is... In some implementations of formula M6, X is... In some implementations of formula M6, X is... .

[0328] In some implementations of formula M6, X is... In some implementations of formula M6, X is... In some implementations of formula M6, X is... .

[0329] In some implementations of formula M6, X is... In some implementations of formula M6, X is... .

[0330] In some implementations of formula M6, X is... In some implementations of formula M6, X is... .

[0331] As described above, in some embodiments of formula M6, Y can 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.

[0332] In some implementations of formula M6, X is... And Y is O. In some implementations of formula M6, X is And Y is S.

[0333] As described above, Z can be selected from O, NH, or NCH3. In some embodiments, Z is O.

[0334] In some implementations of formula M6, X is... And Z is O. In some implementations of formula M6, X is And Z is O. In some implementations of formula M6, X is And Z is O.

[0335] In some implementations of formula M6, X is... And Z is O. In some implementations of formula M6, X is And Z is O.

[0336] In some implementations of formula M6, X is... And Z is O. In some implementations of formula M6, X is And Z is O. In some implementations of formula M6, X is And Z is O.

[0337] In some implementations of formula M6, X is... And Z is O. In some implementations of formula M6, X is And Z is O. In some implementations of formula M6, X is And Z is O. In some implementations of formula M6, X is And Z is O.

[0338] In some implementations of formula M6, X is... And Z is O. In some implementations of formula M6, X is And Z is O. In some implementations of formula M6, X is And Z is O.

[0339] In some implementations of formula M6, X is... And Z is O. In some implementations of formula M6, X is And Z is O. In some implementations of formula M6, X is And Z is O.

[0340] In some implementations of formula M6, X is... And Z is O. In some implementations of formula M6, X is And Z is O. In some implementations of formula M6, X is And Z is O.

[0341] In some implementations of formula M6, X is... And Z is O. In some implementations of formula M6, X is And Y is O. In some implementations of formula M6, X is And Z is O.

[0342] In some implementations of formula M6, X is... And Z is O. In some implementations of formula M6, X is And Z is O.

[0343] In some implementations of formula M6, X is... And Z is O. In some implementations of formula M6, X is And Z is O.

[0344] As described above, for both equations M5 and M6, each R 1 Independently selected from C7-C 11 Alkyl or C7-C 11 Alkenyl. In some embodiments of formula M5 and / or M6, each R 1 Independently selected from C7-C 11 Alkyl groups, such as C7-C 10 Alkyl or C7-C9 alkyl. In certain embodiments of formula M5 and / or M6, each R 1 Independently selected from linear C7-C 11 Alkyl groups, such as linear C7-C 10 Alkyl or linear C7-C9 alkyl. In some embodiments such as formula M5 and / or M6 as described herein, each R 1 Independently selected from (CH2) 6-8 CH3. In some implementations of these and other implementations, R 1 It is (CH2)7CH3. In some embodiments of formula M5 and / or M6, each R 1 Independently selected from linear C7-C 11Alkenyl groups, such as linear C7-C 10 Alkenyl or linear C7-C9 alkenyl. For example, in some embodiments of formula M5 and / or M6, each R 1 It is a linear C8 alkenyl group. In some other embodiments of formula M5 and / or M6, each R 1 Independently selected from branch C7-C 11 Alkyl groups, such as C7-C 10 Alkyl or C7-C9 alkyl. For example, in some embodiments of formula M5 and / or M6, each R 1 It is a branched C8 alkyl group. In certain embodiments of formula M5 and / or M6, each R... 1 Independently selected from branch C7-C 11 Alkenyl groups, such as C7-C 10 Alkenyl or C7-C9 alkenyl. For example, in some embodiments of formula M5 and / or M6, each R 1 It is a branched C8 alkenyl group. In some embodiments of formula M5 and / or M6, wherein R... 1 The ester carbonyl group is branched alkyl or alkenyl, and the position of the branch point is such that the carbonyl group is not in the α position relative to the branch point, but rather in the β position relative to the branch point.

[0345] In some embodiments of formulas M5 and / or M6 as described herein, each R 1 They are the same. In some implementations of formula M5 and / or M6, each R closest to the common branch point 1 They are the same, but those closest to the first common branch point are different from those closest to the second common branch point. In some embodiments of formula M5 and / or M6, each R closest to the common branch point... 1 They are different, but the pair of R closest to the first common branch point 1 It is the same as the pair closest to the second common branch point.

[0346] In some embodiments 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 some embodiments of Formula M6, the ionizable cationic lipid is a racemic mixture. In some embodiments of Formula M6, the ionizable cationic lipid is a mixture of two or more stereoisomers. In some embodiments of Formula M6, at least two of the two or more stereoisomers are diastereomers. In some embodiments of Formula M6, at least two of the two or more stereoisomers are enantiomers.

[0347] In some embodiments as described herein, the ionizable cationic lipid has the structure of Formula 2:

[0348]

[0349] in:

[0350] Y can be O, NH, N-CH3, or CH2.

[0351] n is an integer from 0 to 4.

[0352] X is , , , , , , , or ,

[0353] m is an integer from 1 to 3.

[0354] o is an integer from 1 to 4.

[0355] p is an integer from 1 to 4.

[0356] When p=1:

[0357] Each R is independently C6 to C 16 Straight-chain alkyl; C6 to C 16 Branched alkyl groups; C6 to C 16 Straight-chain alkenyl; C6 to C 16 Branched alkenyl groups; C9 to C 16 Cycloalkyl-alkyl, wherein the cycloalkyl group is a C3 to C8 cycloalkyl group located at any end or within the alkyl chain; or a C8 to C8 cycloalkyl group. 18 Aryl-alkyl, wherein the aryl group is phenyl or naphthyl and is located at either end or inside the alkyl chain;

[0358] When p=2:

[0359] Each R is independently C6 to C 14 Straight-chain alkyl; C6 to C 14 Straight-chain alkenyl; C6 to C 14 Branched alkyl groups; C6 to C 14 Branched alkenyl groups; C9 to C 14 Cycloalkyl-alkyl, wherein the cycloalkyl group is a C3 to C8 cycloalkyl group located at any end or within the alkyl chain; or a C8 to C8 cycloalkyl group. 16 Aryl-alkyl, wherein the aryl group is phenyl or naphthyl and is located at either end or inside the alkyl chain.

[0360] When p=3:

[0361] Each R is independently C6 to C 12 Straight-chain alkyl; C6 to C 12 Straight-chain alkenyl; C6 to C 12 Branched alkyl groups; C6 to C 12 Branched alkenyl groups; C9 to C 12 Cycloalkyl-alkyl, wherein the cycloalkyl group is a C3 to C8 cycloalkyl group located at any end or within the alkyl chain; or a C8 to C8 cycloalkyl group. 14 Aryl-alkyl, wherein the aryl group is phenyl or naphthyl and is located at either end or inside the alkyl chain; and

[0362] When p=4:

[0363] Each R is independently C6 to C 10 Straight-chain alkyl; C6 to C 10 Straight-chain alkenyl; C6 to C 10 Branched alkyl groups; C6 to C 10 Branched alkenyl groups; C9 to C 10 Cycloalkyl-alkyl, wherein the cycloalkyl group is a C3 to C8 cycloalkyl group located at either end or within the alkyl group; or a C8 to C8 cycloalkyl group. 12 Aryl-alkyl, wherein the aryl group is phenyl or naphthyl and is located at either end or inside the alkyl chain.

[0364] In some embodiments as described herein, the ionizable cationic lipid has the structure of Formula 3:

[0365]

[0366] in:

[0367] W is either C=O or CH2.

[0368] n is an integer from 0 to 4.

[0369] X is , , , , , , , or ,

[0370] m is an integer from 1 to 3.

[0371] o is an integer from 1 to 4.

[0372] p is an integer from 1 to 4.

[0373] When p=1:

[0374] Each R cIndependently for C8 to C 18 Straight-chain alkyl; C8 to C 18 Straight-chain alkenyl; C8 to C 18 Branched alkyl groups; C8 to C9 18 Branched alkenyl; C 11 To C 18 Cycloalkyl-alkyl, wherein the cycloalkyl group is a C3 to C8 cycloalkyl group located at any end or within the alkyl chain; or C 10 To C 20 Aryl-alkyl, wherein the aryl group is phenyl or naphthyl and is located at either end or inside the alkyl chain.

[0375] When p=2:

[0376] Each R c Independently for C8 to C 16 Straight-chain alkyl; C8 to C 16 Straight-chain alkenyl; C8 to C 16 Branched alkyl groups; C8 to C9 16 Branched alkenyl; C 11 To C 16 Cycloalkyl-alkyl, wherein the cycloalkyl group is a C3 to C8 cycloalkyl group located at any end or within the alkyl chain; or C 10 To C 18 Aryl-alkyl, wherein the aryl group is phenyl or naphthyl and is located at either end or inside the alkyl chain.

[0377] When p=3:

[0378] Each R c Independently for C8 to C 14 Straight-chain alkyl; C8 to C 14 Straight-chain alkenyl; C8 to C 14 Branched alkyl groups; C8 to C9 14 Branched alkenyl; C 11 To C 14 Cycloalkyl-alkyl, wherein the cycloalkyl group is a C3 to C8 cycloalkyl group located at any end or within the alkyl chain; or C 10 To C 16 Aryl-alkyl, wherein the aryl group is phenyl or naphthyl and is located at either end or inside the alkyl chain, and

[0379] When p=4:

[0380] Each R c Independently for C8 to C 12 Straight-chain alkyl; C8 to C 12 Straight-chain alkenyl; C8 to C 12 Branched alkyl groups; C8 to C9 12 Branched alkenyl; C 11 To C 12Cycloalkyl-alkyl, wherein the cycloalkyl group is a C3 to C8 cycloalkyl group located at either end or within the alkyl group; or C 10 To C 14 Aryl-alkyl, wherein the aryl group is phenyl or naphthyl and is located at either end or inside the alkyl chain.

[0381] R in the text c With R in chemical structure c They are equivalent.

[0382] The ionizable cationic lipids disclosed herein have a branched structure to impart a conical rather than cylindrical shape to the lipids, and such a structure facilitates endosome cleavage activity. Greater endosome cleavage activity leads to more efficient release of bioactive payloads (e.g., one or more nucleic acid molecules).

[0383] The ionizable cationic lipids described herein can be used as components of lipid nanoparticles for the delivery of 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, 6 to 10, 7 to 10, 8 to 10, or 8 to 9. In some 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, or about 10 to about 16, about 10 to about 14, or about 11 to about 18, or about 11 to about 15, or about 11 to about 14. The ionizable cationic lipids described herein may have a cLogD ranging from 9 to 18, for example, from 10 to 18, or 10 to 16, or 10 to 14, or 11 to 18, or 11 to 15, or 11 to 14. In some embodiments, the ionizable cationic lipids have a cLogD ranging from about 13.6 to about 14.4 or 13.6 to 14.4. In some embodiments, the ionizable cationic lipids described herein may have a c-pKa ranging from about 8 to about 11 or 8 to 11 and a cLogD ranging from about 9 to about 18 or 9 to 18. For example, in some embodiments, the ionizable cationic lipids have a c-pKa ranging from about 8.4 to about 8.7 or 8.4 to 8.7 and a cLogD ranging from about 13.6 to about 14.4 or 13.6 to 14.4. These ranges can result in pKa ranges of about 6 to about 7 or 6 to 7 measured in LNPs, which facilitates ionization in endosomes after delivery to cells.

[0384] In some embodiments, a slightly higher alkalinity may be desired and can be obtained from ionizable cationic lipids having c-pKa and cLogD within the ranges disclosed herein. In some embodiments, the cLogD of the ionizable cationic lipids of this disclosure is about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, or within the range defined by any pair of these values. Lipid design also considers the potential biodegradable pathways of the target lipids, such as via esterases in plasma, liver, and other tissues. Another consideration in lipid design is the fate of the ionizable lipid fragments resulting from degradation (such as after esterase cleavage). Preferably, the resulting fragments are rapidly cleared from the body without requiring hepatic oxidative metabolism.

[0385] The synthesis of lipids having structures of M5, CICL, CICL-IE, or M6 is described in U.S. Patent Application Nos. 63 / 632,931 (some M6), 63 / 632,937 (some M5), 63 / 632,940 (CICL-IE, some M5), and 63 / 632,944 (some M6) and U.S. Patent Application Publication No. 2023 / 0320995 (CICL), all of which teach the synthesis of such lipids, as well as the synthesis of specific subgenus and species, and is incorporated herein by reference in its entirety.

[0386] Other ionizable cationic lipids and LNP compositions containing them may be found in WO 2017 / 049245, WO2022 / 112855, WO2013 / 185116, WO2015074085, WO2016,081029, 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 The contents of each of these documents, which are not contradictory to this disclosure, concerning cationic ionizable lipids, LNPs incorporated therein, and nucleic acid delivery mediated by such LNPs, are found in U.S. Patents 2010 / 048536, 9,868,692, 10,435,616, 11,246,933, 11,382,979, 8,058,069, 8,492,359, 8,722,082, 8,822,668, 9,364,435, 9,408,914, 9,504,651, 10,526,284, 10,961,188, 11,141,378, and 11,241,493, and are hereby incorporated herein by reference. In some embodiments, tLNP comprises an antibody or its antigen-binding portion comprising the humanized antigen-binding domain of CT8 of this disclosure as its targeting portion, and further comprises portions from WO2017 / 049245, WO 2022 / 112855, WO 2005 / 007196, WO 2006 / 053430, WO 2007 / 086883, WO2009 / 129387, WO Cationic ionizable lipids of any of the following: 2010 / 048536, U.S. Patent Nos. 9,868,692, 10,435,616, 11,246,933, 11,382,979, 8,058,069, 8,492,359, 8,822,668, 9,364,435, 9,504,651, 11,141,378, and 11,241,493.

[0387] 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 is at least 40 mol% and / or no more than 62 mol% of ionizable cationic lipids. In some embodiments, the LNP of tLNP comprises about 54 mol%, about 58 mol%, or about 62 mol% of ionizable cationic lipids, or a range defined by 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 an even further embodiment, the LNP has at least 40 mol% or no more than 62 mol% of ionizable cationic lipids. In some embodiments, the LNP comprises 54 mol%, 58 mol%, or 62 mol% of ionizable cationic lipids, or a range defined by any pair of these values.

[0388] Phospholipids

[0389] As described above, in various embodiments, the LNP and tLNP comprise phospholipids. Phospholipids are amphiphilic molecules, as understood by those skilled in the art or of ordinary skill. Due to their amphiphilic nature, these molecules are known to form bilayers, and by including them in the LNP and tLNP, as described herein, they can provide film formation, stability, and rigidity. As used herein, phospholipids comprise a hydrophilic head group and two hydrophobic tail groups derived from fatty acids, the hydrophilic head group comprising a functionalized phosphate group. For example, in the various embodiments described herein, the phospholipid comprises 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 can be derived from unsaturated or saturated fatty acids. For example, the hydrophobic tail groups can be derived from C12-C20 fatty acids.

[0390] Regarding the LNP or tLNP of this disclosure, in various embodiments, the phospholipid comprises dimyristoylphosphatidylglycerol (DMPG), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearylphosphatidylcholine (DSPC), distearyl-glycerol-phosphate (18:0 PA, DSGP), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (18:1 PA, DOGP), or arachidonicylphosphatidylcholine (DAPC) or combinations thereof. In various embodiments, the phospholipid is dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), distearylphosphatidylcholine (DSPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylcholine (DPPC), or 1,2-diaarachidonicyl-sn-glycerol-3-phosphate choline (DAPC). In some embodiments, the phospholipid is distearate phosphatidylcholine (DSPC). Phospholipids can facilitate the formation of membranes surrounding a core of an LNP or tLNP, whether monolayer, bilayer, or multilayer. Additionally, phospholipids (such as DSPC, DMPC, DPPC, DAPC) impart structural stability and rigidity to the membrane. Phospholipids (such as DOPE) impart fusion. Other phospholipids (such as DMPG, which acquires a negative charge at physiological pH) promote charge regulation. Therefore, phospholipids constitute means for promoting membrane formation, for imparting membrane stability and rigidity, for imparting fusion, and for charge regulation. Some embodiments specifically include one or more of the above-described phospholipids, while other embodiments specifically exclude one or more of the above-described phospholipids.

[0391] In some embodiments, the LNP or tLNP has about 7 mol% to about 13 mol% phospholipids, about 7 mol% to about 10 mol% phospholipids, or about 10 mol% to about 13 mol% phospholipids. In some embodiments, the LNP has about 7 mol%, about 10 mol%, or about 13 mol% phospholipids. In some cases, the phospholipid is DSPC. In some cases, the phospholipid is DAPC.

[0392] Sterols

[0393] In some embodiments, the disclosed LNP and tLNP contain sterols. A sterol refers to a steroidal subgroup containing at least one hydroxyl (OH) group. More specifically, it is an adenosine derivative in which the H at position 3 is substituted with an OH group, or in other words, but equivalently, a steroid in which the H at position 3 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. Regarding the LNP or tLNP of this disclosure, in various embodiments, the sterol is cholesterol, 20-hydroxycholesterol, 20(S)-hydroxycholesterol, 22-hydroxycholesterol, or phytosterols, or combinations thereof. In further embodiments, the phytosterol includes campesterol, sitosterol, or stigmasterol, or combinations thereof. In some embodiments, the cholesterol is not of animal origin but is obtained through synthesis using phytosterols as a starting point. LNPs containing C-24 alkyl (such as 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. While β-sitosterol and stigmasterol perform well, vitamin D2, D3, and calcipotriol (analogs lacking complete cholesterol bodies) and betulin, lupeol, ursolic acid, and oleanolic acid (containing the 5th ring) should be avoided. Sterols fill the spaces between other lipids in LNPs or tLNPs and influence the shape of LNPs or tLNPs. Sterols also control the flowability of lipid compositions and reduce temperature dependence. Therefore, sterols (such as cholesterol, ergosterol, 20-hydroxycholesterol, 22-hydroxycholesterol, campesterol, fucosterol, β-sitosterol, and stigmasterol) constitute means for controlling LNP shape and flowability or for increasing transfection efficiency. Some embodiments specifically include one or more of the above-mentioned sterols, while other embodiments specifically exclude one or more of the above-mentioned sterols. In designing lipid compositions for LNP or tLNP, in some embodiments, the sterol content can be selected to compensate for different amounts of other types of lipids, such as ionizable cationic lipids or phospholipids.

[0394] In some embodiments, the 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 some embodiments, the LNP or tLNP has about 30.5 mol%, about 33.5 mol%, or about 37.5 mol% of sterols. In some embodiments, the 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, the LNP or tLNP has 30.5 mol%, 33.5 mol%, or 37.5 mol% of sterols. In some cases, the sterol is cholesterol. In some embodiments, the sterol is a mixture of sterols, such as cholesterol and β-sitosterol or cholesterol and 20-hydroxycholesterol. In some cases, the sterol component is about 25 mol% of 20-hydroxycholesterol and about 75 mol% of cholesterol. In some cases, the sterol component is approximately 25 mol% β-sitosterol and approximately 75 mol% cholesterol. In some cases, the sterol component is approximately 50 mol% β-sitosterol and approximately 50 mol% cholesterol. In some cases, the sterol component is 25 mol% 20-hydroxycholesterol and 75 mol% cholesterol. In a further case, the sterol component is 25 mol% β-sitosterol and 75 mol% cholesterol. In an even further case, the sterol component is 50 mol% β-sitosterol and 50 mol% cholesterol.

[0395] assist lipids

[0396] Regarding LNP or tLNP, in some embodiments, the auxiliary lipid is absent or contains 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 lipid containing a quaternary ammonium head group. In some cases, lipids containing a quaternary ammonium head group include 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), or 3β-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol), or combinations thereof. Further cases, in addition to chloride salts of lipids containing a quaternary ammonium head group, include bromide salts, methanesulfonates, and toluenesulfonates.

[0397] PEG-lipids

[0398] Regarding the LNP or tLNP of this disclosure, the PEG-lipid is a lipid conjugated with polyethylene glycol (PEG). In some embodiments described herein, the PEG-lipid is a C14-C20 lipid conjugated with PEG. For example, in various embodiments described herein, the PEG-lipid is a C14-C20 lipid conjugated with PEG, or a C14-C18 lipid conjugated with PEG, or a C14-C16 lipid conjugated with PEG. In some embodiments described herein, the PEG-lipid is a fatty acid conjugated with PEG. The fatty acid of the PEG-lipid can have various chain lengths. For each, in some embodiments, the PEG-lipid is a fatty acid conjugated with PEG, wherein the fatty acid chain length is in the range of C14-C20 (e.g., in the range of C14-C18 or C14-C16). If the fatty acid chain length of the PEG-lipid is less than C14, it will be lost too quickly from the tLNP or LNP; while if the fatty acid chain length is greater than C20, it will easily cause difficulties in the formulation process.

[0399] PEG can be produced in various sizes. In some embodiments, the PEG of the disclosed LNP and tLNP is PEG-1000 to PEG-5000. It should be understood that these sizes of polyethylene formulations are polydisperse, and the nominal size indicates the approximate average molecular weight of the distribution. (Assuming (OCH2CH2)) nIf the molecular weight of a single repeating unit is 44, then a PEG molecule with n=22 will have a molecular weight of 986, a PEG molecule with n=45 will have a molecular weight of 1998, and a PEG molecule with n=113 will have a molecular weight of 4990. n≈22 to 113 is used to represent PEG-lipids containing the PEG portion 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, although some molecules from formulations at the average molecular weight boundary will have n values ​​outside that range. For a single formulation, n≈22 is used to indicate a PEG-lipid containing a PEG portion from PEG-1000, n≈45 is used to indicate a PEG-lipid containing a PEG portion from PEG-2000, n≈67 is used to indicate a PEG-lipid containing a PEG portion from PEG-3000, n≈90 is used to indicate a PEG-lipid containing a PEG portion from PEG-4000, and n≈113 is used to indicate a PEG-lipid containing a PEG portion from PEG-5000. Some embodiments include a PEG portion within a range defined by any pair of the foregoing n or average molecular weight values. In some embodiments of the PEG-lipid, the PEG has a molecular weight (MW) of 500 Da to 5000 Da or 1000 Da to 5000 Da. For example, in some embodiments, the PEG of the PEG-lipid has a molecular weight in the range of 1500 Da to 5000 Da or 2000 Da to 5000 Da. In some embodiments as described herein, the PEG-lipid has a molecular weight in the range of 500 Da to 4000 Da, or 500 Da to 3000 Da, or 1000 Da to 4000 Da, or 1000 Da to 3000 Da, or 1000 Da to 2500 Da, or 1500 Da to 4000 Da, or 1500 Da to 3000 Da, or 1500 Da 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 a molecular weight of 2000 Da (sometimes abbreviated as PEG(2k)). Some embodiments include the PEG moiety of PEG-1000, PEG-2000, or PEG-5000. In some cases, the PEG portion is PEG-2000. Some embodiments contain DSG-PEG, such as DSG-PEG-2000. Some embodiments contain DSPE-PEG, such as DSPE-PEG-2000.Some implementations include DSG-PEG-2000 and / or DSPE-PEG2000.

[0400] Common PEG-lipids are divided into two categories: diacylglycerols and diacylphospholipids. Examples of diacylglycerol PEG-lipids include DMG-PEG (1,2-dimyristoyl-glycerol-3-methoxy polyethylene glycol), DPG-PEG (1,2-dipalmitoyl-glycerol-3-methoxy polyethylene glycol), DSG-PEG (1,2-distearate-glycerol-3-methoxy polyethylene glycol), and DOG-PEG (1,2-dioleoyl-glycerol-3-methoxy polyethylene glycol). Examples of diacylphospholipids include DMPE-PEG (1,2-dimyristoyl-glycerol-3-phosphate ethanolamine-3-methoxy polyethylene glycol), DPPE-PEG (1,2-dipalmitoyl-glycerol-3-phosphate ethanolamine-3-methoxy polyethylene glycol), DSPE-PEG (1,2-distearate-glycerol-3-phosphate ethanolamine-3-methoxy polyethylene glycol), and DOPE-PEG (1,2-dioleoyl-glycerol-3-phosphate ethanolamine-3-methoxy polyethylene glycol).

[0401] In some embodiments, the MW2000 PEG-lipid (e.g., a PEG-lipid containing PEG with a molecular weight of 2000 Da) comprises DMG-PEG2000 (1,2-dimyristoyl-glycerol-3-methoxy polyethylene glycol-2000), DPG-PEG2000 (1,2-dipalmitoyl-glycerol-3-methoxy polyethylene glycol-2000), DSG-PEG2000 (1,2-distearyl-glycerol-3-methoxy polyethylene glycol-2000), DOG-PEG2000 (1,2-dioleoyl-glycerol-3-methoxy polyethylene glycol-2000), DM... PE-PEG200 (1,2-dimyristoyl-glycerol-3-phosphate ethanolamine-3-methoxy polyethylene glycol-2000), DPPE-PEG2000 (1,2-dispalmitoyl-glycerol-3-phosphate ethanolamine-3-methoxy polyethylene glycol-2000), DSPE-PEG2000 (1,2-distearate-glycerol-3-phosphate ethanolamine-3-methoxy polyethylene glycol-2000), DOPE-PEG2000 (1,2-dioleoyl-glycerol-3-phosphate ethanolamine-3-methoxy polyethylene glycol-2000), or combinations thereof. In some embodiments, the PEG unit has a MW of 2000 Da. In some embodiments, MW 2000 PEG-lipids include DMRG-PEG2000 (1,2-dimyristoyl-racemic-glycerol-3-methoxy polyethylene glycol-2000), DPrG-PEG2000 (1,2-dipalmitoyl-racemic-glycerol-3-methoxy polyethylene glycol-2000), DSRG-PEG2000 (1,2-distearate-racemic-glycerol-3-methoxy polyethylene glycol-2000), DorG-PEG2000 (1,2-dioleoyl-glycerol-3-methoxy polyethylene glycol-racemic-2000), and DMPER-PEG200 (1,2-dioleoyl-glycerol-3-methoxy polyethylene glycol-racemic-2000). PEG-2000 (1,2-dipalmitoyl-racemic-glycerol-3-phosphate ethanolamine-3-methoxy polyethylene glycol-2000), DPPEr-PEG2000 (1,2-distearatel-racemic-glycerol-3-phosphate ethanolamine-3-methoxy polyethylene glycol-2000), DSPEr-PEG2000 (1,2-distearatel-racemic-glycerol-3-phosphate ethanolamine-3-methoxy polyethylene glycol-2000), DOPEr-PEG2000 (1,2-dioleoyl-racemic-glycerol-3-phosphate ethanolamine-3-methoxy polyethylene glycol-2000), or combinations thereof. The glycerol in these lipids is chiral. Therefore, in some embodiments, the PEG-lipids are racemic. Alternatively, optically pure enantiomers of the glycerol moiety can be used, i.e., the glycerol moiety is purely chiral. As used herein with respect to the glycerol moiety, optically pure means ≥95% of a single enantiomer (D or L). In some implementations, the enantiomeric excess is ≥98%.In some embodiments, the enantiomeric excess is ≥99%. Other PEG-lipids (including achiral PEG-lipids constructed on symmetrical dihydroxyacetone scaffolds, symmetrical 2-(hydroxymethyl)butane-1,4-diol, or symmetrical glycerol scaffolds) are disclosed in U.S. Provisional Application No. 63 / 362,502, filed April 5, 2022, and PCT / US2023 / 017648 (WO 2023 / 196445), filed April 5, 2023, both entitled “PEG-Lipids and LipidNanoparticles,” the full text of which is incorporated herein by reference.

[0402] The above-described PEG-lipid examples are presented as methoxy polyethylene glycol, but the terminator is not necessarily methoxy. For any unfunctionalized PEG-lipid, in alternative embodiments, the PEG moiety of the PEG-lipid can be capped with methoxy, benzyloxy, 4-methoxybenzyloxy, or hydroxy (i.e., alcohol). The terminal hydroxyl group promotes functionalization. Methoxy, benzyloxy, and 4-methoxybenzyloxy groups are advantageously provided to the PEG-lipid that will be used as a component of the LNP without functionalization. However, all four of these alternatives can be used as (unfunctionalized) PEG-lipid components of the LNP. The 4-methoxybenzyloxy group, which is often used as a protecting group during the synthesis of PEG-lipids, is readily removed to generate the corresponding hydroxyl group. Therefore, the 4-methoxybenzyloxy group provides a convenient route to synthesize alcohols when they are not synthesized directly. Before incorporating the PEG-lipid into the LNP, the alcohol can be used for functionalization so that the binding moiety (e.g., the engineered F(ab') binding moiety or the humanized anti-CD8α binding moiety disclosed herein) can be conjugated thereto as the targeting moiety of the LNP (making it a tLNP). As used in this article, the terminus of the PEG moiety and similar structures refer to the terminus of the PEG moiety that is not connected to lipids.

[0403] The PEG moiety provides a hydrophilic surface on LNPs, inhibiting LNP aggregation or coalescence, thereby enhancing their stability and reducing polydispersity, i.e., reducing the heterogeneity of LNP dispersions. Additionally, the PEG moiety can hinder LNP binding, including binding to plasma proteins. These plasma proteins include apoE, which is understood to mediate hepatic uptake of LNPs, such that inhibition of this binding can lead to an increased proportion of LNPs reaching other tissues. These plasma proteins also include opsonins, such that inhibition of binding reduces recognition by the reticuloendothelial system. The PEG moiety can also be functionalized to act as an attachment site for the targeting moiety. Conjugating the binding moiety (e.g., engineered F(ab') binding moiety or humanized anti-CD8α binding moiety as disclosed herein) to the PEG moiety allows tLNPs to bypass the liver and bind to target tissues or cell types, thereby significantly increasing the proportion of LNPs reaching the target tissue or cell type. Therefore, PEG-lipids can serve as a means of inhibiting LNP binding, and PEG-lipids conjugated to anti-CD8 can serve as a means of targeting CD8+ cells with LNPs.

[0404] As used herein, the term "functionalized PEG-lipid" and similar constructs generally refer to both unreacted and reacted entities. Even after conjugation (formation of tLNP) has occurred, the lipid composition of the LNP can be described by reference to the reactive substance. For example, a lipid composition may be described as containing DSPE-PEG-maleimide and, moreover, an anti-CD8 binding moiety or an engineered F(ab') binding moiety, without explicitly stating that the maleimide has been converted to succinimide (or hydrolyzed succinimide) during the reaction to form the conjugate. Similarly, if the reactive group is bromomaleimide, it will be maleimide after conjugation. These differences in the chemical nomenclature of unreacted and reacted substances should be understood implicitly, even if not explicitly stated. Some embodiments contain DSG-PEG, such as DSG-PEG-2000. Some embodiments contain functionalized DSPE-PEG, such as functionalized DSPE-PEG-2000. Some implementations include DSG-PEG-2000 and functionalized DSPE-PEG-2000. In some cases, the functionalized PEG-lipid is partially functionalized with maleimide, such as DSPE-PEG-2000-MAL.

[0405] In some aspects, the LNP comprises one or more PEG-lipids and / or functionalized PEG-lipids; when both functionalized and unfunctionalized PEG-lipids are present, they may be the same or different; and one or more ionizable cationic lipids; the LNP may also comprise phospholipids, sterols, accessory lipids, or any combination thereof. The term "functionalized PEG-lipid" refers to a PEG-lipid in which the PEG portion has been derivatized with a chemically reactive group that can be used to conjugate a targeting moiety to the PEG-lipid. The functionalized PEG-lipid may react with an anti-CD8 binding moiety or an engineered F(ab') binding moiety, such that the anti-CD8 binding moiety or the engineered F(ab') binding moiety is conjugated to the PEG portion of the lipid. Thus, the conjugated anti-CD8 binding moiety can act as the targeting moiety of the LNP for the CD8+ cells constituting the tLNP. In some embodiments, after the formation of the LNP comprising the functionalized PEG-lipid, the anti-CD8 binding moiety or the engineered F(ab') binding moiety is conjugated to the functionalized PEG-lipid. In other embodiments, the anti-CD8 binding moiety or engineered F(ab') binding moiety is conjugated with a PEG-lipid, and the conjugate is then inserted into a previously formed LNP.

[0406] In some embodiments, the LNP is a tLNP comprising one or more functionalized PEG-lipids conjugated to an anti-CD8 binding moiety or an engineered F(ab') binding moiety. In some embodiments, the tLNP also comprises unfunctionalized or unconjugated PEG-lipids. In some embodiments, the functionalization is a maleimide. In some embodiments, the functionalization is a brominated maleimide or brominated maleimide amide, alkynyl amide, or alkynyl imide moiety 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 moiety, such as engineered F(ab'), as disclosed herein. In some embodiments, the binding moiety is an N- or C-terminal extended polypeptide comprising a binding domain and an accessible thiol group. In some embodiments, the conjugation bond comprises a reaction product of the thiol in the anti-CD8 binding moiety or engineered F(ab') binding moiety with the functionalized PEG-lipid. In some embodiments, the functionalization is a maleimide, an azide, an alkyne, a dibenzocyclooctylene (DBCO), a bromomaleimide or a bromomaleimide amide, an alkynyl amide or an alkynylimide. In some embodiments, the anti-CD8 binding moiety comprises an anti-CD8 antibody or an anti-CD8 binding moiety thereof. In some embodiments, the anti-CD8 binding moiety is a polypeptide comprising a binding domain and an N- or C-terminal extension comprising an accessible thiol group, such as engineered F(ab') as disclosed herein.

[0407] In some embodiments, the PEG-lipid and / or functionalized PEG-lipid comprises a scaffold selected from formula S1, S2, S3, or S4:

[0408]

[0409] in Indicates the ester junction with the fatty acid, and This indicates the ester (S1) or ether (S2, S3, and S4) formation site with the PEG moiety. In some embodiments, the fatty acid ester is C 14 -C 20 Linear alkyl fatty acids. In some embodiments, the PEG moiety is functionalized, and the fatty acid ester is C... 16 -C 20 Straight-chain alkyl fatty acids. For example, straight-chain alkyl fatty acids are C64-C ... 14 C 15 C 16 C 17 C 18 C 19 Or C 20 In some implementations, the fatty acid ester is C 14 -C 20 Symmetrically branched alkyl fatty acids. For example, branched alkyl fatty acids are C16-26-3 ... 14 C 15 C 16 C 17 C 18 C 19 Or C 20 Symmetry refers to the fact that each alkyl branch has the same number of carbons. In some embodiments, the branches are located at positions 3, 4, 5, 6, or 7 of the fatty acid ester. The synthesis and uses of PEG-lipids constructed on scaffolds S1-S4 are disclosed in WO2023 / 196445A1, all of which teaches about PEG-lipids and their uses and is incorporated herein by reference.

[0410] Some embodiments of the disclosed ionizable cationic lipids have head groups with small (<250 Da) PEG moieties. These lipids are not the subject of the term PEG-lipid as used herein. Although these small PEG moieties may affect the lipophilicity of the ionizable cationic lipids, they are generally too small to impede binding to a similar degree as the larger PEG moieties in the PEG-lipids disclosed above. Furthermore, it is understood that PEG-lipids are primarily located in the outer surface thin layer, while the majority of the ionizable cationic lipids reside within the LNP.

[0411] In some embodiments, the LNP or tLNP or the functionalized PEG-lipid of this disclosure comprises one or more fatty acid tails, each fatty acid tail being no shorter than C16 and no longer than C20 for straight-chain fatty acids. For branched-chain fatty acids, tails no shorter than C14 and no longer than C20 are 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 dipalmitoyllipid. In some embodiments, the functionalized PEG-lipid comprises distearate. The fatty acid tail acts as a means of anchoring the PEG-lipid to the tLNP to reduce or eliminate PEG-lipid detachment from the tLNP. This is a useful property regardless of whether the PEG-lipid is functionalized, but it is more significant for functionalized PEG-lipids because a targeting moiety will be attached to it, and if the PEG-lipid (with a conjugated anti-CD8α binding moiety, such as an antibody) detaches from the tLNP, the targeting function may be impaired.

[0412] In some embodiments, the LNP or tLNP comprises about 0.5 mol% to about 3 mol% or 0.5 mol% to 3 mol% of PEG-lipids, which include functionalized and non-functionalized PEG-lipids. In some embodiments, the LNP or tLNP comprises DSG-PEG. In other embodiments, the LNP or tLNP comprises DMG-PEG or DPG-PEG. In some embodiments, the LNP or tLNP comprises DSPE-PEG. In some embodiments, the functionalized and non-functionalized PEG-lipids are not the same PEG-lipid; for example, the non-functionalized PEG-lipid may be diacylglycerol, while the functionalized PEG-lipid may be diacylphospholipid. tLNPs with such a mixture show reduced expression in the liver, possibly due to reduced uptake. In some embodiments, the functionalized PEG-lipid is DSPE-PEG, and the non-functionalized PEG-lipid is DSG-PEG. In some embodiments, the 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-lipids. In some embodiments, the LNP or tLNP comprises about 1.4 mol% or 1.4 mol% of nonfunctionalized PEG lipids. In some embodiments, the LNP or tLNP comprises about 0.1 mol% to about 0.3 mol% or 0.1 mol% to 0.3 mol% of functionalized lipids. In some cases, the functionalized lipid is DSPE-PEG. In some cases, the LNP or tLNP comprises about 0.1 mol%, about 0.2 mol%, or about 0.3 mol% of DSPE-PEG. In some cases, the LNP or tLNP comprises 0.1 mol%, 0.2 mol%, or 0.3 mol% of DSPE-PEG. In some cases, the functionalized PEG-lipid is conjugated with an anti-CD8α binding moiety or engineered F(ab'), as disclosed herein. As used herein, unless the context otherwise specifies, the phrase “conjugated with” and similar constructs are intended to convey a state of being, i.e., a structure, rather than a process.

[0413] Adhesion

[0414] Any suitable chemical approach can be used to conjugate the anti-CD8α binding moiety to PEG-lipids, including maleimide chemistry (see Parhiz et al., Journal of Controlled Release 291:106-115, 2018) and click chemistry (see Kolb et al., Angewandte Chemie International Edition 40(11):2004–2021, 2001; and Evans, Australian Journal of Chemistry 60(6):384–395, 2007). Reagents used in such reactions include lipid-PEG-maleimide, lipid-PEG-cysteine, lipid-PEG-alkyne, lipid-PEG-dibenzocyclooctylene (DBCO), and lipid-PEG-azide. Further conjugation reactions utilize lipid-PEG-bromomaleimide, lipid-PEG-alkanoamide, lipid-PEG-alkynylimide, and lipid-PEG-alkynylene reactions, as disclosed in PCT / US23 / 17648 entitled PEG-Lipids and Lipid Nanoparticles, all of which teaches conjugation chemistry and alternative PEG-lipids and is incorporated herein by reference. On the anti-CD8α binding side of the reaction, existing cysteine ​​thiol groups can be used, or the protein can be derived by adding a sulfur-containing carboxylic acid, for example, to the ε-amino group of lysine to react with maleimide, bromomaleimide (collectively, “maleimide”), alkanoamide, or alkynylimide. Alternatively, an alkyne can be added to the thiol group or ε-amino group of lysine to participate in click chemistry reactions.

[0415] To modify the ε-amino group of the lysine residue in the anti-CD8α binding moiety to react with maleimide-functionalized PEG-lipids, the anti-CD8α binding moiety (e.g., antibody) can be reacted with N-succinimide-S-acetylthioacetate (SATA). The SATA is then deprotected, for example, using 0.5 M hydroxylamine, followed by removal of unreacted components via a G-25 Sephadex Quick Spin Protein column (Roche Applied Science, Indianapolis, IN). The reactive thiol group on the anti-CD8α binding moiety is then conjugated to the maleimide moiety on the LNP of this 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 cryopreserved at -80°C until needed. Others conjugate antibodies with free functionalized PEG-lipids and then incorporate the conjugated lipids into a pre-formed LNP. However, it was found that incorporating functionalized PEG-lipids into LNPs during LNP formation and subsequently conjugating the anti-CD8α binding moiety with functionalized PEG-lipids in LNPs was more controllable and produced more consistent results.

[0416] Several site-specific conjugation methods also exist. Particularly applicable, but not exclusively, to truncated forms of antibodies, these typically involve C-terminal extensions of natural or artificial sequences containing particularly accessible cysteine ​​residues. For example, partial reduction of cystine bonds in antibodies with tris(2-carboxy)phosphine (TCEP) can also generate thiol groups for conjugation, which can be site-specific when using a suitable antibody fragment under controlled conditions. Potential cysteine ​​residues that can be reduced with TCEP to conjugate with LNPs (particularly in F(ab')) are shown in Table 17. Cysteine, glutathione (GSH), mercaptoethylamine (MEA), and dithiobutylamine (DTBA) can also be used instead of TCEP for reduction. The use of the latter two is described in (Crivianu-Gaita et al., Biochem Biphys Rep. 2: 23-28, (2015)). β-mercaptoethanol and dithiothreitol (DTT) can also be used under well-controlled conditions. The various engineered (Fab') constructs disclosed herein are capable of forming F(ab')2 at least to some extent. However, they are referred to as F(ab') throughout the text, which is consistent with their use as non-dimerizing molecules to be conjugated with LNPs and to act as the targeting part of the tLNP thus formed.

[0417] Alternatively, the C-terminal extension may contain the sorting enzyme A substrate sequence LXTG (SEQ ID NO: 197) (where X is any amino acid), which can then be functionalized and conjugated to PEG-lipids in a reaction catalyzed by sorting enzyme A, including via click chemistry (see, for example, Moliner-Morro et al., 2020, Biomolecules 10(12):1661, all of which teaches about antibody conjugation mediated by sorting enzyme A reactions and / or click chemistry is incorporated herein by reference). The use of click chemistry for conjugating targeting moieties (such as antibodies of various forms) is disclosed, for example, in WO2024 / 102,770, all of which teaches about the conjugation of targeting moieties to LNPs without contradiction to this disclosure is incorporated herein by reference in its entirety.

[0418] For intact antibodies and other forms containing the Fc region, site-specific conjugation to either (or both) of the two specific lysine residues (Lys248 and Lys288) can be achieved using AJICAP. ® One of the reagents is used to achieve this without any alteration or extension of the natural antibody sequence (see, for example, Matsuda et al., 2021, Molecular Pharmaceutics 18:4058-4066; Fujii et al., 2023, Bioconjugate Chemistry 34(4):728-738 [https: / / doi.org / 10.1021 / acs.bioconjchem.3c00040], and WO2019 / 240287, all of which teach about the conjugation of antibodies with AJICAP reagents and are incorporated herein by reference). AJICAP reagents are modified affinity peptides that bind to a specific locus on the Fc and react with adjacent lysine residues 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 be conjugated by, for example, a maleimide or haloamide reaction. Functionalization with azides or dibenzocyclooctyne (DBCO) for click chemistry conjugation is also possible. This technique and similar techniques are further described in US20200190165 (corresponding to WO2018199337), US20210139549 (corresponding to WO2019 / 240287), and US20230248842 (corresponding to WO2020184944), all of which teach about such modified affinity peptides and their uses and are incorporated herein by reference in their entirety.

[0419] The term "affinity peptide" refers to a peptide that has the ability to specifically bind to other molecules and has a high affinity for them. In some embodiments, the affinity peptide binds to a specific locus on the Fc region of an antibody. In some embodiments, the affinity peptide is modified with chemically reactive groups that allow it to form covalent bonds with adjacent amino acid residues in the antibody, such as specific lysine residues, such as Lys248 or Lys288 in IgG1.

[0420] Therefore, in some embodiments, the anti-CD8α binding moiety is conjugated to the PEG portion of the PEG-lipid via a thiol-modified lysine residue. In some embodiments, the conjugation is via a cysteine ​​residue in the natural or added antibody sequence. In such embodiments, regardless of whether a thiolized lysine or a thiol of cysteine ​​is used, the thiol in the antibody can be conjugated to the maleimide group of the maleimide-modified PEG-lipid in the LNP using a maleimide-thiol reaction. In other embodiments, the conjugation is via a sorting enzyme A substrate sequence. In still other embodiments, the conjugation is via a specific lysine residue (Lys248 or Lys288) in the Fc region. In some embodiments of such embodiments, the humanized anti-CD8 antibody is linked to the LNP using an N-succinimide-S-acetylthioacetate (SATA)-maleimide conjugation chemistry to form a targeted LNP (tLNP). Advantageously, the antibody is first modified with SATA to introduce a thiol group at an accessible lysine residue, thereby allowing conjugation with maleimide. (Some lysine residues may be buried inside the protein, making them inaccessible to the SATA reagent). The biantibody and F(ab')2 can be conjugated by first partially reducing the cysteine ​​bonds in the antibody with tris(2-carboxy)phosphine (TCEP) to generate a thiol group for partial conjugation with maleimide via LNP.

[0421] Nucleic acid molecules

[0422] In some embodiments, the disclosed LNP and tLNP include a payload that comprises or is composed of one or more nucleic acid molecules. In some embodiments, the LNP or tLNP payload contains only one nucleic acid substance, while in other embodiments, the LNP or tLNP payload contains multiple nucleic acid substances, such as two, three, or four nucleic acid substances. For example, in embodiments where the payload comprises a nucleic acid encoding a CAR or immune cell connective (ICE), the payload may comprise or consist of the following: 1) a single nucleic acid type encoding a single type of CAR or ICE; 2) a single nucleic acid type encoding two or more types of CAR or ICE (or a mixture of CAR and ICE), such as a tracking antigen; 3) a single nucleic acid type encoding two or more types of CAR or ICE (or a mixture of CAR and ICE), such as bicistronic or polycistronic mRNA, wherein at least one CAR and / or ICE is specific to a tracking antigen different from the other tracking antigens; 4) two or more nucleic acid types encoding two or more types of CAR or ICE (or a mixture of CAR and ICE), wherein each CAR and / or ICE is specific to the same tracking antigen; or 5) two or more nucleic acid types encoding two or more types of CAR or ICE (or a mixture of CAR and ICE), wherein at least one CAR and / or ICE is specific to a target antigen different from the other target antigens. When two or more CARs and / or ICEs are specific to the same target antigen, they may be specific to the same or different epitopes of the same tracking antigen. Further variations will be apparent to those skilled in the art (e.g., multiple bicistronic or polycistronic nucleic acids, nucleic acids encoding TCRs, etc.). The nucleic acid can be RNA or DNA. The nucleic acid can be polycistronic, such as bicistronic.

[0423] In some embodiments, the nucleic acid molecule is mRNA, self-replicating RNA, circular RNA, siRNA, miRNA, DNA, gene editing components (e.g., guide RNA, tracr RNA, sgRNA), gene writing components, mRNA encoding gene or base editing proteins, zinc finger nucleases, TALENs, CRISPR nucleases (such as Cas9), DNA molecules to be inserted or used as repair templates, or combinations 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 contains or encodes an opsonizing agent. In some embodiments, the mRNA (linear, circular, or self-replicating) contains a miRNA binding site. In some embodiments, the mRNA encodes a chimeric antigen receptor (CAR). In other embodiments, the mRNA encodes a gene editing, base editing, or gene writing protein. In some embodiments, the nucleic acid is a guide RNA. In some embodiments, an LNP or tLNP comprises mRNA encoding a gene editing, base editing, or gene writing protein and one or more guide RNAs. CRISPR nucleases can have altered activities; for example, they can be modified to act as nicking enzymes instead of producing double-stranded cuts, or to bind to sequences specified by guide RNA but without enzymatic activity. Base-editing proteins are typically fusion proteins containing a deaminase domain and a sequence-specific DNA-binding domain (such as inactive CRISPR nucleases).

[0424] In some implementations, the reprogramming agent comprises an immune receptor (e.g., a chimeric antigen receptor or a T-cell receptor) or an immune cell connector (e.g., a bispecific T-cell connector (BiTE), a bispecific killer cell connector (BiKE), a trispecific killer cell connector (TriKE), a dual affinity retargeting antibody (DART), a TRIDENT (connecting two DART units or a DART unit and a Fab domain), a macrophage connector (e.g., BiME), an innate cell connector, etc.).

[0425] In some embodiments, the nucleic acid is RNA, such as mRNA, and the RNA contains at least one modified nucleoside. In some embodiments, the modified nucleoside is pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methyluridine, N6-methyladenosine, 2'-O-methyluridine, or 2-thiouridine. In some embodiments, all uridines are substituted with modified nucleosides. Further disclosure of modified nucleosides and their uses can be found in U.S. Patent No. 8,278,036, the teachings of which are incorporated herein by reference.

[0426] In some embodiments, the reprogramming agent encodes a gene / genome editing component or a gene / genome editing component. In some embodiments, the gene / genome editing component is the guide RNA of an RNA-guided nuclease or other nuclease 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-guided nuclease, a gene or base editing protein, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a large-scale nuclease, a transposase, or a CRISPR nuclease (e.g., Cas9 or Cas12). In some embodiments, the gene / genome editing component is the DNA to be inserted or DNA that serves as a template in gene or genome editing (e.g., a template for repairing double-strand breaks).

[0427] In some embodiments comprising multiple reagents, the nucleic acid may be polycistronic. In other embodiments comprising multiple reagents or components, each reagent or component is encoded or contained as a separate nucleic acid substance. In some embodiments involving multiple payload nucleic acid substances, two or more nucleic acid substances are encapsulated together in a single LNP substance. In other embodiments, a subset of the payload nucleic acid substances to be delivered (e.g., a single nucleic acid substance) is encapsulated in one LNP or tLNP substance, while another subset of the nucleic acid substances is encapsulated in another LNP or tLNP substance. Different (t)LNP substances may differ only in the payload they contain. Different (t)LNP substances may be combined in a single formulation or pharmaceutical composition for administration.

[0428] In any of the above embodiments, the nucleic acid molecular payload of the LNP or tLNP of this disclosure encodes a CAR, TCR, or ICE that is specific to a particular antigen, such as 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 receptor ‡ CEACAM5* †‡ CLL1 ‡ CSPG4* ‡ Kappa*, Lambda*, FCRL5 †‡ GPRC5D †‡ CTSK, PD-1 (CD279) †‡ CD319 (SLAMF7)* †‡ CD248 (TEM1) ‡ , ULBP1, ULBP2, CD319 (SLAMF7)* †‡ GPRC5D †‡ Tight junction protein 6 (CLDN6), tight junction protein 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 †‡ .

[0429] Tolerance

[0430] Conventional LNPs are primarily delivered to the liver. Hepatotoxicity is a major dose-limiting parameter observed with drugs containing LNPs. For example, ONPATTRO contains the ionizable lipid MC3. ® The NOAEL (no adverse reaction dose) observed after multiple administrations in rats was only 0.3 mg / kg. (SARS-CoV-2 vaccine COMIRNATY) ® The baseline LNPs used, comprising the ionizable cationic lipid ALC-0315, induced elevated levels of liver enzymes and acute-phase proteins in rats at a single dose of ≥1 mg / kg. This elevation was partially reversed only by antibody attachment to the baseline LNPs, and the reversal was greater if the antibody directed the LNPs to some other tissue (i.e., tLNPs). However, the use of the highly biodegradable ionizable cationic lipid CICL-1 (whose catabolism should be similar to those disclosed herein) resulted in a greater reduction in liver delivery and associated liver enzyme and acute-phase protein levels for LNPs, antibody-conjugated LNPs, and tLNPs.

[0431] Methods for preparing LNP or tLNP

[0432] In some aspects, this disclosure provides a method for preparing LNPs or tLNPs, the method comprising mixing an aqueous solution of a nucleic acid (or other negatively charged payload) and an alcoholic solution of a lipid in proportions disclosed herein. In certain embodiments, the mixing is rapid.

[0433] The aqueous solution can be buffered to a pH of about 3 to about 5, for example, but not limited to, citrate or acetate buffer. In various embodiments, the alcohol can be ethanol, isopropanol, tert-butanol, or a combination thereof. In some embodiments, rapid mixing is achieved by pumping the two solutions through a T-joint or using an impingement jet mixer. Microfluidic mixing via a staggered herringbone mixer (SHM) or hydrodynamic mixer (microfluidic hydrodynamic focusing), microfluidic bifurcation mixer, and microfluidic baffle mixer can also be used. After LNP formation, it is diluted with a buffer (e.g., phosphate, HEPES, or Tris) with a pH range of 6 to 8.5 to reduce the alcohol (ethanol) concentration. The diluted LNP is purified by dialysis or ultrafiltration or percolation using tangential flow filtration (TFF) against a buffer (e.g., phosphate, HEPES, or Tris) with a pH range of 6 to 8.5 to remove the alcohol. Alternatively, size exclusion chromatography can be used. Once the alcohol has been completely removed, the buffer solution is replaced with a similar buffer containing a cryoprotectant (e.g., glycerol or a sugar such as sucrose, trehalose, or mannose). The LNP is concentrated to the desired concentration, then filtered through a 0.2 μm filter, such as a polyethersulfone (PES) or modified PES filter, and filled into glass vials, stoppered, capped, and frozen for storage. In an alternative embodiment, a lyophilization protectant is used, and the LNP is lyophilized for storage rather than as a cryo-liquid. Other methods for preparing LNPs can be found, for example, in U.S. Patent Application Publications Nos. US2020 / 0297634, US2013 / 0115274, and International Patent Application Publication No. WO2017 / 048770, all of which teach the production of LNPs and are incorporated herein by reference.

[0434] One aspect is a method for preparing tLNPs, which includes rapidly mixing an aqueous solution of a nucleic acid (or other negatively charged payload) and an alcoholic solution of a lipid as disclosed for LNPs. In some embodiments, the lipid mixture includes a functionalized PEG-lipid for subsequent conjugation to a target moiety. As used herein, a functionalized PEG-lipid refers to a PEG-lipid whose PEG moiety has been derivatized with a chemically reactive group (such as maleimide, N-hydroxysuccinimide (NHS) ester, Cys, azides, alkynes, etc.) that can be used to conjugate the target moiety to the PEG-lipid, and thus to an LNP containing the PEG-lipid. In other embodiments, the functionalized PEG-lipid is inserted into the LNP after the initial formation of the LNP from other components. In either type of embodiment, the target moiety is conjugated to the functionalized PEG-lipid after the formation of the functionalized PEG-lipid containing the LNP. Conjugation schemes can be found, for example, Parhiz et al., 2018, J. Controlled Release 291:106-115 and Tombacz et al., 2021, Molecular Therapy 29(11):3293-3304, all of which teach the following about the conjugation of PEG-lipids with the binding moiety and are incorporated herein by reference. Alternatively, the targeting moiety may be conjugated with the PEG-lipid prior to the insertion of the pre-formed LNP.

[0435] In some embodiments of the tLNP preparation method, the method includes:

[0436] i) The initial LNP is formed by mixing all components of the tLNP (excluding one or more functionalized PEG-lipids and one or more targeted fractions) in proportions disclosed herein;

[0437] ii) Preconjugated tLNPs are formed by mixing initial LNPs with one or more functionalized PEG-lipids; and

[0438] iii) A tLNP is formed by concatenating a pre-concatenated tLNP with one or more target portions.

[0439] In some embodiments of the tLNP preparation method, the method includes:

[0440] i) Preconjugated tLNPs are formed by mixing all components of tLNP (including one or more functionalized PEG-lipids, in addition to one or more targeting moieties) in proportions disclosed herein; and

[0441] ii) Forming a tLNP by concatenating a pre-concatenated tLNP with one or more target portions.

[0442] In some embodiments of the tLNP preparation method, the method includes:

[0443] i) Forming one or more conjugated functionalized PEG-lipids by conjugating one or more functionalized PEG-lipids with one or more targeted moieties; and

[0444] ii) tLNP is formed by mixing all components of tLNP (including one or more conjugated functionalized PEG-lipids) in proportions disclosed herein.

[0445] In some embodiments of the tLNP preparation method, the method includes:

[0446] i) Forming one or more conjugated functionalized PEG-lipids by conjugating one or more functionalized PEG-lipids with one or more targeting moieties;

[0447] ii) To form LNP by mixing all components of tLNP (excluding one or more conjugated functionalized PEG-lipids); and

[0448] iii) tLNP is formed by mixing the initial LNP with one or more conjugated functionalized PEG-lipids.

[0449] After conjugation, as disclosed above for LNP, tLNP is purified and stored by dialysis, tangential flow filtration or size exclusion chromatography.

[0450] Encapsulation efficiency of LNP or tLNP is typically determined by adding a nucleic acid-binding fluorescent dye to both intact and lysed aliquots of the final LNP or tLNP formulation to measure the amounts of unencapsulated nucleic acid and total nucleic acid, respectively. Encapsulation efficiency is usually expressed as a percentage and calculated as 100 × (TU) / T, where T is the total amount of nucleic acid and U is the amount of unencapsulated nucleic acid. In various embodiments, encapsulation efficiencies are ≥80%, ≥85%, ≥90%, or ≥95%.

[0451] Anti-CD8 tLNP

[0452] This disclosure covers any of the above-described embodiments of an anti-CD8α binding agent conjugated with the tLNP formulation disclosed herein. For example, in some aspects, the targeted lipid nanoparticles (tLNPs) comprise: (a) a lipid formulation comprising an ionizable cationic lipid (such as CICL or a variant thereof disclosed herein), phospholipids, sterols, functionalized PEG-lipids, and nonfunctionalized PEG-lipids (e.g., any of those listed in Table 14), and (b) a humanized anti-CD8α antibody or antigen-binding fragment thereof conjugated to the lipid, wherein the humanized anti-CD8α antibody or 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 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: 58, and VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 4; VH-CDR2 containing the amino acid sequence of SEQ ID NO: 59 and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 4; or (iv) VH-CDR1 containing the amino acid sequence of SEQ ID NO: 2, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 60 and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 4; and (b) VL-CDR1 containing the amino acid sequence of SEQ ID NO: 6, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 7; and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 8.

[0453] In another embodiment, the targeted lipid nanoparticle (tLNP) comprises: (a) a lipid formulation comprising an ionizable cationic lipid (such as CICL or a variant thereof disclosed herein), phospholipids, sterols, functionalized PEG-lipids, and nonfunctionalized PEG-lipids (e.g., any one listed in Table 14), and (b) an anti-CD8α antibody or antigen-binding fragment thereof conjugated to the lipid, wherein the anti-CD8α antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90% to 100% identical to the amino acid sequence of SEQ ID NO: 10, 11, 12, 13, 14, 27, 28, 29, 35, or 36, and 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: 3, 58, 59, or 60. The amino acid sequence of SEQ ID NO: 4; and a light chain variable region (VL) comprising at least 90% to 100% identical amino acid sequences to the amino acid sequences of SEQ ID NO: 16, 17, 18 or 39, 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.

[0454] As used herein, "LNP formulation" or "tLNP formulation" refers to the complete corresponding composition (e.g., all lipids comprising an LNP or all lipids comprising a tLNP together with a targeting portion, each optionally encompassing a payload, such as a nucleic acid molecule), and also includes a buffer, carrier, solvent, or other excipient. In some embodiments, a humanized anti-CD8α antibody of this disclosure or its antigen-binding fragment, or a targeting LNP (tLNP) of this disclosure, is conjugated to such an anti-CD8α antibody or antigen-binding fragment, which may be formulated into a composition or pharmaceutical composition together with a pharmaceutically acceptable carrier, excipient, or stabilizer.

[0455] In some 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 "pharmaceutically acceptable carrier" means one or more non-toxic materials that do not interfere with the effectiveness of the bioactivity of the active ingredient. Such formulations may conventionally 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 substances suitable for human administration. Other contemplated carriers, excipients, and / or additives that may be used 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 other drug carriers, excipients, and / or additives suitable for the formulations described herein are known in the art, for example, as listed in "Remington: The Science & Practice of Pharmacy," 23rd edition, Lippincott Williams & Wilkins, (2005) and "Physician's Desk Reference," 71st edition, Medical Economics, Montvale, NJ (2005). Pharmaceutically acceptable carriers suitable for the desired or required administration modality, solubility, and / or stability can be selected.

[0456] 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 may be local or systemic. The method of administration is determined by the physician and depends in part on the site of the medical condition. In most cases, administration results in the release of the humanized anti-CD8α antibody described herein or a polypeptide containing its antigen-binding domain into the bloodstream.

[0457] In other embodiments, CD8-targeting tLNPs conjugated to the humanized anti-CD8α antibody antigen-binding fragment of this disclosure are administered parenterally, such as via intravenous infusion. Other embodiments utilize other routes of administration, including subcutaneous, intraperitoneal, intranodal, and intratumoral. In most cases, administration results in the binding of tLNPs to CD8-positive cells (e.g., T cells) and the release of payloads (such as nucleic acid molecules like RNA) encapsulated in the tLNPs into the cells.

[0458] Methods for delivering payloads into cells using anti-CD8 tLNP

[0459] In some aspects, this document discloses methods for delivering nucleic acids (or other negatively charged payloads) into CD8+ expressing cells (CD8+ cells), methods comprising contacting CD8+ cells with a CD8-targeting tLNP of any of the foregoing aspects. Various embodiments of the methods for delivering the payload to CD8+ cells limit each other. Each of the various genera, subgenera, and / or species of LNPs or tLNPs disclosed herein (including those based on the inclusion or exclusion of specific lipids, specific lipid compositions, and / or specific payloads) can be used to define the scope of methods for delivering the payload to CD8+ cells. In some embodiments, contact occurs ex vivo. In some embodiments, contact occurs in vivo. In some cases, in vivo contact includes intravenous, intramuscular, subcutaneous, intranodal, or intralymphatic administration. In further embodiments, hepatocyte transfection is reduced compared to tLNPs containing conventional ionizable cationic lipids (such as ALC-0315) (Table 14). In some embodiments, LNPs or tLNPs are administered 1 to 3 times weekly for 1, 2, 3, or 4 weeks. In some implementations, as disclosed above, toxicity is limited to (or largely limited to) Grade 0, Grade 1, or Grade 2.

[0460] Compared to widely used existing LNP compositions, such as those containing ALC-0315, the LNP and tLNP compositions and formulations disclosed herein exhibit reduced toxicity. In various embodiments, toxicity can be described as observable toxicity, substantial toxicity, serious toxicity, or acceptable toxicity or dose-limiting toxicity (such as, but not limited to, maximum tolerated dose (MTD)). Observable toxicity refers to an effect that is negligible or minor, although a change is observed. Substantial toxicity refers to a negative impact on the patient's overall health or quality of life. In some cases, substantial toxicity can be mitigated or resolved through other ongoing medical interventions. Serious toxicity refers to an effect that requires acute medical intervention and / or dose reduction or treatment interruption. The acceptability of toxicity will be affected by the specific disease being treated and its severity, as well as the availability of mitigating medical interventions. In some embodiments, toxicity is limited to (or largely limited to) observable toxicity. In some embodiments, toxicity is limited to (or largely limited to) grade 0, 1, or 2.

[0461] 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. Nucleotide structures, and especially mRNA structures, particularly those encoding specific polypeptides, are well-suited for delivery via LNP or tLNP and are disclosed in U.S. Application 18 / 934,237 (Attorney General's File No. 23-1871-US), filed November 1, 2024. All teachings in each of these documents regarding nucleic acid payloads for in vivo transfection and their design are incorporated herein by reference.

[0462] In some embodiments, the payload comprises nucleic acid encoding an immune receptor or immune cell connector, and the delivery method is also a method of reprogramming immune cells expressing CD8+ surface molecules. In some embodiments, the payload comprises nucleic acid encoding or being a BRM, and the delivery method is also a method of providing an opsonizing agent. In various embodiments, the BRM or opsonizing agent is a γ-chain receptor cytokine, such as IL-2, IL-7, IL-15, IL-15 / 15Rα, IL-21; an immunomodulatory cytokine, such as IL-12, IL-18; a chemokine, such as RANTES, IP10, MIG; or another BRM, such as Flt3, GM-CSF, and G-CSF.

[0463] In some embodiments, the payload comprises nucleic acid encoding a gene / genome editing enzyme and / or guide RNA or other components of a gene / genome editing system, and the delivery method is also a method of reprogramming cells. In some cases, the cells are immune cells expressing CD8+ surface molecules. In some cases, the cells are hematopoietic stem cells (HSCs). In some cases, the cells are mesenchymal stem cells (MSCs). In some embodiments that include delivery of the payload to immune cells, the anti-CD8 binding portion binds to lymphocyte CD8+ surface molecules.

[0464] In some embodiments, including delivery of the payload to immune cells, anti-CD8 tLNP binds to lymphocytes expressing CD8+.

[0465] Treatment

[0466] The anti-CD8 binding agents disclosed herein and tLNPs conjugated with such anti-CD8 binding agents can be used to treat diseases (e.g., CBD1032, CBD1033, CBD1035, CBD1037, CBD1039, CBD1047, CBD1049, etc., and conjugates of such binding agents with LNPs). Those anti-CD8 binding agents and CD8-specific tLNPs disclosed herein provide targeted methods for drug delivery strategies. Therefore, certain embodiments provide a method for treating a disease (or its symptoms) comprising administering a therapeutically effective amount of an anti-CD8 binding agent or CD8-specific tLNP, or a composition comprising these substances, to a mammal (e.g., a human) in need.

[0467] "Treatment" and / or "treating" refers to any indicator of success in the 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 may include reducing the frequency with which a patient experiences symptoms of a disease, defect, symptom, or adverse condition. Treatment may be used herein to refer to a method of treatment or improvement that results in a certain level of treatment or improvement of a disease or condition, and may consider a range of outcomes for that purpose, including complete prevention of the condition.

[0468] "Prevention" or "preventing" refers to preventing a patient's disease or condition, such as the production of autoimmune antibodies. For example, if an individual at risk of developing an autoimmune attack or other related symptoms is treated with the methods disclosed herein and subsequently does not develop an autoimmune attack or other related symptoms, then the disease is prevented in that individual, at least for a period of time. Prevention can also refer to preventing the recurrence of a disease or condition in a patient who has previously been treated for it, for example, by preventing relapse.

[0469] A therapeutically effective dose (also known as an effective dose) can be an amount of a composition comprising an anti-CD8 binding agent or a CD8-specific tLNP sufficient to provide a beneficial effect to the individual administering the composition or otherwise reduce harmful, non-beneficial events. A therapeutically effective dose can be a dose that produces one or more desired or desirable (e.g., beneficial) effects for which the dose is administered, and such administration occurs once or multiple times over a given period of time. The exact dose may depend on the purpose of treatment and can be determined by those skilled in the art using known techniques and the teachings provided herein.

[0470] The anti-CD8 binding agent or CD8-specific tLNP of this disclosure, which can be used in therapy, can be formulated and dosed in accordance with good medical practice, taking into account the disease or condition to be treated, the individual patient's condition, the site of delivery of the composition, the method of administration, and other factors known to the practitioner. The composition can be prepared according to the preparation instructions described herein.

[0471] The compositions can be used in the methods described herein and can be administered to subjects in need using techniques known to those skilled in the art. These compositions may be suitable as a therapy affecting a subject's disease or condition. Those skilled in the art will understand that the amount, duration, and frequency of administration of the pharmaceutical composition to a subject in need depends on a variety of factors, including, for example, the subject's health status, the patient's specific disease or condition, the grade or level of the patient's specific disease or condition, and any other treatments the subject is receiving or has received.

[0472] The anti-CD8 binding agents or CD8-specific tLNPs, compositions, and methods disclosed herein can be used as single agents to treat or prevent diseases such as autoimmune diseases (e.g., idiopathic inflammatory myopathy, such as antisynergistic syndrome) and cancer. Alternatively, the anti-CD8 binding agents or CD8-specific tLNPs, compositions, and methods disclosed herein can be used in combination therapy with a second therapeutic agent to treat or prevent diseases such as autoimmune diseases and cancer.

[0473] In some aspects, this disclosure provides methods for treating diseases or conditions, including administering the anti-CD8 binding agent or CD8-specific tLNP of this disclosure to a subject in need. In some embodiments, the subject is a human. In some embodiments, the antibody or tLNP of this disclosure is administered systemically. In some embodiments, the antibody or tLNP of this disclosure is administered via intravenous or subcutaneous infusion or injection. In some embodiments, the antibody or tLNP of this disclosure is administered locally. In some embodiments, the antibody or tLNP of this disclosure is administered via intraperitoneal or intralesional infusion or injection. Certain embodiments of the LNP and tLNP disclosed herein are capable of treating diseases or conditions as described in paragraphs

[00307] -

[00312] .

[0474] In some implementations, the disease or condition is an autoimmune disease. Examples of autoimmune diseases include, but are not limited to, myocarditis, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sidnam's chorea, myasthenia gravis, systemic lupus erythematosus, fibrotic alveolitis, multiple sclerosis, rheumatic fever, polyglandular syndrome, agranulocytosis, autoimmune hemolytic anemia, bullous pemphigoid, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis / polymyalgia rheumatica, pernicious anemia, rapidly progressive glomerulonephritis, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, allergic reactions, insulin-resistant diabetes mellitus, psoriasis, diabetes mellitus, Addison's disease, and Graves' disease. Diabetes mellitus, endometriosis, celiac disease, Crohn's disease, allergic purpura, ulcerative colitis, Goodpassu syndrome, thromboangiitis obliterans, Sjögren's syndrome, aplastic anemia, rheumatoid arthritis, sarcoidosis, scleritis, T-cell-mediated or B-cell-mediated autoimmune diseases, B-cell-mediated (antibody-mediated) autoimmune diseases, necrotizing myopathy, chronic inflammatory demyelinating polyneuropathy (CIDP), neuromyelitis optica (NMO)-associated myositis, neuromyelitis optica spectrum disorders, pemphigus vulgaris, systemic sclerosis, antisynthetic enzyme syndrome (idiopathic inflammatory myopathy), lupus nephritis, membranous nephropathy, Fanconi anemia, and vasculitis.

[0475] In some implementations, autoimmune diseases are either T-cell-mediated or B-cell-mediated autoimmune diseases. In some cases, B-cell-mediated autoimmune diseases include myositis (such as antisynthetic enzyme antibody-associated myositis), lupus nephritis, membranous nephropathy, systemic lupus erythematosus, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, neuromyelitis optica spectrum disorder (NMOSD), myasthenia gravis, pemphigus vulgaris, rheumatoid arthritis, dermatomyositis, immune-mediated necrotizing myopathy (IMNM), antisynthetic enzyme syndrome, polymyositis, systemic sclerosis, and diffuse cutaneous myositis. Systemic sclerosis, localized cutaneous systemic sclerosis, antisynthetic enzyme syndrome (idiopathic inflammatory myopathy), stiff-person syndrome, myeloid oligodendrocyte glycoprotein autoantibody-associated disease (MOGAD), amyloid light chain amyloidosis, multiple sclerosis, relapsing-remitting multiple sclerosis, secondary progressive multiple sclerosis, primary progressive multiple sclerosis, inactive secondary progressive multiple sclerosis, Sjögren's syndrome, IgA nephropathy, IgG4-related disease, or Fanconi anemia. In some implementations, B-cell-mediated autoimmune diseases are myositis, lupus nephritis, membranous nephropathy, scleroderma, systemic lupus erythematosus, myasthenia gravis, ANCA vasculitis, multiple sclerosis, or pemphigus vulgaris. In some implementations, B-cell-mediated autoimmune diseases are myositis, lupus nephritis, membranous nephropathy, or scleroderma. In some implementations, B-cell-mediated autoimmune diseases are myositis. In some cases, myositis is antisynthetic enzyme myositis. In some implementations, B cell-mediated autoimmune diseases are systemic lupus erythematosus, myasthenia gravis, ANCA vasculitis, multiple sclerosis, or pemphigus vulgaris.

[0476] In some implementations, the disease or condition is rejection of allogeneic organ or tissue transplants. Pre-existing antibodies and / or B cells, in their role as antigen-presenting cells, can promote rapid immune rejection through known mechanisms; therefore, depleting large numbers of B cells can help prevent allogeneic graft rejection.

[0477] In some implementations, the disease or symptom is cancer. Examples of cancer include, but are not limited to, carcinoma, sarcoma, and blood cancers. In some implementations, blood cancers are lymphoma, leukemia, or myeloma. In some cases, blood cancers are B-lineage or T-lineage cancers. In some cases, B-lineage cancers are multiple myeloma, diffuse large B-cell lymphoma, acute myeloid leukemia, mantle cell lymphoma, follicular lymphoma, B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, or myelodysplastic syndrome. In some implementations, the cancer is a sarcoma. In some implementations, the cancer is carcinoma, such as breast cancer, colon cancer, ovarian cancer, lung cancer, testicular cancer, or pancreatic cancer. In some implementations, the cancer is melanoma.

[0478] In some implementations, the disease or condition is a genetic disease or condition, such as a single-gene inherited disease. In other cases, the genetic disease or condition is a hemoglobinopathic disorder, such as sickle cell disease or beta-thalassemia.

[0479] In some implementations, the disease or condition is a fibrotic disease or condition. In some cases, the fibrotic disease is cardiac fibrosis, arthritis, idiopathic pulmonary fibrosis, and non-alcoholic steatohepatitis (also known as metabolic dysfunction-related steatohepatitis). In other cases, the condition involves tumor-associated fibroblasts.

[0480] Methods for treating tLNPs containing nucleic acids encoding chimeric antigen receptors (CARs)

[0481] In some embodiments, the tLNP of this disclosure comprises a nucleic acid encoding a chimeric antigen receptor (CAR). Receptors are chimeric because they combine antigen binding and T cell activation functions into a single receptor. Currently, five generations of CARs are generally accepted. The “first-generation” CAR used in this invention comprises an antigen-binding domain fused to a transmembrane domain, such as a single-stranded variable fragment (scFv) or VHH, which is fused to a cytoplasmic / intracellular domain of the T cell receptor chain. “First-generation” CARs typically have an intracellular signaling (or activation) domain from the CD3ζ chain, which is the primary signaling medium from the endogenous T cell receptor (TCR). “First-generation” CARs can achieve de novo antigen recognition in a single fusion molecule and induce activation of CD4+ and CD8+ T cells via their CD3ζ chain signaling domain, independent of HLA-mediated antigen presentation. The intracellular signaling domain of CD3ζ, in which one or two of the three ITAM motifs have been disrupted, can modulate the balance between effector and memory programs (Feucht et al., 2019 Nat Med 25(1):82-88). The intracellular signaling domain of CD3ε or the low-affinity receptor FcyRIIIA (CD16A) of IgG can be used as a substitute for CD3ζ. In some embodiments, the intracellular signaling domain of CD3ε comprises the sequence KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO: 115). In some embodiments, the intracellular signaling domain of FcγRIIIA (CD16A) comprises the sequence FcγRIIIA:KTNIRSSTRDWKDHKFKWRKDPQDK (SEQ ID NO: 116). In some embodiments, these intracellular signaling domains constitute a means for signal transduction or for activation.

[0482] The "second-generation" CAR used in this invention comprises an antigen-binding domain fused to a transmembrane domain, such as scFv or VHH, which is fused to an intracellular signaling domain capable of activating T cells and a co-stimulatory domain designed to enhance T cell potency and persistence (Sadelain et al., 2013, Cancer Discov. 3:388-398). Thus, CAR design can combine antigen recognition with signal transduction, both physiologically performed by two separate complexes (i.e., the TCR heterodimer and the CD3 complex). The "second-generation" CAR includes intracellular domains of various co-stimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40, CD27, etc.) from the cytoplasmic tail of the CAR to provide additional signals to the cell. The "second-generation" CAR can provide both co-stimulation (e.g., via the CD28 or 4-1BB domain) and activation (e.g., via the CD3ζ signaling domain). Preclinical studies have shown that the "second-generation" CAR can enhance the antitumor activity of CAR-T cells. For example, in clinical trials in patients with chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL), “second-generation” CAR-modified T cells targeting the CD19 molecule have demonstrated robust efficacy (Davila et al., 2012, Oncoimmunol. 1(9):1577-1583). In some implementations, these co-stimulatory domains constitute a means for co-stimulation.

[0483] The “third-generation” CAR provides multiple co-stimulation (e.g., by including both the CD28 domain and the 4-1BB domain) and activation (e.g., by including the CD3ζ activation domain).

[0484] In addition to providing co-stimulation (e.g., through the CD28 or 4-1BB domains) and activation (e.g., through the CD3ζ signaling domain), the "fourth-generation" CAR also provides constitutive or inducible chemokine components.

[0485] In addition to providing co-stimulation (e.g., through the CD28 or 4-1BB domains) and activation (e.g., through the CD3ζ signaling domain) and constitutive or inducible chemokine components, the "fifth-generation" CAR also provides intracellular domains of cytokine receptors (e.g., IL-2Rβ).

[0486] Further variations in the basic CAR structure and the sources of various domains are described in Zabel et al., Immunol Lett 2019 212:53-69. All content of that document that is consistent with the contents of this disclosure regarding the CAR structure and its functional domains is incorporated herein by reference.

[0487] a) Signal peptide

[0488] In some embodiments, the CAR may include a signal peptide at its N-terminus. Non-limiting examples of signal peptides include the CD8α signal peptide, the IgK signal peptide, and the granulocyte-macrophage colony-stimulating factor receptor subunit α (GMCSFR-α, also known as colony-stimulating factor 2 receptor subunit α (CSF2RA)) signal peptide and variants thereof, the amino acid sequences of which are provided in Table 1 below.

[0489] Table 1. Exemplary sequences of signal peptides

[0490]

[0491] b) Extracellular binding domain

[0492] CARs contain an extracellular binding domain, also known as a binder or binding moiety. In some embodiments, the extracellular binding domain may include one or more antibodies specific to one or more tracking antigens. The antibody may be an antibody fragment, such as scFv, or a single-domain antibody fragment, such as VHH. In some embodiments, the scFv may contain a heavy chain variable region (VHH) of the antibody linked via a linker. H ) and light chain variable region (V L V H and V L They can be connected in any order, i.e., V H -Connector-V L or V L -Connector-V H Non-limiting examples of connectors include the Whitlow connector, (G4S)n (SEQ ID NO: 123, where n can be a positive integer, such as 1, 2, 3, 4, 5, 6, etc.), and variations thereof. In some embodiments, the antigen may be an antigen specifically or preferentially expressed on tumor cells, or an antigen characterized by an autoimmune or inflammatory disease.

[0493] Exemplary tracking antigens that can be specifically recognized by CAR, TCR, or ICE include, but are not limited to, B cell maturation agents (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 myeloma); and tight junction protein 6 (CLDN6), tight junction protein 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 †‡ (Related to solid tumors). Antigens associated with B-cell leukemia can also be used for B-cell depletion in non-oncology applications, though CD19 (present on pro-B cells, pre-B cells, immature B cells, naïve B cells, germinal center B cells, memory B cells, and short-lived plasmablasts (sometimes called short-lived plasma cells)) and BCMA (present on memory B cells, short-lived plasmablasts, and long-lived plasma cells) are of particular interest. (* indicates exemplary antibodies with the indicated specificity, from which the binding moiety can be derived, and can be found in Table 9 or Table 10 of U.S. Patent No. 11,326,182B2.) † Exemplary antibodies with the indicated specificity are shown, from which the binding moiety can be derived; these can be found in Wilkinson & Hale, 2022. Both references cited above are incorporated herein by reference. ‡Exemplary antibodies with the specificity shown are described, from which the binding moiety can be derived and are available in the Therapeutic Antibody Database (TABS) at tabs.craic.com. Other suitable antibodies are available in Appendix A. Many of these target antigens are themselves receptors and, if expressed on immune cells, can bind to their ligands. Thus, in some embodiments, the extracellular binding domain of the CAR contains a ligand of the receptor expressed on the target cell. In a further embodiment, the extracellular binding domain of the CAR contains a ligand-binding domain of the receptor against the 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 have variant sequences to enhance the function of the extracellular binding domain. The advantages of the aspects and embodiments disclosed herein are independent of the specificity of the binding moiety. Therefore, the binding specificity is generally unknown in the disclosed aspects and embodiments. In some embodiments, a specific binding specificity may be required. A broader discussion of antibodies that recognize the multiple individual antigens listed above can be found in WIPO Publication WO2024040195A1 and U.S. Patent Application No. 18 / 731,223, all of which teach about antibodies and related molecules that can be used to provide binding moieties that recognize target antigens and are incorporated herein by reference.

[0494] c) Hinge structural domain

[0495] In some embodiments, the CAR may include a hinge domain, also known as a spacer region. The terms “hinge” and “spacer region” are used interchangeably in this disclosure. Non-limiting examples of hinge domains include the CD8α hinge domain, the CD28 hinge domain, the IgG4 hinge domain, the IgG4 hinge-CH2-CH3 domain, and variants thereof, the amino acid sequences of which are provided in Table 2 below.

[0496] Table 2. Exemplary sequence of hinge domains

[0497]

[0498] d) Transmembrane domain

[0499] In some embodiments, the CAR may comprise a transmembrane domain. In other embodiments, the transmembrane domain may comprise transmembrane regions of the following: 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, including the human form of each of these sequences. Table 3 provides several exemplary amino acid sequences of transmembrane domains.

[0500] Table 3. Exemplary sequences of transmembrane domains

[0501]

[0502] e) Intracellular domains

[0503] In some implementations, the CAR may include intracellular signal transduction domains. All generations of CARs include intracellular domains that provide activation or stimulation, such as those from CD3ζ, CD3ε, or CD16A. Second- and third-generation CARs each added one or more intracellular domains to provide co-stimulatory functions, such as those from CD28 or 4-1BB.In some embodiments, the intracellular signal transduction domain may include one or more signal transduction domains selected from the following: 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-α / 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, Ikaros, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / 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 function-associated antigen-1 (LFA-1), NKG2C, CD3ζ, immune receptor tyrosine-based activation motifs (ITAM), ligands that specifically bind to CD83, and their functional variants, including human forms of each of these domains.In some implementations, the intracellular signaling domain comprises one or more signaling domains selected from the group consisting of: CD3ζ domain, ITAM, CD28 domain, 4-1BB domain, or functional variants thereof. Table 4 provides the amino acid sequences of several exemplary intracellular signaling domains. 4-1BB, also known as CD137, delivers an effective co-stimulatory signal to T cells, promoting T lymphocyte differentiation and enhancing T lymphocyte long-term survival. CD28 is another co-stimulatory molecule on T cells. CD3ζ binds to the T cell receptor (TCR) to generate a signal and contains an activation motif (ITAM) based on the tyrosine residue of the immune receptor. The CD3ζ signaling domain refers to amino acid residues from the cytoplasmic domain of the ζ chain that are sufficient to functionally deliver the initial signal required for T cell activation. In some embodiments, such as in the case of tisagenlecleucel as described below, the CD3ζ signaling domain of SEQ ID NO: 129 may have a mutation at amino acid position 14, for example, a glutamine (Q) to lysine (K) mutation (see SEQ ID NO: 130).

[0504] Table 4. Exemplary sequences of intracellular signal transduction domains

[0505]

[0506] f) Exemplary CAR Construction

[0507] In some embodiments, as described in the therapeutic uses and methods disclosed herein, CARs are used to treat diseases or conditions associated with target cells expressing antigens targeted by the CAR. For example, in some embodiments, anti-CD19, anti-CD20, or anti-BCMA CARs can be used to target and treat B-cell malignancies or B-cell-mediated autoimmune conditions or diseases. In other embodiments, anti-FAP CARs can be used to target 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 (anti-CD19), US 10,287,350 (anti-CD19), US2021 / 0363245 (anti-CD19 and anti-CD20), US 10,543,263 (anti-CD22), US 10,426,797 (anti-CD33), US 10,844,128 (anti-CD123), US 10,428,141 (anti-ROR1), and US2021 / 0087295 (anti-FAP). To the extent that they do not contradict this disclosure, all teachings in each of these documents regarding the general structure and function of CARs, as well as regarding the antigen specificity and tracking indications of CARs, are incorporated herein by reference.

[0508] In some implementations, the binding domain from the antibody can be used to construct a CAR to track and treat solid tumors or fibrosis. Exemplary binding domains are available from antibodies such as anti-LRRC 15 (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 anti-ITGA11 (WO 2008 / 075038, US 2011 / 0256061). Other antibodies that can be used to construct CARs for tracking and treating solid tumors or fibrosis include 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.

[0509] The mRNAs encoding CARs disclosed herein include both a mature CAR and a signal peptide. A mature CAR minimally comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the CAR also includes one or more co-stimulatory domains in the intracellular portion of the CAR. In some embodiments, the CAR also includes 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 multiple antigen-binding domains. In some embodiments of the mRNAs disclosed herein, the CAR is an anti-CD19 CAR, an anti-CD20 CAR, an anti-BCMA CAR, or an anti-FAP CAR.

[0510] i) Anti-CD19 CAR

[0511] In some embodiments, two CAR conformations are used for the anti-CD19 CAR: CAR1 and CAR2. The CAR1 mRNA encodes an amino acid sequence consisting of the following domains in N-terminal to C-terminal order: 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; 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 was originally disclosed in US Patent No. 10,287,350 (WO2015 / 187528) as SEQ ID NO: 199, the CAR1 amino acid sequence and its synthesis thereof are incorporated herein by reference. The amino acid sequence of the mature CAR1 protein (i.e., without the signal peptide) is shown in SEQ ID NO: 198. Compared to similar anti-CD19 CAR molecules incorporating CD28 hinges and transmembrane domains, incorporating CD8α hinges and transmembrane domains into CAR1 helps alleviate cytokine release syndrome (cytokine storm), but in vivo CARs can benefit from stronger signaling provided by CD28 hinges and transmembrane domains.

[0512] In some embodiments comprising an anti-CD19 CAR, the anti-CD19 CAR includes an anti-CD19 binding domain. Some embodiments of an anti-CD19 CAR including an anti-CD19 binding domain also include a CD28 hinge, transmembrane and co-stimulatory domains, and a CD3ζ signaling domain. Some embodiments of an anti-CD19 CAR including an anti-CD19 binding domain also include a hinge and transmembrane domain from CD8α, a CD28 co-stimulatory domain, and a CD3ζ chain signaling domain. In some embodiments, the anti-CD19 binding domain includes 47G4 scFv. In some embodiments, CAR-T cells containing an anti-CD19 CAR containing a CD28 hinge, transmembrane and co-stimulatory domain exhibit more homing cell killing than CAR-T cells containing an anti-CD19 CAR containing a CD8α hinge and transmembrane domain and a CD28 co-stimulatory domain.

[0513] In some embodiments, the CAR2 mRNA used encodes an amino acid sequence consisting of the following domains in N-terminal to C-terminal order (SEQ ID NO: 201): CD8α signal peptide (SP), anti-CD19 scFv derived from mAb 47G4 (light chain variable domain, VL; linker, L; heavy chain variable domain, VH), CD28 hinge, CD28 transmembrane (TM), CD28 co-stimulatory domain (co-stim), and CD3ζ signaling domain (stim). The amino acid sequence of the immature CAR2 protein (i.e., with the signal peptide) is disclosed in Genbank: QHQ73565.1 and is provided as SEQ ID NO: 201. In contrast to conventional CAR-T cells containing an integrated DNA sequence encoding CAR, the combination of 47G4 scFv and the CD28 hinge and transmembrane domain provides the advantage of transient in vivo transfection of CAR2. Using the same UTR and codon optimization methods, CAR2 was expressed at a higher mRNA level than CAR1, and T cells expressing CAR2 eliminated more CD19. + cell.

[0514] Further examples of anti-CD19 CARs include those containing a CD19-binding moiety derived from the mouse antibody FMC63. FMC63 and the derived scFv have been described in Nicholson et al., 1997, Mol. Immun. 34(16-17):1157-1165 and PCT applications WO 2018 / 213337 and WO 2015 / 187528, the entire contents of each of which (all teachings concerning anti-CD19 CARs and their uses) are incorporated herein by reference.

[0515] Table 5. Exemplary sequences of anti-CD19 scFv and components

[0516]

[0517] In some cases, anti-CD19 CARs are those found in the following: tesalenocin (Vairy et al., 2018, Drug Des Devel Ther. 12: 3885-3898), lisocabtagene maraleucel, or axicabtagene ciloleucel and brexucabtagene autoleucel (Cappell et al., 2023, Nat Rev Clin Oncol 20: 359-371), all of which use the same CAR. The entire contents of each of the foregoing references in this paragraph (all teachings concerning the design, structure, and activity of anti-CD19 CARs) are incorporated herein by reference.

[0518] Table 6. Exemplary sequences of CD19 CAR*

[0519]

[0520]

[0521]

[0522] *The nucleotide sequence is presented here (and throughout the text) as a DNA sequence; t should be understood as “u” for the corresponding RNA sequence.

[0523] Table 7. Annotations of the telsalonid CD19 CAR sequence

[0524]

[0525] Table 8. Annotations of the Liki Myron CD19 CAR sequence

[0526]

[0527] Table 9. Annotations of Akylonex CD19 CAR sequences

[0528]

[0529] The 47G4-based CAR is disclosed in U.S. Patent No. 10,287,350, and all the contents of that document that teach about anti-CD19 CAR and its uses are incorporated herein by reference. In some implementations, the extracellular binding domain of the CD19 CAR is derived from antibodies specific to CD19, including, for example, SJ25C1 (Bejcek et al., 1995, CancerRes. 55:2346-2351), HD37 (Pezutto et al., 1987, J. Immunol. 138(9):2793-2799), 4G7 (Meeker et al., 1984, Hybridoma 3:305-320), B43 (Bejcek et al., 1995 Cancer Res 55(11):2346-2351), BLY3 (Bejcek et al., 1995 Cancer Res 55(11):2346-2351), B4 (Freedman et al., 1987, Blood 70:418-427), B4 HB12b (Kansas & Tedder, 1991, J. Immunol. 147:4094-4102; Yazawa et al., 2005, Proc. Natl. Acad. Sci. USA 102:15178-15183; Herbst et al., J. Pharmacol. Exp. Ther. 335:213-222 (2010)), BU12 (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 may contain the V of any antibody. H V L And / or one or more CDRs.

[0530] Table 10. Exemplary sequences of 47G4-based anti-CD19 CAR

[0531]

[0532] ii) Anti-CD20 CAR

[0533] CD20 is an antigen found on the surface of B cells as early as the pre-B phase, and its level gradually increases until the B cells mature; it is also an antigen found on cells in most B-cell tumors. CD20-positive cells are sometimes also found in cases of Hodgkin's disease, myeloma, and thymoma. Examples of anti-CD20 CARs include those containing a CD20-binding moiety derived from an antibody specific for CD20, including, for example, MB-106 (Fred Hutchinson Cancer Research Center, see Shadman et al., 2019, Blood 134(Supplement 1):3235), UCART20 (Cellectis, www.cellbiomedgroup.com), or C-CAR066 (Cellular Biomedicine Group, see Liang et al., 2021, J. Clin. Oncol. 39(15)Supplement 1:2508) Leu16 and 2.1.2. In some implementations, the extracellular binding domain of the CD20 CAR includes an scFv derived from a Leu16 monoclonal antibody, which contains a heavy chain variable region (V) of Leu16 linked via a linker. H ) and light chain variable region (V L (See Wu et al., 2001, Protein Engineering. 14(12):1025-1033), such as CAR22 and CAR25 as described herein. In some embodiments, the extracellular binding domain of the CD20 CAR comprises an scFv derived from the monoclonal antibody 2.1.2, which contains the heavy chain variable region (V) of 2.1.2 linked via a linker. H ) and light chain variable region (V L ), such as CAR7 as described herein. Other antibodies that provide an anti-CD20 binding domain include IF5, 1.5.3, rituximab, octotuzumab, teimomab, octotuzumab, tosimob, onituzumab, vetotuzumab, uttotuzumab, and ozenalizumab. The entire contents of each of the foregoing references in this paragraph (all teachings concerning the design, structure, and activity of anti-CD20 CARs) are incorporated herein by reference.

[0534] In some embodiments, CAR25 is provided herein as a CAR conformation for use as an anti-CD20 CAR. The CAR25 mRNA encodes an amino acid sequence consisting of the following domains in N-terminal to C-terminal order: mouse Ig-κ signal peptide (Igksp), an anti-CD20 scFv derived from Leu16 mAb (light chain variable domain, VL; linker, L; heavy chain variable domain, VH), an IgG4 hinge, a CD28 transmembrane domain (TM), a 4-1BB co-stimulatory domain (co-stim), and a CD3ζ signaling domain (stim). The amino acid sequence of the mature CAR25 protein (i.e., without the signal peptide) is shown in SEQ ID NO: 19.

[0535] In some embodiments comprising an anti-CD20 CAR, the anti-CD20 CAR comprises Leu16 scFv. In some embodiments, the anti-CD20 CAR comprising Leu16 scFv further comprises an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain. Examples of such anti-CD20 CARs include, but are not limited to, CAR25 (SEQ ID NO: 19, or having a signal peptide, SEQ ID NO: 20). In some embodiments, the anti-CD20 CAR comprising Leu16 scFv further comprises an IgG4 hinge, a CD28 transmembrane and costimulatory domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain. Examples of such anti-CD20 CARs include CAR22 (SEQ ID NO: 21), or having a signal peptide (SEQ ID NO: 22).

[0536] Table 11. Exemplary sequences of anti-CD20 scFv and components

[0537]

[0538]

[0539] In some embodiments 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 also comprises a CD28 hinge, a transmembrane and co-stimulatory domain, and a CD3ζ signaling domain. Examples of such anti-CD20 CARs include, but are not limited to, CAR7 (SEQ ID NO: 214), or CAR7 having a signal peptide (SEQ ID NO: 215).

[0540] iii) Anti-BCMA CAR

[0541] In some implementations, anti-CD8 tLNP encapsulates nucleic acid encoding an anti-BCMA chimeric antigen receptor (CAR). BCMA is a member of the tumor necrosis family receptors (TNFR) expressed on cells of the B-cell lineage, with the highest expression on terminally differentiated B cells or mature B lymphocytes. BCMA is involved in mediating plasma cell survival to maintain long-term humoral immunity. BCMA expression has recently been shown to be associated with many cancers, such as multiple myeloma, Hodgkin's lymphoma, and non-Hodgkin's lymphoma, various leukemias, and glioblastoma. Examples of anti-BCMA CARs include those containing the BCMA-binding moiety derived from the mouse monoclonal antibody C11D5.3, as described in Carpenter et al., 2013, Clin. Cancer Res. 19(8):2048-2060. See also PCT Application Publication No. WO 2010 / 104949. In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from another mouse monoclonal antibody, C12A3.2, as described in Carpenter et al., 2013, Clin. Cancer Res. 19(8):2048-2060, and PCT application publication WO2010104949. In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from a mouse monoclonal antibody highly specific for human BCMA, referred to as BB2121 in Friedman et al., 2018, Hum. Gene Ther. 29(5):585-601. See also PCT application publication WO2012163805. In some embodiments, the extracellular binding domain of the BCMA CAR comprises single variable segments (VHHs) of two heavy chains that can bind to two epitopes of BCMA, as described in Zhao et al., 2018, J. Hematol. Oncol. 11(1):141, also known as LCAR-B38M. See also PCT Application Publication WO 2018 / 028647. In some embodiments, the extracellular binding domain of the BCMA CAR comprises a fully human heavy chain variable domain (FHVH), as described in Lam et al., 2020, Nat. Commun. 11(1):283, also known as FHVH33. See also PCT Application Publication WO 2019 / 006072. In some embodiments, the extracellular binding domain of the BCMA CAR comprises scFv derived from CT103A (or CAR0085), as described in U.S. Patent No. 11,026,975 B2. Other anti-BCMA CARs are disclosed in U.S. Patent Application Publication Nos. 2020 / 0246381 and 2020 / 0339699.The entire contents of each of the aforementioned references in this paragraph (all teachings on the design, structure, and activity of anti-BCMA CARs) are incorporated herein by reference.

[0542] Table 12. Exemplary sequences of anti-BCMA binders and components

[0543]

[0544]

[0545]

[0546] iv) Anti-FAP CAR

[0547] In some embodiments comprising an anti-FAP CAR, the anti-FAP CAR comprises an antibody 4G5-based scFv (see WO2021 / 061708 and WO2021 / 061778). In some embodiments, the anti-FAP CAR comprising an antibody 4G5-based scFv further comprises a hinge and transmembrane domain from CD8, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain; examples of anti-FAP CARs include the CAR disclosed in WO2021 / 061778.

[0548] Table 13. Exemplary sequences of CAR based on anti-FAP 4G5

[0549]

[0550] In some implementations, anti-CD8 tLNP encapsulates nucleic acids encoding an anti-GPRC5D chimeric antigen receptor (CAR). GPRC5D is a G protein-coupled receptor with no known ligand, and its function in human tissues is unclear. However, this receptor is expressed in myeloma cell lines and bone marrow plasma cells from patients with multiple myeloma. GPRC5D has been identified as an immunotherapeutic target in multiple myeloma and Hodgkin's lymphoma. Examples of anti-GPRC5D CARs include CARs 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 CARs containing a GPRC5D binding moiety derived from an antibody specific for GPRC5D, such as taquituzumab (Pillarisetti et al., Blood 135:1232-43 (2020)) or voritumumab. In some embodiments, the extracellular binding domain of the anti-GPRC5D CAR comprises an scFv derived from a 6D9 mouse antibody specific for human GPRC5D (see creative-biolabs.com / car-t / anti-gprc5d-6d9-h-41bb-cd3-car-pcdcar1-26380.htm). In some implementations, the extracellular binding domain of the GPRC5D CAR comprises an scFv of an anti-GPRC5D antibody linked to a 4-1BB or CD28 co-stimulatory domain and a CD3ζ signaling domain, as described in 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 Rodriguez-Otero et al., Blood Cancer J. 14(1): 24 (2024). The entire contents of each of the foregoing references in this paragraph (all contents teaching the design, structure, and activity of anti-GPRC5D CARs and anti-GPRC5D antibodies that may provide an antigen-binding domain for a CAR or immune cell connector) are incorporated herein by reference, and each example constitutes a means for binding GPRC5D.In any of the above-described tLNP implementations, some implementations include tLNPs encapsulating a GPRC5DCAR payload encoded by RNA and having a T-cell targeting portion (such as an anti-CD8 antibody).

[0551] In some implementations, anti-CD8 tLNP encapsulates nucleic acids 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, contact with FCRL5 stimulates B cell proliferation; therefore, FCRL5 has been identified as an immunotherapeutic target for this disease. Examples of anti-FCRL5 CARs include CARs 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 an scFv 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 an scFv derived from a mouse antibody specific to human FCRL5. Such antibodies include 7D11, F25, F56, and F119, as described in Polson et al., Int. Immunol., 18(9): 1363-1373 (2006); Franco et al., J. Immunol. 190(11): 5739-5746 (2013); Ise et al., Clin. CancerRes. 11(1): 87-96 (2005); and 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 includes a binding moiety derived from an antigen-binding domain of an anti-FCRL5 antibody or nanobody, including civastimab, 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 includes a binding moiety derived from an antibody-drug conjugate targeting FCRL5, such as the binding moiety described in Elkins et al., Mol. Cancer Ther. 11(10): 2222-2232 (2012).In some embodiments, the extracellular binding domain of the anti-FCRL5 CAR is coupled with a co-stimulatory domain (such as a 4-1BB or CD28 co-stimulatory domain) and a signal transduction domain (such as a CD3ζ signal transduction domain). The entire contents of each of the foregoing references in this paragraph (all teachings on the design, structure, properties, and activity of anti-FCRL5 CARs and anti-FCRL5 antibodies that may provide an antigen-binding domain for a CAR or immune cell connector) are incorporated herein by reference. Each example constitutes a means for binding FCRL5. In any of the above-described tLNP embodiments, some embodiments include tLNPs encapsulating an RNA-encoded FCRL5 CAR payload and having a T-cell targeting portion (such as an anti-CD8 antibody).

[0552] Each CAR described herein, specific to a particular antigen, constitutes a means for antigen recognition of that antigen, and all CARs described herein collectively constitute a means for antigen recognition. This function can also be described as antigen recognition by immune cells, or antigen recognition by T cells, etc.

[0553] In some implementations, the ORF may encode a gene-editing nuclease, such as a gene-editing nuclease encoding: RNA-directed nuclease, gene or base-editing protein, leader editing protein, gene-writing protein (e.g., modified or modularized non-long terminal repeat (LTR) retrotransposon), retrotransposase, RNA writer, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), wide-range nuclease, transposase, retrotransposon, reverse transcriptase (e.g., M-MLV reverse transcriptase), nicking enzyme or inactive nuclease (e.g., Cas9, nCas9, dCas9), DNA recombinase, CRISPR nuclease (e.g., Cas9, Cas12, Cas13, Cas3, CasMINI, Cas7-11, CasX), DNA nicking enzyme, Cas9 nicking enzyme (e.g., D10A or H840A), or any fusion or combination thereof. Genome, gene, and base editing technologies have been reviewed in 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. To the extent that they do not conflict with this disclosure, all the contents of each of these documents that teach about the components and uses of this technology are incorporated herein by reference.

[0554] In some embodiments of any of the foregoing aspects, 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 some embodiments, the poly(A) sequence has about 90 adenosine residues. In some embodiments, the poly(A) sequence has about 100 adenosine residues. In some embodiments, the poly(A) sequence has about 110 adenosine residues. In some embodiments, the poly(A) sequence has about 130 adenosine residues. Each CAR described herein that is specific to a particular antigen constitutes a means for antigen recognition of that antigen, and all CARs described herein collectively constitute a means for antigen recognition. This function may also be described as antigen recognition by immune cells, or antigen recognition by T cells, etc.

[0555] Each of the various genera, subgenera, and / or species of LNPs or tLNPs disclosed herein (including those based on the inclusion or exclusion of specific lipids, specific lipid compositions, specific payloads, and / or specific humanized anti-CD8 antibodies) can be used to define the scope of implementation schemes for each treatment method.

[0556] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0557] To facilitate an understanding of the principles of this disclosure, reference will now be made to embodiments, and these embodiments will be described using specific language. However, it should be understood that this is not intended to limit the scope of the disclosure, and such changes and further modifications to the disclosure as illustrated herein are contemplated, as would normally occur to those skilled in the art to which this disclosure pertains.

[0558] Various exemplary embodiments of the compositions and methods according to the invention are now described in the following non-limiting examples. The examples provided are for illustrative purposes only and are not intended to limit the scope of the invention in any way. In fact, various modifications to the invention, other than those shown and described herein, will be apparent to those skilled in the art from the foregoing description and the following examples, as well as from the appended claims.

[0559] Table 14. LNP Compositions

[0560]

[0561]

[0562] Example

[0563] Materials and methods

[0564] Generation of humanized anti-CD8α binding agents / antibodies

[0565] This article discloses the sequence of the CT8 antibody (also referred to herein as CBD1017p). The VH and VL sequences were compared with known human germline sequence libraries from the human VH and human VLκ genes (IMGT). ® The International Immunogenetic Information System (www.imgt.org; Founder and Director: Marie-Paule Lefranc, Montpellier, France) uses the IMGT human VH gene (F+ORF, 273 germline sequences) and IMGT human VLκ gene (F+ORF, 74 germline sequences) databases used by the NCBI IgBLAST program. Recipient human germlines are selected from those most sequence-closest to the parental antibody.

[0566] For VH, the human germline IGHV1-46*01 was used as the receptor sequence, and the human heavy chain IGHJ6 (allele 1) linker region (J gene) was selected from IMGT. ® International Immunogenetic Information System ® The human-connected region sequence compiled by www.imgt.org (founder and director: Marie-Paule Lefranc, Montpellier, France) (see...) Figure 1A ).

[0567] For VL, the human germline IGKV1-39*01 was used as the receptor sequence, and the human light chain IGKJ2 (allele 1) linker region (J gene) was selected from IMGT. ® International Immunogenetic Information System ® The human-connected region sequence compiled by www.imgt.org (founder and director: Marie-Paule Lefranc, Montpellier, France) (see...) Figure 1B ).

[0568] CDRs are defined according to the AbM definition (see Dr. Andrew C. Martin's website on bioinf for a table comparing CDR definitions). It is envisioned that the human germline framework positions (i.e., non-CDR residues in VH and VL) be altered to the corresponding parental mouse sequences to optimize the binding of humanized antibodies to CD8-specific antigens. Potential variations for each humanized sequence are described in... Figure 1A and Figure 1B The bid was successful.

[0569] The CBD1017p VH and VL sequences were also compared with known human germline sequence libraries from the human VH and VL κ genes using an online IMGT / BlastSearch implementation. 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 was used. ® Manual checks using online modeling software (Schrödinger Inc.) are used to obtain consensus-modified phylogenetic sequences, which are then used as the hypothetical humanization. Figure 1C and Figure 1D The starting point of the position.

[0570] Binding affinity (K) determined by biolayer interferodynamics. D )Measurement

[0571] Biolayer interference kinetics assays were performed using the GatorBio Gator Plus BLI system. Baselines were calculated using a kinetic buffer containing PBS with 0.1% Tween 20 and 0.2% BSA. 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 loaded with 100 mM biotinylated recombinant CD8α protein (CDA-H82E3, AcroBiosystems) at a rotation speed of 400 rpm to a threshold response of 10 nm. The sensor was then incubated in kinetic buffer for 300 s to obtain baseline measurements prior to each association. Each anti-CD8α Fab fragment was diluted 2-fold in kinetic buffer at concentrations ranging from 100 nM to 3.12 nM. The antigen-loaded sensor was incubated in the diluted solution for 300 s to capture the Fab fragment, thus recording the association phase. Finally, the sensor was incubated in kinetic buffer for 900 s to record the dissociation phase. Except for the antigen loading step, the rotation speed for all steps was 1000 rpm. During the association and dissociation phases, the assays were performed at 30°C or 37°C. The sensor data were subtracted from the baseline, and global curve fitting using a 1:1 monovalent binding model was performed on the kinetic data from six different analytical concentrations using GatorOne software (version v2.10) to determine the kinetic rate constant (k) of the analyzed antibody. on and k off ) and equilibrium dissociation constant (K D ).

[0572] The binding kinetics of CBD1033 Fab and its parent CBD1017ch Fab against human CD8αα homodimer, human CD8αβ heterodimer, and cynomolgus CD8αα homodimer were evaluated using the same methods employed in the BLI evaluation of the humanized Fab variant. To evaluate the binding kinetics against the cynomolgus CD8αβ heterodimer protein, the antigen protein was directly coupled to the tip of an amine-reactive biosensor (Gator Bio) according to the manufacturer's protocol. Briefly, the sensor was activated for 300 s using a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (ThermoFisher Scientific) and 10 mM N-hydroxysulfosuccinimide sodium salt (sulfon-NHS) (Sigma-Aldrich) in water. The cynomolgus CD8αβ protein, diluted to 100 mM in 10 mM sodium acetate buffer (pH 6.0), was covalently captured for 300 s to an approximate threshold of 5 nm, and then the sensor was inactivated for 300 s with 1 M ethanolamine (ETA) (Sigma-Aldrich) solution (pH 8.5). The sensor was then washed in kinetic buffer for 600 s. The washing step was repeated for 120 s for baseline calculation before measuring binding kinetics. The antibody fragment was diluted 3-fold in the kinetic buffer at concentrations ranging from 1000 nM to 1.37 nM. As above, the sensor with captured antigen was incubated in the diluted solution for 1200 s to capture the Fab fragment, thus recording the association phase. The sensor was then incubated in the kinetic buffer for 1200 s to record the dissociation phase. All steps were performed at a shaking speed of 1000 rpm and at a temperature of 30 °C. Kinetic parameters were determined using a 1:1 monovalent (Langmuir) binding model using the previous method.

[0573] 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 loaded with 50 mM biotinylated Fab sample CBD1033.37 or CBD1033.24 up to a threshold response of 10 nm. The sensor was then briefly blocked for 60 s with kinetic buffer containing 50 mM biotin and 3% BSA, followed by a further 120 s incubation in kinetic buffer to obtain stable baseline measurements before each association. Customized recombinant human CD8α mouse IgG2a Fc fusion protein was diluted 2-fold in kinetic buffer at concentrations ranging from 100 nM to 1.56 nM. The binding agent-loaded sensor was incubated in the diluted solution for 1200 s to capture the CD8α protein, thus recording the association phase. Finally, the sensor was incubated in kinetic buffer for another 1200 s to record the dissociation phase. All steps were performed at a rotation speed of 1000 rpm. During association and dissociation, the assays were performed at a temperature of 37°C. Sensor data were subtracted from the baseline, and global curve fitting using a 1:1 monovalent binding model was performed on kinetic data from six different analytical concentrations using GatorOne software (version v2.10) to determine the kinetic rate constant (k) of the analyzed binder. on and k off ) and equilibrium dissociation constant (K D ).

[0574] Binding affinity (K) through biolayer interference steady-state analysis D )Measurement

[0575] To assess the affinity of intact antibodies, steady-state assays were used because the divalent nature of intact antibodies and CD8α complicates kinetic analysis. 50 nM CD8α-His (i.e., CD8α with a C-terminal oligohistidine tag; Acro) was immobilized on a Ni-NTA sensor probe. Binding was analyzed serially with three-fold antibody dilutions: 60 nM, 20 nM, 6.67 nM, 2.22 nM, 0.74 nM, and 0.25 nM. The antigen-loaded sensor was incubated in dilution solution for 300 s to capture intact IgG antibodies, thus recording the association phase. The sensor was incubated in kinetic buffer for 900 s to record the dissociation phase. Except for the antigen loading step, all steps were performed at a rotation speed of 1000 rpm, with a rotation speed of 400 rpm for the antigen loading step to control antigen density. The assay was performed at 30 °C during both the association and dissociation phases. The sensor data was subtracted from the baseline and further analyzed in GatorOne software (version 2.10) to determine the antibody's equilibrium dissociation constant (K0) by fitting the steady-state signal to a 1:1 binding model. D ).

[0576] Binding affinity (K) determined by surface plasmon resonance method D )Measurement

[0577] Surface plasmon resonance (SPR) assays were performed at 25 °C using a Biacore 8K SPR instrument (Cytiva) in run buffer containing 1xHBS-N (Cytiva) and 0.05% Tween-20. CBD1033 antibody was captured by anti-human IgG (Fc) antibody (Cytiva) immobilized on a CM5 sensor chip (Cytiva) at densities of 40–50 and 80–100 response units (RU) for kinetic measurements with CD8αα and CD8αβ, respectively. To measure binding kinetics, serially 2-fold dilutions of recombinant CD8 protein prepared in run buffer were injected into the flow cell at 30 µL / min, ranging from 200 nM to 6.25 nM (serial 1:2 dilutions). Association data were collected for 180 s, followed by a 1200 s dissociation step. At the end of each binding cycle, the sensor surface was regenerated with 3 M MgCl2 buffer. For the cynomolgus CD8αβ heterodimer, binding kinetics were evaluated using the same experimental parameters, except that the concentration range was 400 nM to 12.5 nM. Sensing plots were generated, and background was subtracted using blank run buffer. Binding kinetic parameters were analyzed and determined using the BIAcore evaluation software (Cytiva) with a standard 1:1 monovalent binding (Langmuir) model.

[0578] Expression and purification of disulfide bond engineered F(ab')

[0579] Using a proprietary expression protocol, disulfide-engineered F(ab') analogues were transiently expressed in an engineered CHO-K1 cell line (Wuxi Biologics). After 7 days, the culture supernatant was harvested by centrifugation and filtration. Initially, KanCap was used... ™F(ab') analogues were captured from the filtered supernatant using affinity chromatography on G resin (Kaneka) and eluted with 50 mM citrate buffer (pH 3.5). The antibody-containing eluent was exchanged for PBS (pH 6.5) containing 10 mM EDTA by dialysis. Fab molecules were then 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 every 30 minutes, and the purity of intact Fab was checked by SDS-PAGE analysis. The reduced F(ab') analogues were then diluted with 20 mM NaAc (pH 5.0), captured by cation exchange chromatography on SP Sepharose HighPerformance resin (Cytiva), and eluted with a gradient from 0 M to 1 M NaCl. The purified F(ab') protein was then dialyzed against 20 mM histidine-HCl (pH 5.5) and 240 mM sucrose. The purity of the final F(ab') analogue was assessed by SDS-PAGE, analytical SEC-HPLC and LC-MS.

[0580] ECs that specifically bind to the antigen CD8 50 Measurement

[0581] In order to obtain Figure 3A The EC shown confirms specific binding to the antigen. 50 The results showed that HEK293T cells overexpressing CD8 were dissociated in Versine solution (PBS buffer at pH 7.4, supplemented with 0.5 mM EDTA), and 2 x 10⁻⁶ cells were added. 5Total cells were transferred to 96-well V-bottom plates (Corning). Cells were washed twice with cell staining buffer (#420201, Biolegend). Each anti-CD8α antibody containing human IgG1 isotypes and Fc silencing mutations L234A, L235A, and P329A was serially diluted from 100 µg / mL to 6.1 ng / mL in cell staining buffer via a four-fold antibody titration series. A 1:4000 dilution of eFluor 780 cell-fixing viable dye (eBioscience) was also included in the antibody solution. Cells were stained on ice for 30 minutes with each diluted antibody solution. Cells were then washed three times with cell staining buffer, and primary antibody binding was detected by staining on ice for 30 minutes with a 1:100 dilution of anti-human Fc BV421 conjugate antibody (#410704, Biolegend). After staining with the secondary detection antibody, cells were washed three times with cell staining buffer, fixed in FluoroFix buffer (Biolegend), and stored at 4°C protected from light until analysis. Flow cytometry analysis was performed on each sample using an Agilent NovoCyte flow cytometer. Flow cytometry data were analyzed using Flowjo 10 (Becton, Dickinson & Company) to measure the geometric median fluorescence intensity (gMFI) of binding on cells for each antibody. gMFI values ​​were normalized to the gMFI measured in samples stained only with the secondary detection antibody and plotted against antibody concentration. Binding curves were constructed using GraphpadPrism software, and EC50 was determined by nonlinear curve fitting. 50 value.

[0582] In order to obtain Figure 6 The results shown were obtained by culturing SupT1 (ATCC #CRL-1942) and HPB-ALL (DSMZ; ACC-483) in RPMI medium supplemented with 10% fetal bovine serum (#97068-085; Avantor). To determine the binding of EC from anti-CD8α antibodies... 50 The value will be 2x10. 5Total cells were transferred to 96-well V-bottom plates (Corning). Cells were washed twice with cell staining buffer (#420201, Biolegend). Anti-CD8α antibody was serially diluted fourfold from 60 µg / mL to 57 pg / mL in cell staining buffer. Cells were stained on ice for 30 min with each diluted antibody solution. Cells were then washed three times with cell staining buffer and primary antibody binding was detected by staining on ice for 30 min with 1:200 diluted anti-human Fc BV421 conjugate antibody (#410704, Biolegend). The detection antibody solution also contained 1:4000 diluted eFluor 780 cell-fixing viable dye (eBioscience). After staining with the secondary detection antibody, cells were washed three times with cell staining buffer, fixed in FluoroFix buffer (Biolegend), and stored at 4°C protected from light until analysis. Flow cytometry analysis was performed on each sample using an Agilent NovoCyte flow cytometer. Flow cytometry data were analyzed using Flowjo10 (Becton, Dickinson & Company) to measure the geometric median fluorescence intensity (gMFI) of cell binding for each antibody. The gMFI values ​​were normalized to the gMFI measured in samples stained with secondary detection antibodies only and plotted against antibody concentrations. Binding curves were constructed using Graphpad Prism software, and EC50 was determined by nonlinear curve fitting. 50 value.

[0583] In order to obtain Figure 8A and Figure 8B The results shown are 5x10 4Expanded primary T cells (human, cynomolgus monkey, or rhesus monkey) were thawed and transferred to 96-well V-bottom plates (Corning). Cells were washed twice with cell staining buffer (#420201, Biolegend). Each anti-CD8α antibody was serially diluted from 60 µg / mL to 0.057 ng / mL in cell staining buffer using a four-fold antibody titration series. Cells were stained on ice for 30 minutes with each diluted antibody solution. Cells were then washed three times with cell staining buffer, and primary antibody binding was detected by staining on ice for 30 minutes with a 1:200 diluted anti-human Fc BV421 conjugate antibody (#410704, Biolegend). The detection antibody solution also contains a 1:200 dilution of anti-CD3 SP34-2 Alexa Fluor 700 conjugate antibody (#557917, BD Pharmagen), a 1:200 dilution of anti-CD4 OKT4 BV650 conjugate antibody (#317436, Biolegend), and a 1:5000 dilution of eFluor 780 cell-fixing viability dye (eBioscience). After staining with the secondary detection antibody, the cells were washed three times with cell staining buffer and fixed on eBioscience. ™ Samples were fixed in IC fixation buffer and stored at 4°C protected from light until analysis. Flow cytometry analysis was performed on each sample using an Agilent NovoCyte flow cytometer. Flow cytometry data were analyzed using Flowjo 10 (Becton, Dickinson & Company) to measure the geometric median fluorescence intensity (gMFI) of binding on cells for each antibody. gMFI values ​​were normalized to the gMFI measured in samples stained with secondary detection antibodies only and plotted against antibody concentrations. Binding curves were constructed using Graphpad Prism software, and EC50 was determined by nonlinear curve fitting. 50 value.

[0584] tLNP formation

[0585] Initial LNPs were formed by mixing an aqueous solution of mCherry mRNA and an ethanolic solution of lipids in a ratio of CICL1:DSPC:CHOL:DSG-PEG(2k):DSPE-PEG(2k)-MAL [58:10:30.5:1.4:0.1] (the F9 composition mentioned in Table 14). Stepwise phosphate and Tris buffer dilutions and tangential flow filtration (TFF) purification were then performed.

[0586] Intact antibodies were conjugated to LNPs using an N-succinimide-S-acetylthioacetate (SATA)-maleimide conjugation chemistry. The antibody was modified with SATA (Sigma-Aldrich) to introduce thiol groups at accessible lysine residues, allowing conjugation to maleimide. SATA was deprotected using 0.5 M hydroxylamine, followed by removal of unreacted fractions via a G-25 Sephadex QuickSpin Protein column (Roche Applied Science, Indianapolis, IN). The reactive thiol groups on the antibody were then conjugated to the maleimide portion of the LNP using a thioether conjugation chemistry. The conjugated tLNPs (LNPs conjugated with the targeting antibody) were purified using a Sepharose CL-4B gel filtration column (Sigma-Aldrich) or TFF (tangential flow filtration). The tLNPs were frozen at -80°C until use.

[0587] The biantibody and F(ab')2 are conjugated as follows: First, the cystine bonds in the antibody are partially reduced with tris(2-carboxy)phosphine (TCEP) to generate a thiol group for conjugation via maleimide of LNP, as described in the previous paragraph. Fab and Fab' molecules, engineered to have free thiols, are similarly conjugated to maleimide of LNP.

[0588] The conjugation reaction between anti-CD8 F(ab') and maleimide-PEG-biotin (MPB)

[0589] Prior to the conjugation reaction, PBS (pH 7.4) and 10 mM EDTA were prepared as conjugation buffers. The reduced F(ab') analog buffer was exchanged for the conjugation buffer and diluted to a concentration of 30 µM. The diluted F(ab') solution was then reacted with a 10-fold molar excess of EZ-Link. ™ Maleimide-PEG 11 - Biotin (Thermo Fisher) was reacted with gentle shaking at room temperature for 1 hour. The reaction was then quenched by adding a 50-fold molar excess of N-acetylcysteine ​​(Sigma Aldrich) and incubated with gentle shaking at room temperature for another 1 hour. The reaction mixture buffer from Fab was exchanged for 20 mM Tris, 150 mM NaCl (pH 7.4) using a Zeba 7k MWCO column (Thermo Fisher). Protein Simple was then used to... ™ Immunoblotting was performed using the Jess system (Bio-Techne), and biotin conjugation was validated using HRP-conjugated streptavidin detection. The purity of the biotin conjugation was further analyzed using analytical SEC-HPLC, LC-MS, and peptide mapping.

[0590] Transfection rate and mCherry expression measurement

[0591] In order to obtain Figures 4A to 5C , Figures 17A to 18B and Figure 24A The results shown demonstrate the isolation of primary human T cells from different donors from leukocyte-removed samples using the Easysep Human T Cell Isolation Kit (#17951 Stem Cell Technologies) and cryopreservation in CryoStor CS10 cryopreservation medium (#210102 Biolife Solutions). ™ OpTmizer ™ Complete T-cell culture medium was prepared by supplementing serum-free T-cell expansion medium (#A1048501 Thermo Fisher) with 5% heat-inactivated human AB serum (#HP1022HI Valley Biomedical), 1% G...

Claims

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) comprising an amino acid sequence having at least 90% identity with the amino acid sequence of the frame region of SEQ ID NO: 9 or 31, wherein the 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 comprising 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 frame region of SEQ ID NO: 15 or 37, wherein the VL comprises: CDR1 (VL-CDR1) comprising the amino acid sequence RASESVX3GFGX1SFMN (SEQ ID NO: 49), VL-CDR2 comprising the amino acid sequence LASX2LES (SEQ ID NO: 50), and VL-CDR3 comprising the amino acid sequence QQX2X2EX3PYT (SEQ ID NO: 51). 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.

2. The isolated antibody or its antigen-binding fragment according to claim 1, wherein X1 of VH-CDR2 is S, Q or A.

3. The isolated antibody or its antigen-binding fragment according to claim 1 or 2, wherein X2 of VL-CDR1 is S or A.

4. The isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the VH comprises 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.

5. The isolated antibody or antigen-binding fragment thereof according to claims 1 and 3, wherein the VL comprises the amino acid sequence of SEQ ID NO: 64 or SEQ ID NO:

65.

6. The isolated antibody or its antigen-binding fragment according to claim 1, comprising: (a) A human heavy chain variable region (VH), wherein the VH comprises: a heavy chain CDR1 (VH-CDR1) comprising the amino acid sequence of SEQ ID NO: 2, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 3, 58, 59 or 60, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 4; and (b) Light chain variable region (VL), wherein the VL comprises: CDR1 (VL-CDR1) containing the amino acid sequence of SEQ ID NO: 6, 227 or 228, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 7 and VL-CDR3 containing the amino acid sequence of SEQ ID NO:

8.

7. The isolated antibody or its antigen-binding fragment according to claim 1 or 6, wherein: (a) The 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, and wherein the VH-CDR1 comprises the amino acid sequence of SEQ ID NO: 2, the VH-CDR2 comprises the amino acid sequence of SEQ ID NO: 3, and the VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 4; and (b) The VL comprises an amino acid sequence having at least 90% identity with the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, wherein the VL-CDR1 comprises the amino acid sequence of SEQ ID NO: 6, the VL-CDR2 comprises the amino acid sequence of SEQ ID NO: 7, and the VL-CDR3 comprises the amino acid sequence of SEQ ID NO:

8.

8. The isolated antibody or its antigen-binding fragment according to claim 1 or 6, wherein: (a) The 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, and wherein the VH-CDR1 comprises the amino acid sequence of SEQ ID NO: 2, the VH-CDR2 comprises the amino acid sequence of SEQ ID NO: 3, and the VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 4; and (b) The 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, and wherein the VL-CDR1 comprises the amino acid sequence of SEQ ID NO: 6, the VL-CDR2 comprises the amino acid sequence of SEQ ID NO: 7, and the VL-CDR3 comprises the amino acid sequence of SEQ ID NO:

8.

9. The isolated antibody or its antigen-binding fragment according to claim 1 or claim 6, The VH therein comprises an amino acid sequence having at least 90% identity with the amino acid sequences 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 contains an amino acid sequence that has at least 90% identity with the amino acid sequences 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.

10. The isolated antibody or antigen-binding fragment thereof according to claim 1, claim 6 or claim 9, wherein: (a) The 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 the VL comprises the amino acid sequence of SEQ ID NO: 16; (b) The 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 the VL comprises the amino acid sequence of SEQ ID NO: 17; (c) The 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 the VL comprises the amino acid sequence of SEQ ID NO: 18; or (d) The VH contains 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 the VL contains the amino acid sequence of SEQ ID NO:

39.

11. The isolated antibody or antigen-binding fragment thereof according to claim 1 or 6, wherein: (a) The VH comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 11, and wherein the VH-CDR1 comprises the amino acid sequence of SEQ ID NO: 2, the VH-CDR2 comprises the amino acid sequence of SEQ ID NO: 3, and the VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 4; and (b) The VL comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 17, wherein the VL-CDR1 comprises the amino acid sequence of SEQ ID NO: 6, the VL-CDR2 comprises the amino acid sequence of SEQ ID NO: 7, and the VL-CDR3 comprises the amino acid sequence of SEQ ID NO:

8.

12. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein VH and VL are bound to form scFv or a biantibody.

13. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, comprising a κ constant region, a λ constant region, a human IgG1 constant region, a human IgG2 constant region, a human IgG3 constant region or a human IgG4 constant region.

14. The isolated antibody or antigen-binding fragment thereof according to claim 13, comprising a silenced Fc region.

15. The isolated antibody according to claim 14, wherein the silenced Fc region comprises SEQ ID NO: 43 or 44.

16. The isolated antibody according to claim 15, comprising a heavy chain having the amino acid sequence of SEQ ID NO:

61.

17. The isolated antibody according to claim 13 or 14, wherein it is a complete antibody.

18. The isolated antibody or antigen-binding fragment thereof according to claim 13, wherein the κ constant region has the amino acid sequence of SEQ ID NO:

41.

19. The isolated antibody according to claim 13, wherein the human IgG1 constant region has the amino acid sequence of SEQ ID NO: 42, SEQ ID NO: 43 or SEQ ID NO:

44.

20. The antigen-binding fragment according to claim 13, wherein it is an F(ab), F(ab'), or an F(ab') analogue.

21. The antigen-binding fragment of claim 20, comprising a human IgG1 F(ab') constant region having the amino acid sequence of SEQ ID NO:

76.

22. The antigen-binding fragment according to claim 21, wherein the F(ab') heavy chain has the amino acid sequence of SEQ ID NO:

78.

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

79.

24. The antigen-binding fragment according to claim 23, wherein the F(ab') heavy chain has the amino acid sequence of SEQ ID NO:

80.

25. The antigen-binding fragment according to any one of claims 20 to 24, comprising a κ constant region having the amino acid sequence of SEQ ID NO:

41.

26. The antigen-binding fragment of claim 20, wherein the F(ab') analog comprises IgG1 or IgG4CH1 F174C substitution and Cκ S162C substitution.

27. The antigen-binding fragment of claim 26, wherein the F(ab') analog further comprises Cκ C214S substitution, Cκ C214S substitution and IgG1 hinge C233S substitution, or Cκ C214S substitution and IgG1 hinge truncation at T238.

28. The antigen-binding fragment of claim 26, wherein the F(ab') analog further comprises IgG4 CH1C127S substitution, or IgG4 CH1 C127S substitution and Cκ C214S substitution.

29. The antigen-binding fragment according to claim 20, wherein the IgG1 constant region 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.

30. The antigen-binding fragment of claim 27, comprising a heavy chain having an 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.

31. The antigen-binding fragment according to claim 27, comprising a heavy chain having an amino acid sequence having SEQ ID NO: 92, SEQ ID NO: 98 or SEQ ID NO:

102.

32. The antigen-binding fragment of claim 27, comprising a heavy chain having an 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.

33. The antigen-binding fragment according to claim 20, wherein the IgG4 constant region has the amino acid sequence of SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 93 or SEQ ID NO:

103.

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

104.

35. The antigen-binding fragment according to claim 26, wherein the Cκ has the amino acid sequence of SEQ ID NO: 89 or SEQ ID NO:

100.

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

112.

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

101.

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

112.

39. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 38, wherein the humanized antibody or antigen-binding fragment thereof has an aggregation temperature ≥60°C (T0). agg ) and a chain-breaking temperature of ≥65℃ (T M ).

40. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 39, wherein the humanized antibody or antigen-binding fragment thereof has a low tendency for self-interaction.

41. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 40, wherein the humanized antibody or antigen-binding fragment thereof lacks multireactivity with any of the following: (a) Double-stranded DNA and insulin; (b) Baculovirus particles; (c) Human cell surface and secretory proteins; or Any combination of (d)(a) to (c).

42. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 41, wherein the humanized antibody or antigen-binding fragment thereof has minimal to undetectable off-target binding.

43. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 42, comprising a thiolated lysine residue at Lys248 or Lys288 in the constant region.

44. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, wherein it is F(ab), F(ab'), F(ab')2, scFv, biantibody or microantibody.

45. An F(ab') analogue comprising VH and VL of the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 11.

46. ​​An F(ab') analogue comprising a repositioned interchain disulfide bond and an antigen-binding domain that binds to the CT8 epitope of CD8.

47. An F(ab') analogue comprising an antigen-binding domain that competitively binds to an epitope bound by an anti-CD8 antibody CT8, TRX2, or YTC182.

20.

48. The F(ab') analogue according to claim 46 or claim 47, comprising means for binding the CT8 epitope or means for binding an epitope identical to that bound by CT8, TRX2 and / or YTC182.

20.

49. The F(ab') analogue according to any one of claims 46 to 48, comprising IgG1 or IgG4 CH1F174C substitution and Cκ S162C substitution.

50. The F(ab') analogue according to claim 49, further comprising Cκ C214S substitution, or Cκ C214S substitution and IgG1 hinge C233S substitution, or Cκ C214S substitution and IgG1 hinge truncation at T238.

51. The F(ab') analogue according to claim 49, further comprising IgG4 CH1 C127S substitution, or IgG4 CH1 C127S substitution and Cκ C214S substitution.

52. The F(ab') analogue according to any one of claims 46 to 51, comprising VH and VL, The 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; and The VL comprises: CDR1 (VL-CDR1) having the amino acid sequence of SEQ ID NO: 223, VL-CDR2 having the amino acid sequence of SEQ ID NO: 224, and VL-CDR3 having the amino acid sequence of SEQ ID NO:

225.

53. The F(ab') analogue according to any one of claims 46 to 51, comprising VH and VL of YTC182.

20.

54. The F(ab') analogue according to any one of claims 46 to 51, comprising VH and VL of CT8.

55. The F(ab') analogue according to any one of claims 46 to 51, comprising: (a) A heavy chain variable region (VH) comprising an amino acid sequence having at least 90% identity with the amino acid sequence of the frame region of SEQ ID NO: 9 or 31, wherein the 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 comprising 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 frame region of SEQ ID NO: 15 or 37, wherein the VL comprises: CDR1 (VL-CDR1) comprising the amino acid sequence RASESVX3GFGX1SFMN (SEQ ID NO: 49), VL-CDR2 comprising the amino acid sequence LASX2LES (SEQ ID NO: 50), and VL-CDR3 comprising the amino acid sequence QQX2X2EX3PYT (SEQ ID NO: 51). 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.

56. A lipid nanoparticle (LNP) comprising a separated antibody or antigen-binding fragment thereof conjugated to the LNP according to any one of claims 1 to 55.

57. The LNP of claim 56, wherein the LNP comprises a lipid formulation, the lipid formulation comprising: Approximately 35 mol% to approximately 65 mol% of ionizable cationic lipids having the following structure Where R is , , or , From about 0.5 mol% to about 3 mol% of PEG-lipids, wherein the PEG-lipids comprise functionalized PEG-lipids and non-functionalized PEG-lipids. Phospholipids of approximately 7 mol% to approximately 13 mol%, and Sterols, approximately 27 mol% to approximately 50 mol%. The antibody or its antigen-binding fragment is conjugated with the functionalized PEG-lipid.

58. The LNP of claim 56 or 57, wherein the LNP comprises a lipid composition, the lipid composition comprising: a) about 40 mol% to about 62 mol% of ionizable cationic lipids, about 7 mol% to about 13 mol% of phospholipids, about 30 mol% to about 50 mol% of sterols, about 0.5 mol% to about 3 mol% of total functionalized and nonfunctionalized PEG-lipids, and about 0.1 mol% to 0.3 mol% of functionalized PEG-lipids; b) Approximately 50 mol% of CLC, approximately 10 mol% of phospholipids, approximately 38.5 mol% of sterols, approximately 1.4 mol% of nonfunctionalized PEG-lipids, and approximately 0.1 mol% of functionalized PEG-lipids; c) Approximately 58 mol% CLC1, approximately 10 mol% phospholipids, approximately 30.5 mol% sterols, approximately 1.4 mol% nonfunctionalized PEG-lipids, and approximately 0.1 mol% functionalized PEG-lipids; or d) Approximately 62 mol% of CLCL, approximately 10 mol% of phospholipids, approximately 26.5 mol% of sterols, approximately 1.4 mol% of nonfunctionalized PEG-lipids and approximately 0.1 mol% of functionalized PEG-lipids.

59. The LNP according to claim 57 or claim 58, wherein R of the CICL is The phospholipid is distearylphosphatidylcholine (DSPC), the sterol is cholesterol, the nonfunctionalized PEG-lipid is 1,2-distearyl-glycerol-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000 (DSPE-PEG(2k)), and the functionalized PEG-lipid is DSPE-PE(2k)-maleimide (DSPE-PE(2k)-MAL).

60. The LNP according to any one of claims 57 to 59, wherein the isolated humanized monoclonal antibody or its antigen-binding fragment is covalently linked to a functionalized PEG-lipid via a modified lysine or cysteine ​​residue of the antibody or its binding fragment.

61. A lipid nanoparticle (LNP) comprising a separated antibody or antigen-binding fragment thereof conjugated to the LNP according to any one of claims 1 to 44.

62. A lipid nanoparticle (LNP) comprising an F(ab') analogue conjugated to the LNP according to any one of claims 45 to 55.

63. A lipid nanoparticle (LNP) conjugated with an F(ab') analogue containing repositioned interchain disulfide bonds.

64. A composition comprising an isolated antibody or an antigen-binding fragment thereof according to any one of claims 1 to 44, an F(ab') analogue according to any one of claims 45 to 55 or an LNP according to any one of claims 56 to 63, and a pharmaceutically acceptable carrier or excipient.

65. A method of delivering a payload to CD8-positive cells, comprising contacting the CD8-positive cells with an LNP according to any one of claims 56 to 63 or a composition according to claim 64.

66. The method of claim 65, wherein delivering the payload comprises transfecting the CD8-positive cells.

67. The method of claim 66, wherein the payload comprises mRNA, circular RNA, self-amplifying RNA, or guide RNA.

68. The method of claim 65, wherein the contact is performed in vivo, in vitro, or ex vivo.

69. The method of claim 65, wherein the payload mediates the reprogramming of the CD8 positive cells.

70. The method of claim 69, wherein the payload comprises a nucleic acid encoding an immune receptor or an immune cell connector.

71. The method of claim 69, wherein the payload comprises nucleic acid encoding gene / genome editing enzymes and / or other components of the guide RNA or gene / genome editing system.

Images