WO2025059589A1

WO2025059589A1 - Immune cell engaging molecules

Info

Publication number: WO2025059589A1
Application number: PCT/US2024/046790
Authority: WO
Inventors: Jason Price; James M. Olson; Bianka HARO; Raymond RUFF; Ian BLUMENTHAL
Original assignee: Seattle Childrens Hospital
Current assignee: Seattle Childrens Hospital
Priority date: 2023-09-13
Filing date: 2024-09-13
Publication date: 2025-03-20
Anticipated expiration: 2026-03-13

Abstract

Multi-specific immune cell engaging molecules are provided herein. These immune cell engaging molecules target and kill cancer antigen-positive and cancer antigen-negative cancer cells, use cancer supporters as therapeutic targets, and, in certain instances, overcomes checkpoint inhibition at a tumor site.

Description

IMMUNE CELL ENGAGING MOLECULES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63/582,480 filed September 13, 2023, which is incorporated herein by reference in its entirety as if fully set forth herein.

REFERENCE TO SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is provided in XML format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the file containing the Sequence Listing is 3AR7167.XML. The file is 226,736 bytes, was created on September 13, 2024, and is being submitted electronically via Patent Center.

FIELD OF THE DISCLOSURE

[0003] The present disclosure provides a multi-specific immune cell engaging molecule. These immune cell engaging molecules target and kill cancer antigen-positive and cancer antigennegative cancer cells, use cancer supporters as therapeutic targets, and, in certain instances, overcome checkpoint inhibition at a tumor site.

BACKGROUND OF THE DISCLOSURE

[0004] Despite advances in cancer treatments, mortality associated with the disease remains too high. For example, despite improvements in outcome for many pediatric patients, cancer remains the leading cause of death past infancy among children in the United States. Thus, the need for effective new therapies for cancers, including childhood cancers, is unquestioned.

[0005] Progress has been made in genetically engineering T cells of the immune system to target and kill unwanted cell types, such as cancer cells. For example, T cells have been genetically engineered to express molecules having extracellular components that bind particular target antigens (e.g., cancer markers) and intracellular components that direct actions of the T cell when the extracellular component has bound the target antigen. As an example, the extracellular component can be designed to bind target antigens found on cancer cells and, when bound, the intracellular component directs the T cell to destroy the bound cancer cell. Examples of such molecules include engineered T cell receptors (eTCR) and chimeric antigen receptors (CAR).

[0006] While TCR and/or CAR-modified T cells provide a major advantage in that they can create immune memory against cancer cells that can attack recurrent or progressive cancer cells as they emerge over time, this immune memory can lead to autoimmune toxicities when they recognize targets on normal tissue as abnormal or foreign.

[0007] Targeting cancer cells with antibodies raised high expectations as a potent means of eliminating tumor cells with limited non-specific toxicities. T lymphocytes have the ability to engage in close proximity to cancer cells and as such can induce anti-cancer T-cell mediated cytotoxicity. However, cancer cells have the ability to induce cancer-specific T-cell tolerance which significantly limits cancer-mediated immune responses.

[0008] Bispecific T-cell engagers (BTEs) and bispecific antibodies targeting both a cancerspecific antigen and T-cells have shown significantly greater anti-cancer activity compared to simple antibodies. The reason for this enhanced anti-cancer activity is mediated by the ability of the antibody to bind T-cells in proximity of the cancer and thus increase the cancer-specific T-cell mediated cytotoxicity.

[0009] The described cancer treatment methodologies fail to address the significant issues of tumor heterogeneity and mechanisms that cancer cells use to avoid detection or destruction by the immune system or to become resistant to different therapeutic treatments. Tumor heterogeneity refers to the fact that not all cancer cells within a tumor express the particularly targeted cancer marker. Mechanisms that cancer cells use to avoid detection or destruction by the immune system and/or use to become resistant to therapeutic treatments include endogenous immune checkpoint inhibition and the upregulation of drug efflux pumps. Immune checkpoint inhibition refers to a cancer cell’s ability to negatively modulate a patient’s immune system to avoid detection and destruction. For example, programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) checkpoints on T-cells negatively regulate immune function and prevent overreaction (i.e., promote immune system self-recognition). This mechanism is exploited by tumor cells to escape immune attacks. Immunotherapies such as PD- 1 inhibition (e.g., anti-PD1 antibodies) and CTLA-4 (e.g., CTLA-4 antibodies) can block checkpoint activity, thereby facilitating T-cell identification and destruction of cancer cells. Drug efflux pumps can be upregulated by cancer cells in response to chemotherapy, and are used by the cancer cells to pump chemotherapeutic drugs out of the cells, contributing to treatment resistance.

[0010] Thus, there remains a need in the art for more effective cancer therapies, especially for those with heterogeneous tumors and cancer cells, that employ mechanisms to avoid detection or destruction by the immune system and/or to become resistant to therapeutic treatments.

SUMMARY OF THE DISCLOSURE

[0011] The present disclosure provides cancer therapeutics based on immune cell engaging molecules (ICEm). The ICEm can: (i) target and kill cancer antigen-positive cancer cells; (ii) use molecules that cancer cells express to promote their survival as a therapeutic target; (iii) target what would have originally been antigen-negative cancer cells; and, in certain examples, (iv) overcome checkpoint inhibition at a tumor site.

[0012] In particular embodiments, the ICEm include a binding domain that binds a cancer marker, a binding domain that binds an immune cell activating epitope (ICAE), and a binding domain that binds a cancer supporter. In particular embodiments, the ICEm targets cancer cells by including at least two binding domains that bind a cancer marker (e.g., ROR1). This feature results in targeted delivery of the therapeutic to a cancer site. In particular embodiments, having one binding domain that binds an ICAE binds and activates immune cells for destruction of the cancer cell and avoids unintended off-target immune activation. The cancer supporter binding domain targets molecules that are upregulated by cancer cells in response to the immune system or therapeutics and often provide a mechanism for cancer cells to evade destruction by the immune system or therapeutic. Use of a cancer supporter binding domain on the ICEm uses the molecules that are upregulated on cancer cells to evade treatment as therapeutic targets. Therefore, the mechanism which cancer cells were using to evade treatment can be used to further target the cancer cells. For example, the cancer supporter can be PD-L1. Using PD-L1 in an ICEm turns a cancer supporter into a therapeutic target, and also serves to reduce or reverse immune checkpoint inhibition at the cancer site. In this instance, the more PD-L1 the cancer cell produces to try to avoid destruction by the immune system, the more therapeutic targets it presents. In certain examples, these benefits are provided by a single molecule.

[0013] Certain examples of ICEm utilize single domain binding domains. For example, single domain antibodies (also referred to as nanobodies) are small, naturally occurring single domain antigen binding proteins, possessing numerous properties advantageous to their production and use.

[0014] A particular example of an ICEm includes a multispecific T cell engager (MTE). An example of an MTE (e.g., MTE1) includes an aglycosylated Fc scaffold presenting two cancer marker binding domains (e.g., anti-ROR1 binding domains), an anti-CD3 binding domain, and an anti-PD-L1 binding domain. The MTE can crosslink cancer marker (e.g., ROR1) and PD-L1 positive cancer cells with CD3 positive T cells to direct the killing of the cancer cells. Additionally, the MTE can block the PD1/PD-L1 immune checkpoint signaling axis. Furthermore, the MTE can upregulate the PD-L1 target on nearby cancer antigen/PD-L1 negative cancer cells through initial engagement of the cancer marker and CD3. Without being bound by theory, this initial activity triggers T cell production of interferon-gamma (IFN-y) which in turn drives upregulation of PD-L1 on nearby cells. This mechanism allows for complete targeting of tumors/cancers with heterogeneous targeted cancer marker expression levels. This MTE thus achieves multiple objectives. It binds targeted cancer marker/PD-L1 positive tumors for elimination by endogenous T cells, blocks inhibitory immune checkpoint signaling, and locally converts target negative cells into target positive cells.

[0015] Additional benefits of the disclosed ICEm over many currently available therapies include that the disclosed ICEm can be provided as an “off-the-shelf therapy that can be administered universally to patients with a particular cancer without the need for personalized genetic therapies, such as CAR-modified T-cell therapies that can lead to autoimmune toxicities.

BRIEF DESCRIPTION OF THE FIGURES

[0016] Some of the drawings submitted herewith may be better understood in color. Applicant considers the color versions of the drawings as part of the original submission and reserves the right to present color images of the drawings in later proceedings.

[0017] FIG. 1. 3D cartoon depiction of an immune cell engaging molecule (ICEm) (left) and proof- of-concept design (right). Alphafold 3 generated model imaged with Blender software. The ICEm design includes a tumor-selective targeting arm (i.e. , cancer marker binding domain), immune cell engagement arm (i.e., immune cell activating epitope (ICAE) binding domain), and a checkpoint blockade and targeting arm (i.e., cancer supporter binding domain). The cancer marker binding domain(s) include binder(s) with specificity for tumor associated antigens, the ICAE binding domain includes binder(s) with specificity for immune activating targets, and the cancer supporter binding domain includes binders with specificity for tumor associated checkpoints that are upregulated by tumor cells in response to immune activity. The proof-of-concept design includes 2 anti-ROR1 variable heavy chain only antibodies (VHHs) for tumor-selective targeting, an anti- CD3 single chain variable fragment (scFv) for immune engagement, and an anti-PD-L1 miniprotein for checkpoint blockade and targeting. The various binding domains of the design are scaffolded by a human knob/hole I gG 1 Fc with effector silencing mutations (LALAPG).

[0018] FIG. 2. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) multi-specific T cell engagers (MTEs). Cartoon representation of exemplary ROR1 :PD-L1:CD3 targeting multispecific designs (MTE 1-5).

[0019] FIG. 3. K562 +ROR1-GFP overexpression cell line. T cell Killing Assay of exemplary multispecific design (MTE 1-5). K562 cells overexpressing full-length ROR1-eGFP fusion protein were incubated with T cells at a 5:1 E:T and 0.5 or 5ug/ml_ of indicated MTE. K562 cell viability was assessed at 96 hrs by flow cytometry. MTE 1 and 2 effectively direct the killing of K562 +ROR1 cells. MTE that lack CD3 targeting are unable to direct the killing of K562 +ROR1 cells alone (MTE 3 and 4), but may be paired with other MTE. K562 endogenously express low levels of PD-L1 and thus MTE 4, which lacks ROR1 targeting but retains PD-L1 targeting, can direct the killing of K562 cells but to a lesser degree than ROR1 containing designs.

[0020] FIG. 4. IFN-y dependent killing mechanism. Cartoon description of ROR1 Dependent tumor cell killing of endogenously ROR1 and PD-L1 null cells directed by MTE 1. MTE 1 can direct the killing of ROR1(+) tumor cells in monoculture which stimulates T cells to secrete IFN-y, MTE 1 does not direct the killing of T-47D cells which lack ROR1 and PD-L1 targets in monoculture (upper diagram). MTE 1 can direct the killing of endogenously ROR1 (-) PD-L1(-) cells when cocultured with ROR1 (+) positive cells through initial killing ROR1(+) cells driving IFN-y production which in turn induces PD-L1 expression on endogenous ROR1(-) PD-L1 (-) cells converting them to ROR1(-) PD-L1 (+) and rendering them susceptible to MTE 1 directed T cell killing (lower diagram).

[0021] FIG. 5. IFN-y inhibitor TCK assay. T cell killing assay of T-47D cells with 5ug/ml MTE 1 ± IFN-y inhibitor at varying E:T ratios. T cell killing data was captured every 4 hours via incucyte and demonstrates IFN-y dependent lysis of T-47D cells when incubated with MTE 1 at very high E:T (15:1).

[0022] FIG. 6. MTE 1 Production - Size exclusion chromatography (SEC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Production data of exemplary ROR1 :PD-L1:CD3 targeting multi-specific design 1 by size exclusion chromatography (top) and SDS PAGE (bottom). Protein demonstrates uniform dispersity and expected retention volume by size exclusion. SDS PAGE analysis shows a single band at the anticipated migration size that forms two expected bands upon reduction.

[0023] FIGs. 7A, 7B. ICEm mechanism. 7A) Cartoon depiction of immune cell (e.g., T cell) redirected killing of a heterogenous tumor with a traditional ROR1:CD3 bispecific vs ROR1 :PDL1 :CD3 MTE. 7B) Cartoon depiction of IFN-y dependent MTE mechanism. Tumor cells universally upregulate immune checkpoints including PD-L1 in response to interferon gamma (IFN-y) exposure as a mechanism to evade native immune surveillance and clearance. The MTE design takes advantage of this defense mechanism by inclusion of a cancer supporter binding domain. Cancer marker and ICAE binding domains direct initial killing of tumor cells and cause release of IFN-y in the local tumor microenvironment. This initial activity seeds PD-L1 expression on nearby tumor cells and makes them secondary targets of the MTE through engagement with the aPD-L1 binding domain of the MTE. Traditional bispecific T cell engagers (BTEs) including cancer marker and ICAE binding domains, such as the depicted ROR1:CD3 BTE, are ineffective against tumors with heterogenous expression of the cognate tumor associated marker. [0024] FIGs. 8A-8E. ZR-75 T cell killing (TCK) assay. ZR-75-1 cell line was obtained from ATCC (CRL-1500) and transduced with an iRFP or ROR1-GFP expressing lentivirus using the Daedalus expression platform (PMID: 21911364) to generate the ZR-75 and ZR-75^+ROR1 cell lines respectively. Cryopreserved, healthy human donor PBMCs were obtained from Bloodworks NW, CD3⁺ T cells were isolated with a magnetic negative selection kit (Stemcell Technologies #17951) and activated with CD3/CD28 Dynabeads (ThermoFisher #11131 D) according to manufactures protocol. Activated T cells (ATCs) were cryopreserved day 10 post initiation of activation/expansion protocol. T cell killing (TCK) assays were carried out by incubating ZR-75 or ZR-75^+ROR1 target cells as a mono or mixed co-culture with ATCs at a 5:1 Effector(E):Target(T) ratio in a 96 well plate. 0.2ug/mL ROR1:CD3, PDL1 :CD3 BTEs or ROR1 :PDL1 :CD3 MTE were added as indicated at Time = 0. Experimental wells were recorded over time with an Incucyte instrument (Sartorius) and tumor cell count over time was calculated with Incucyte 2022B Rev2 software. Tumor cell fold expansion was normalized by experimental well and “% TCK activity” was calculated with following equation: [fold tumor expansion(no drug) - fold tumor expansion(w/drug)] / fold tumor expansion(no drug). Calculated values were averaged across experimental replicates and plotted vs time with standard error indicated. 8A) ZR-75 mono-culture (left), ZR-75^+ROR1 mono-culture (center), and ZR-75(10% ZR-75^+ROR1) co-culture (right). ZR-75-1 is a breast cancer cell line that at baseline expresses undetectable levels of ROR1 and PD-L1. ZR-75-1 cells upregulate PD-L1 expression on cell surface after exposure to IFN-y. ZR-75^+ROR1 were engineered to overexpress ROR1. Traditional ROR1 :CD3 (MTE2; SEQ ID NOs: 130 and 132) and PDL1 :CD3 (MTE4; SEQ ID NOs: 147 and 148) BTEs and ROR1 :PDL1:CD3 MTE (MTE1 ; SEQ ID NOs: 130 and 128) are unable to target and eliminate ZR-75 cells grown in monoculture in an in vitro TCK assay (8A, left). ROR1 :CD3 BTE and ROR1 :PDL1 :CD3 MTE are able to target and eliminate ZR-75^+ROR1 cells grown in mono-culture (8A, center). Only the ROR1 :PDL1 :CD3 MTE can target and eliminate ZR-75 cells when grown in co-culture with 10% ZR-75^+ROR1 cells (8A, right). 8B) ZR-75 normalized tumor cell growth ± PDL1 :CD3 BTE in ZR- 75/ZR-75^+ROR1 co-culture TCK assay (left), ZR-75 normalized tumor cell growth ± ROR1 :CD3 BTE in ZR-75/ZR-75^+ROR1 co-culture TCK assay (center), and ZR-75 normalized tumor cell growth ± ROR1 :PDL1 :CD3 MTE in ZR-75/ZR-75^+ROR1 co-culture TCK assay (right). Traditional PDL1 :CD3 and ROR1 :CD3 BTEs fail to target and eliminate ROR1 and PD-L1 target negative at baseline ZR-75 cell growth in co-culture assay with 10% ZR-75^+ROR1 cells indicated by similar ZR-75 fold change cell density ± BTE in the assay. ROR1 :PDL1 :CD3 MTE is able to target and eliminate ROR1 and PD-L1 target negative at baseline ZR-75 cell growth in co-culture assay with 10% ZR- 75^+RORI cells indicated by decrease in ZR-75 fold change cell density ± MTE in the assay. 8C) ZR-75 co-culture T cell killing (TCK) assay with varied ratios of ZR-75:ZR-75^+ROR1. As little as 5% ZR-75^+ROR1 cells present in the co-culture potentiates MTE-mediated killing of ZR-75 cells that are ROR1/PD-L1 negative at baseline. Increasing percent of ZR-75^+ROR1 decreases the lag time before ZR-75 cell targeting and elimination. 8D) ZR-75 cell % TCK activity over time in ZR-75/ZR- 75+^RO^RI (I QO/_O) co-culture TCK assay. 8E) ZR-75^+ROR1 cell % TCK activity over time in ZR-75/ZR- 75^+ROR1 (10%) co-culture TCK assay. 0.2ug/mL ROR1 :PDL1 :CD3 MTE was added ± 20ug/mL IFN-y inhibitor (Biolegend # 506531). as indicated at Time = 0. ROR1 :PD-L1 :CD3 MTE-mediated killing of ZR-75 cells in a ZR-75/ZR-75^+ROR1(10%) co-culture TCK assay is inhibited in the presence of IFN-y inhibitor. ROR1 :PD-L1 :CD3 MTE-mediated killing of ZR-75^+ROR1 cells in a ZR- 75/ZR-75^+ROR1 (10%) co-culture TCK assay is weakly inhibited in the presence of IFN-y inhibitor. This result supports the proposed IFN-y dependent mechanism.

[0025] FIGs. 9A-9C. MDA-MB-231/T47D TCK assay. 9A) ROR1/PD-L1 expression levels at baseline and after IFN-y exposure (top). Fluorescence microscopy if iRFP labeled MDA-MB-231 and GFP labeled T47D cells in mono and co-culture (bottom). 9B) Mono-culture MDA-MB-231 and T47D TCK assay with ROR1 :PDL1 :CD3 MTE. 9C) Co-culture MDA-MB-231 and T47D TCK assay with ROR1 :PDL1 :CD3 MTE. MDA-MB-231 (ATCC, HTB-26) and T47D (ATCC, HTB-133) breast cancer cell lines were obtained from ATCC and labeled with iRFP and GFP expressing lentivirus respectively using the Daedalus expression platform (PMID: 21911364). Cryopreserved, healthy human donor PBMCs were obtained from Bloodworks NW, CD3⁺ T cells were isolated with a magnetic negative selection kit (Stemcell Technologies #17951) and activated with CD3/CD28 Dynabeads (ThermoFisher #11131 D) according to manufactures protocol. Activated T cells (ATCs) were cryopreserved day 10 post initiation of activation/expansion protocol. T cell killing (TCK) assays were carried out by incubating MDA- MB-231 or T47D target cells as a mono or mixed co-culture with ATCs at a 5:1 Effector(E):Target(T) ratio in a 96 well plate. 2ug/ml_ ROR1 :PDL1 :CD3 MTE was added as indicated at Time = 0. Experimental wells were recorded over time with an Incucyte instrument (Sartorius) and tumor cell count over time was calculated with Incucyte 2022B Rev2 software. Tumor cell fold expansion was calculated as described above. ROR1 :PDL1 :CD3 MTE targets and eliminates native ROR1/PD-L1 positive MDA-MB-231 cells but not native ROR1/PD-L1 negative T47D cells in mono-culture TCK assay. In co-culture assay ROR1 :PDL1:CD3 MTE targets and eliminates MDA-MB-231 cells followed by elimination of T47D cells consistent with the proposed IFN-y dependent mechanism.

[0026] FIG. 10. PC3 preclinical prostate cancer model. Average tumor volume vs days on study ± ROR1 :PD-L1 :CD3 MTE (MTE6). Male and female NSG mice were subcutaneously implanted with 5 x 10⁶ PC3 prostate cancer cells (ATCC, CRL-1435) in the right flank. When tumors reached 100-200 mm³ (30 days post-implantation), mice were randomly assigned to two treatment groups (n = 7 per group): vehicle control and ROR1 :PDL1 :CD3 MTE (MTE6). Treatments were administered intravenously twice weekly (1 nmol MTE or 200 L vehicle), along with weekly infusions of 7.5 x 10⁶ ATCs. Tumor volumes and body weights were measured three times a week. Mice were euthanized when tumors reached 1500 mm³, body weight loss exceeded 20%. ROR1 :PDL1 :CD3 MTE targets and eliminates PC3 tumors in a preclinical prostate cancer model. [0027] FIG. 11 . Alternative IFN-y upregulated targets on tumor cells. Cartoon depiction of an MTE highlighting the cancer supporter binding domain. Selection methodology and list of alternative targets identified (right).

[0028] FIG. 12. Alternative tumor selective targets. Cartoon depiction of MTE highlighting (Circling) the cancer marker binding domain (left). List and pairs of example alternative targets identified (right).

[0029] FIG. 13. Table showing example list of genes used to select the cancer supporter. The list starts with the reduced set of genes with properties including: expression that is altered by exposure to IFNy, expressed on the cell surface, and demonstrates expression beyond hematopoietic lineages. This list of genes is further reduced in the middle column to include those expressed on solid tumors. This list of genes is further reduced in the right column to include those genes that the literature suggests may provide selective benefit to a tumor.

[0030] FIG. 14. Description of methods used for identification of cancer supporter therapeutic targets. RNA sequencing identified set of mRNAs differentially upregulated after IFN-y exposure in DMG tumor cells and healthy PMBCs. Candidate genes were selected with the following criteria, i) upregulated in DMG cells but not PBMCs and ii) annotated as surface expressed protein. Diffuse midline glioma (DMG) primary tumor cells (PBT29-FH) and healthy PBMCs were treated with IFN-y or control for 48hrs. RNA was isolated with Trizol following the manufacturers' protocol and cleaned up with Qiagen RNeasy Kit. Triplicate samples for each condition were submitted for Illumina 150bp paired-end mRNA sequencing. Genes differentially upregulated in the IFN-y treated samples were identified with DESeq2 software (Love et al., Genome Biol. 2014, 15(12):550). Genes upregulated in DMG cells but not PBMCs and annotated to be expressed on the cell surface were selected as candidate targets.

[0031] FIG. 15. Description of general methods used for identification of alternative cancer supporter therapeutic targets that are applicable to any tumor type. Genomic, proteomic, and animal immunization followed by mAb discovery/isolation can be used to identify candidate targets following the above scheme. Tumor cells and normal tissue can be derived from in vitro cell lines or in vivo tissue. In some cases, IFN-y treatment can be replaced with other immune signals released during an active immune response such as TNFa, IL-2, IL-7, IL-15. Additionally, IFN-y treatment can be replaced with supernatant from or co-culture of activated T cells.

[0032] FIG. 16. Representative gene list according to selection methods disclosed herein.

[0033] FIG. 17. Sequences supporting the disclosure including MTE1 (SEQ ID NOs: 130 and 128) and its coding sequences (SEQ ID NOs: 219 and 220); MTE2 (SEQ ID NOs: 130 and 132) and its coding sequences (SEQ ID NOs: 221 and 222); MTE4 (SEQ ID NOs: 147 and 148) and its coding sequences (SEQ ID NOs: 223 and 224); and MTE6 (SEQ ID NOs: 149 and 150) and its coding sequences (SEQ ID NOs: 225 and 226).

DETAILED DESCRIPTION

[0034] Despite advances in cancer treatments, mortality associated with the disease remains too high. For example, despite improvements in outcome for many pediatric patients, cancer remains the leading cause of death past infancy among children in the United States. Thus, the need for effective new therapies for cancers, including childhood cancers, is unquestioned.

[0035] Progress has been made in genetically engineering T cells of the immune system to target and kill unwanted cell types, such as cancer cells. For example, T cells have been genetically engineered to express molecules having extracellular components that bind particular target antigens (e.g., cancer markers) and intracellular components that direct actions of the T cell when the extracellular component has bound the target antigen. As an example, the extracellular component can be designed to bind target antigens found on cancer cells and, when bound, the intracellular component directs the T cell to destroy the bound cancer cell. Examples of such molecules include engineered T cell receptors (eTCR) and chimeric antigen receptors (CAR).

[0036] While TCR and/or CAR-modified T cells provide a major advantage in that they can create immune memory against cancer cells that can attack recurrent or progressive cancer cells as they emerge over time, this immune memory can lead to autoimmune toxicities when they recognize targets on normal tissue as abnormal or foreign.

[0037] Targeting cancer cells with antibodies raised high expectations as a potent means of eliminating tumor cells with limited non-specific toxicities. T lymphocytes have the ability to engage in close proximity to cancer cells and as such can induce anti-cancer T-cell mediated cytotoxicity. However, cancer cells have the ability to induce cancer-specific T-cell tolerance which significantly limits cancer-mediated immune responses.

[0038] Natural antibodies have been engineered into various formats in attempts to increase their therapeutic efficacy. For example, bispecific T-cell engaging antibodies bind both a targeted cancer antigen on cancer cells and a T cell activating epitope (e.g., CD3), with the goal of bringing T cells to cancer cells to destroy the cancer cells. See, for example, US 2008/0145362. Some have explored use of such multi-specific antibodies in combinations that bind two different T cell activating epitopes (e.g., CD3 and CD28) or to bind two different targeted cancer antigens (e.g., PD-L1 and ROR1) and two different ? cell activating epitopes (e.g., CD3 and 4-1 BB).

[0039] The described cancer treatment methodologies fail to address the significant issues of tumor heterogeneity and mechanisms that cancer cells use to avoid detection or destruction by the immune system or to become resistant to different therapeutic treatments. Tumor heterogeneity refers to the fact that not all cancer cells within a tumor express the particularly targeted cancer marker. Mechanisms that cancer cells use to avoid detection or destruction by the immune system and/or use to become resistant to therapeutic treatments include endogenous immune checkpoint inhibition and the upregulation of drug efflux pumps. Immune checkpoint inhibition refers to a cancer cell’s ability to negatively modulate a patient’s immune system to avoid detection and destruction. For example, programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) checkpoints on T-cells negatively regulate immune function and prevent overreaction (i.e., promote immune system self-recognition). This mechanism is exploited by tumor cells to escape immune attacks. Immunotherapies such as PD- 1 inhibition (e.g., anti-PD1 antibodies) and CTLA-4 (e.g., CTLA-4 antibodies) can block checkpoint activity, thereby facilitating T-cell identification and destruction of cancer cells. Drug efflux pumps can be upregulated by cancer cells in response to chemotherapy, and are used by the cancer cells to pump chemotherapeutic drugs out of the cells, contributing to treatment resistance.

[0040] The present disclosure provides cancer therapeutics based on immune cell engaging molecules (ICEm). The ICEm can: (i) target and kill cancer antigen-positive cancer cells; (ii) use molecules that cancer cells express to promote their survival as a therapeutic target; (iii) target what would have originally been antigen-negative cancer cells; and, in certain examples, (iv) overcome checkpoint inhibition at a tumor site.

[0041] In particular embodiments, an ICEm includes a cancer marker binding domain, an immune cell activating epitope (ICAE) binding domain, and a cancer supporter binding domain. In particular embodiments, the cancer marker binding domain includes a binding domain that binds receptor tyrosine kinase like orphan receptor 1 (ROR1), prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), mesothelin, CD19, CD20, CD33, Wilms tumor protein (WT1), CD123, CD22, CD133, B-cell maturation antigen (BCMA), human epidermal growth factor receptor 2 (HER2), carbonic Anhydrase IX (CAIX), carcinoembryonic antigen (CEA), or GD2. In particular embodiments, the cancer marker binding domain includes a binding domain that binds R0R1. In particular embodiments, the ICEm includes 2 cancer marker binding domains. In particular embodiments, the 2 cancer marker binding domains bind different cancer markers. In particular embodiments, the 2 cancer marker binding domains bind the same cancer marker. In particular embodiments, the 2 cancer marker binding domains bind ROR1. In particular embodiments, the ICAE binding domain binds CD3. In particular embodiments, the cancer supporter binding domain binds a molecule that is upregulated in cancer and supports its evasion of the immune system and/or treatment. In particular embodiments, a cancer supporter is selected to include the following criteria: induced by IFNy, expressed on the cell surface, expressed beyond hematopoietic lineages, expressed on tumors, and provides selective advantage. In particular embodiments, the cancer supporter includes PD-L1 .

[0042] In particular embodiments, the ICEm includes a first polypeptide chain and a second polypeptide chain; wherein the first polypeptide chain includes a first cancer marker binding domain, a first multimerization domain, and a cancer supporter binding domain; and wherein the second polypeptide chain includes a second cancer marker binding domain, a second multimerization domain, and an ICAE binding domain. In particular embodiments, the ICEm includes a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain includes a first cancer marker binding domain, an lgG1 Fc knob, and a cancer supporter binding domain. In particular embodiments, the first polypeptide chain includes linkers, a signal peptide, and/or a tag. In particular embodiments, the second polypeptide chain includes a cancer marker binding domain, an IgG 1 Fc hole, and an ICAE binding domain. In particular embodiments, the second polypeptide chain includes linkers, a signal peptide, and/or a tag. In particular embodiments, an ICEm includes Fc silencing mutations. In particular embodiments, the Fc silencing mutations include an aglycosylated Fc domain or LALAPG mutations.

[0043] In particular embodiments the first polypeptide chain includes an anti-ROR1 binding domain, a linker, an lgG1 Fc knob, a linker, and an anti-PD-L1 binding domain. In particular embodiments the first polypeptide chain further includes a linker, a signal peptide, and/or a tag. In particular embodiments, the second polypeptide chain includes an anti-ROR1 binding domain, a linker, an lgG1 Fc hole, a linker, and an anti-CD3 binding domain. In particular embodiments the second polypeptide chain further includes a linker, a signal peptide, and/or a tag. In particular embodiments the first polypeptide chain includes the sequence as set forth in SEQ ID NO: 127 or SEQ ID NO: 128. In particular embodiments, the second polypeptide chain includes the sequence as set forth in SEQ ID NO: 129 or SEQ ID NO: 130. In particular embodiments, the first polypeptide chain includes the sequence as set forth in SEQ ID NO: 149 and the second polypeptide chain includes the sequence as set forth in SEQ ID NO: 150. In particular embodiments, the first polypeptide chain is encoded by the sequence as set forth in SEQ ID NO: 219 and the second polypeptide chain is encoded by the sequence as set forth in SEQ ID NO: 220. In particular embodiments, the first polypeptide chain is encoded by the sequence as set forth in SEQ ID NO: 225 and the second polypeptide chain is encoded by the sequence as set forth in SEQ ID NO: 226. In particular embodiments, an ICEm includes Fc silencing mutations.

[0044] In particular embodiments, the cancer marker binding domain results in targeted delivery of the therapeutic to a cancer site. In particular embodiments, the ICEm includes only one binding domain that binds an ICAE, thereby avoiding unintended off-target immune activation. Finally, the cancer supporter binding domain makes a protein that cancer cells upregulate or select for to promote their survival into a therapeutic target and also reduces or reverses immune checkpoint inhibition at the cancer site. In certain examples, these benefits are provided by a single molecule. In other embodiments, these benefits can be provided by a combination of molecules.

[0045] Certain examples of the ICEm utilize single domain antibodies as binding domains. Single domain antibodies (also referred to as nanobodies) are small naturally occurring single domain antigen binding proteins, possessing numerous properties advantageous to their production and use. For example, nanobodies are generally highly soluble, stable, lack glycans and are readily cloned and expressed in bacteria (Muyldermans, 2013, Annual review of biochemistry 82, 775- 797). They have low immunogenicity (Bannas et al., 2017, Frontiers in immunology 8, 1603; Jovcevska and Muyldermans, 2020, BioDrugs 34, 11-26; Revets et al., 2005, Expert Opin Biol Ther 5, 111-124.) and can be readily modified to be “humanized” (including Fc addition), to change clearance rates, to add cargos such as drugs or fluorophores or to combine and improve characteristics by multimerization (Chanier and Chames, 2019, Antibodies (Basel) 8; Duggan, 2018, Drugs 78, 1639-1642; Vincke et al., 2009, J Biol Chem 284, 3273-3284).

[0046] A particular example of an ICEm includes a multi-specific T cell engager (MTE). An example of an MTE (e.g., MTE1) includes an aglycosylated Fc scaffold presenting two cancer marker binding domains (e.g., anti-ROR1 binding domains), an anti-CD3 binding domain, and an anti-PD-L1 binding domain. The MTE can crosslink cancer marker (e.g., ROR1) and PD-L1 positive cancer cells with CD3 positive T cells to direct the killing of the cancer cells. Additionally, the MTE can block the PD1/PD-L1 immune checkpoint signaling axis. Furthermore, the MTE can upregulate the PD-L1 target on nearby cancer antigen/PD-L1 negative cancer cells through initial engagement of the cancer marker and CD3. Without being bound by theory, this initial activity triggers T cell production of interferon-gamma (IFN-y) which in turn drives upregulation of PD-L1 on nearby cells. This mechanism allows for complete targeting of tumors/cancers with heterogeneous targeted cancer marker expression levels. This MTE thus achieves multiple objectives. It binds targeted cancer marker/PD-L1 positive tumors for elimination by endogenous T cells, blocks inhibitory immune checkpoint signaling, and locally converts target negative cells into target positive cells.

[0047] The present disclosure also provides design considerations for preparing an ICEm. The design considerations include selecting a cancer marker binding domain that binds a cancer marker expressed by a tumor within the subject; selecting an ICAE binding domain that binds and activates an immune cell; and selecting a cancer supporter binding domain that binds a cancer supporter. In particular embodiments, the cancer supporter is selected from a list of molecules winnowed using a method to select a cancer supporter. The method to select a cancer supporter includes limiting a list of molecules to those altered by exposure to a molecule released in response to immune cell activation (e.g., IFNy) or therapeutics; limiting the list of molecules to those expressed on the cell surface; limiting the list of molecules to those expressed outside the hematopoietic lineage; limiting the list of molecules to those expressed on tumors or tumor cells; limiting the list of molecules to those that provide a selective benefit to the tumor or tumor cell. The design considerations will include considerations for the number of binding domains to include for each of the cancer marker binding domain, ICAE binding domain, and cancer supporter binding domain. The design consideration will include considerations for the relative placement of each binding domain, Fc mutations, multimerization domains, tags, signal peptides, and linkers.

[0048] Although having all the binding domains on a single molecule has benefits over a combination treatment, an example of a combination treatment includes a first ICEm including a cancer marker binding domain and an ICAE binding domain and a second ICEm including a ICAE binding domain and a cancer support binding domain. As an example, FIG. 2 shows various MTE. A combination treatment could include MTE2 and MTE4.

[0049] Additional benefits of the disclosed ICEm over many currently available therapies include that the disclosed ICEm can be provided as an “off-the-shelf’ therapy that can be administered universally to patients with a particular cancer without the need for personalized genetic therapies, such as CAR-modified T-cell therapies that can lead to autoimmune toxicities.

[0050] Aspects of the disclosure are now described in additional detail and with additional options to practice the disclosure as follows: (I) Binding Domains; (II) Cancer Markers and Binding Domains Thereof; (III) Immune Cell Activating Epitopes and Binding Domains Thereof; (IV) Cancer Supporter and Binding Domains Thereof; (V) Multimerization Domains and Linkers; (VI) Variants & Nucleic Acid Sequences; (VII) Expression of Immune Cell Engaging Molecules; (VIII) Modifications to Provide Administration Benefits; (IX) Compositions for Administration; (X) Kits; (XI) Methods of Use; (XII) Examples and Exemplary Embodiments; and (XIII) Closing Paragraphs. These headings are provided for organizational purposes only and do not limit the scope or interpretation of the disclosure.

[0051] (I) Binding Domains. In certain examples, an ICEm includes three or more binding domains, wherein each binding domain binds a target antigen. Binding domains include any substance that binds to a cellular marker to form a complex. The choice of binding domain can depend upon the type and number of cellular markers that define the surface of a target cell. In particular embodiments, a binding domain includes any molecule that can bind a marker or antigen. In particular embodiments, the binding domain includes a protein. In particular embodiments, the protein includes an antibody or a peptide (e.g., miniprotein). In particular embodiments, the binding domains includes antibodies, peptides (e.g., miniproteins, peptide aptamers), receptor ligands, receptors (e.g., T cell receptors), nucleic acid aptamers, or fragments thereof.

[0052] As used herein, the term "antibody" refers to a monomeric or multimeric protein comprising one or more polypeptide chains that comprise antigen-binding sites. An antibody binds specifically to an antigen and may be able to modulate the biological activity of the antigen. As used herein, the term "antibody" can include "full length antibody" and "antibody fragments." The terms "binding site" or "antigen-binding site" as used herein denotes the region(s) of an antibody molecule to which a ligand actually binds. The term "antigen-binding site" includes an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL), or in the case of heavy chain only antibodies, an antibody heavy chain variable region.

[0053] As is understood by those of ordinary skill in the art, a conventional antibody includes two heavy chains and two light chains. Each heavy chain includes a variable region and a first, second, and third constant region, while each light chain includes a variable region and a constant region. Mammalian heavy chains are classified as a, 6, E, y, and p, and mammalian light chains are classified as A or K. Immunoglobulins including the a, 6, E, y, and p heavy chains are classified as immunoglobulin (lg)A, IgD, IgE, IgG, and IgM. The complete antibody forms a “Y” shape. The stem of the Y includes the second and third constant regions (and for IgE and IgM, the fourth constant region) of two heavy chains bound together and disulfide bonds (inter-chain) are formed in the hinge. Heavy chains y, a and b have a constant region composed of three tandem (in a line) Ig domains, and a hinge region for added flexibility; heavy chains p and E have a constant region composed of four immunoglobulin domains. The second and third constant regions are referred to as “CH2 domain” and “CH3 domain”, respectively. Each arm of the Y includes the variable region and first constant region of a single heavy chain bound to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding.

[0054] Light and heavy chain variable regions contain a “framework” region interrupted by three hypervariable regions, also called “complementarity determining regions” or “CDRs”.

[0055] CDR sets can be based on, for example, Kabat numbering (Kabat et al. (1991) “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme)); Chothia (Al-Lazikani et al. (1997) JMB 273:927-948 (“Chothia” numbering scheme)); Martin (Abinandan et al. (2008) Mol Immunol. 45:3832-3839 (“Martin” numbering scheme)); Gelfand (Gelfand and Kister (1995) Proc Natl Acad Sci USA. 92:10884-10888; Gelfand et al. (1998) Protein Eng. 11 :1015-1025; Gelfand et al. (1996) Proc Natl Acad Sci USA. 93:3675-3678; Gelfand et al. (1998) J Comput Biol. 5:467- 477 (“Gelfand” numbering scheme)); Contact (MacCallum et al. (1996) J. Mol. Biol. 262:732-745 (Contact numbering scheme)); IMGT (Lefranc et al. (2003) Dev Comp Immunol 27(1):55-77 (“IMGT” numbering scheme)); AHo (Honegger and Pluckthun (2001) J Mol Biol 309(3):657-670 (“AHo” numbering scheme)); North (North et al. (2011) J Mol Biol. 406(2) :228-256 (“North” numbering scheme)); or other numbering schemes. Software programs and bioinformatical tools, such as ABodyBuilder (Leem et al. (2016) MAbs 8(7): 1259-1268), PIGSPro (Lepore et al. (2017) Nucleic Acids Res 45(W1):W17-W23), Kotai Antibody Builder (Yamashita et al. (2014) Bioinformatics 30(22): 3279-3280), Rosetta Antibody (Weitzner et al. (2017) Nature Protocols 12:401-416), Paratome (Kunik et al. (2012) Nucleic Acids Res 40:W521-W524), Antibody i-Patch (Krawczyk et al. (2013) Protein Eng Des Sei 26(10):621-629), and proABC-2 (Ambrosetti et al. (2020) Bioinformatics 36(20):5107-5108 can also be used to determine CDR sequences.

[0056] The sequences of the framework regions of different light or heavy chains are relatively conserved within a species, such as humans. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space. The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1 , CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, the CDRs located in the variable domain of the heavy chain of the antibody are referred to as CDRH1, CDRH2, and CDRH3, whereas the CDRs located in the variable domain of the light chain of the antibody are referred to as CDRL1 , CDRL2, and CDRL3. Antibodies with different specificities (i.e., different combining sites for different antigens) have different CDRs. Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs).

[0057] References to “V_H” or “VH” refer to the variable region of an immunoglobulin heavy chain. References to “V_L” or “VL” refer to the variable region of an immunoglobulin light chain.

[0058] Antibodies that specifically bind an antigen can be prepared using methods of obtaining monoclonal antibodies, methods of phage display, methods to generate human or humanized antibodies, or methods using a transgenic animal or plant engineered to produce human antibodies. Phage display libraries of partially or fully synthetic antibodies are available and can be screened for an antibody or fragment thereof that can bind to the target antigen. Phage display libraries of human antibodies are also available. Once identified, the amino acid sequence or polynucleotide sequence coding for the antibody can be isolated and/or determined. Many relevant antibodies are also publicly known and commercially available.

[0059] In particular embodiments, antibodies specifically bind to a surface molecule on a cell (e.g., cancer cell or immune cell) and do not cross react with nonspecific components such as bovine serum albumin or other unrelated antigens.

[0060] “Antibody fragment” refers to at least one portion of an antibody that retains the ability to specifically bind an antigen. Examples of antibody fragments include Fab, Fab', F(ab')2, Fv fragments, single chain variable (scFv) antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment including VH and constant CH1 domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid variable heavy only (VHH) domains, multi-specific antibodies formed from antibody fragments such as a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody (Harlow et al. , 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879- 5883; Bird et al., 1988, Science 242:423-426). An antigen binding fragment can also be incorporated into a single domain antibody, maxibody, minibody, nanobody, intrabody, diabody, triabody, tetrabody, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson (2005) Nature Biotechnology 23:1126-1136).

[0061] “scFv” refers to an engineered fusion protein including the VH and VL of an antibody linked via a linker and capable of being expressed as a single chain polypeptide. The scFv retains the specificity of the intact antibody from which it is derived. In particular embodiments, a linker connecting the variable regions can include glycine-serine linkers, including, for example, those shown as SEQ ID NOs: 103-116 or described elsewhere herein. In particular embodiments, an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may include VL-linker-VH or may include VH- linker-VL.

[0062] "Single domain antibody (sdAb)", or also referred to as "VHH antibody", refers to an antibody molecule having an antigen binding ability, including a heavy chain variable region without a light chain. From a structural point of view, a single domain antibody can also be considered an antigen-binding fragment of an antibody molecule. In various embodiments, the antibody includes a VHH. Camelids (camels, dromedary, and llamas) contain, in addition to conventional heavy and light chain antibodies, two-chain antibodies (containing only variant heavy chains). The dimeric antibodies are coded for by a distinct set of VH segments referred to as VHH genes. The VH and VHH are interspersed in the genome (i.e., they appear mixed in between each other). The identification of an identical D segment in a VH and VHH cDNA suggests the common use of the D segment for VH and VHH. Natural VHH-containing antibodies are missing the entire CH1 domain of the constant region of the heavy chain. The exon coding for the CH1 domain is present in the genome but is spliced out due to the loss of a functional splice acceptor sequence at the 5' side of the CH1 exon. As a result the VDJ region is spliced onto the CH2 exon. When a VHH is recombined onto such constant regions (CH2, CH3), an antibody is produced in which the half-antibody is a single chain instead of a light chain/heavy chain pair (i.e., an antibody of two heavy chains without a light chain interaction). Binding of an antigen is different from that seen with a conventional antibody, but high affinity is achieved the same way, i.e., through hypermutation of the variable region and selection of the cells expressing such high affinity antibodies. In particular embodiments, the sdAb is derived from a camelid, shark, or a cow. In particular embodiments, the sdAb is humanized. In particular embodiments, the sdAb is recombinantly produced.

[0063] In particular embodiments, VHH are produced by immunizing a transgenic mouse in which endogenous murine antibody expression has been eliminated and camelid transgenes have been introduced. VHH mice are disclosed in US8,883,150, US8,921 ,524, US8.921.522, US8,507,748, US8,502,014, US 2014/0356908, US2014/0033335, US2014/0037616, US2014/0356908, US2013/0344057, US2013/0323235, US2011/01 18444, and US2009/0307787. The VHH mice are immunized and the resulting primed spleen cells fused with a murine myeloma cells to form hybridomas. In other embodiments, VHH are produced by immunizing llamas with a desired antigen, and isolating sequences encoding the VHH regions of resulting antigen-binding antibodies. In alternative embodiments, VHH are isolated using a phage display library. See, for example, WO 91/17271 ; WO 92/01047; and WO 92/06204. [0064] The Immunoglobulin New Antigen Receptors (IgNARs) are an unconventional subset of antibodies identified in fish. In domain structure, IgNAR proteins are reportedly similar to other immune effector molecules, being disulphide-bonded homodimers of two polypeptide chains having five constant domains (CNARs) and one variable domain (VNAR) (Greenberg 1995). However, unlike conventional antibodies, there are no associated light chains and the individual variable domains are independent in solution and do not appear to associate across a hydrophobic interface (as seen for conventional VH/VL type antibodies) (Roux 1998).

[0065] IgNARs have minimally variable loop regions analogous to conventional CDR1 and CDR2 loops, with diversity being concentrated in an elongated loop region analogous to a conventional CDR3 loop (Greenberg 1995; Nuttall 2001; Diaz 2002). The elongated loop region can reportedly vary in length from 5 to 23 residues in length, though the modal classes are more in the order of 15 to 17 residues (Nuttall 2003). This is significantly larger than for conventional murine and human antibodies, but approximate to the extended CDR3 loops found in camelid single VH antibodies (Wu 1993; Muyldermans 1994).

[0066] Cattle possess exceptional antibodies with ultra-long complementarity-determining regions (ulCDRs) that can include 40-70 amino acids. The bovine ulCDR is folded into a stalk and a disulfide-rich knob domain. The binding to the antigen is via the 3-6 kDa knob. There exists an immense sequence and structural diversity in the knob that enables binding to different antigens. Isolated bovine knobs, also referred to as picobodies are small antigen-binding domains derived from a conventional antibody. In particular embodiments, binding domains disclosed herein include picobodies.

[0067] In addition to antibodies, binding domains disclosed herein can include peptides. A peptide is a compound including two or more amino acids linked in a chain and can include miniproteins or peptide aptamers.

[0068] Miniproteins refer to a diverse group of proteins characterized by small (1-10 kDa) size, stability, and versatility in drug-like roles. Miniproteins may retain the potency and specificity advantages of antibodies while avoiding some of their liabilities. Miniproteins may be cysteine reinforced and include Avimers, Kunits, and cysteine-dense peptide (CDPs). Miniproteins may have a hydrophobic core and include Affibodies, Adnectin + Centyrins, Nanofittins + Affitins, and Fynomers. Miniproteins may be chemically stabilized and include p-hairpins, stapled peptides, and bicycles. For additional information about the characterization of miniproteins, see e g., Crook et al., Trends Biochem Sci. 45(4): 332-346, 2020; Crook et al., Sci. Transl. Med. 14(645) 2022; Bryan et al., Proc Natl Acad Sci USA, 18(29):e2102164118, 2021.

[0069] A “peptide aptamer” refers to a polypeptide, generally between 2-20 amino acid residues in length, capable of binding target proteins and interfering with their function in living cells and organisms. They include conformationally-constrained random sequence peptide loops (called 'variable regions') displayed by a scaffold protein. They bind their cognate targets with a strong affinity and, usually, a high specificity, which allows them to discriminate between closely related members within a protein family, or even between different allelic variants of a given protein. So far, peptide aptamers have mostly been selected through yeast two-hybrid screening experiments, for their ability to bind a given target protein.

[0070] In particular embodiments, the binding domains disclosed herein can include receptor ligands, receptors, or fragments thereof. Receptors are proteins expressed by a cell that transmit signals upon binding to their cognate receptor ligand. Several receptor ligand and receptor pairs are known in the art and include PD-L1 and PD-1 , cytotoxic T-lymphocyte antigen 4 (CTLA-4) and B7-1 or B7-2, FasL and Fas, tumor necrosis factor alpha (TNFo) and tumor necrosis factor receptor (TNFR)1 or TNFR2, transforming growth factor beta (TGF ) and TGF receptors, interleukin (I L)-2 and IL-2R, CXCL12 and CXCR4, vascular endothelial growth factor (VEGF) and VEGFR, and CD47 and signal regulatory protein alpha (SIRPa). Other receptor ligand and receptor pairs include: estrogen and estrogen receptor, androgen and androgen receptor, glucocorticoid and glucocorticoid receptor, p53 and mouse double minute 2 homolog (MDM2), B- cell lymphoma 2 (BCL-2) and BCL-2 associated X protein (BAX), and Wnt and p-catenin

[0071] A nucleic acid aptamer is a single stranded nucleic acid molecule that structurally bind to targets with high affinity and specificity. The unique properties, including high specific affinity, structural stability and ease of synthesis, make aptamers promising therapeutic and diagnostic agents. In addition, nucleic acid aptamers have less batch-to-batch variation (than antibodies) because they are manufactured by chemical synthesis. Nucleic acid aptamers are often usually generated by a process called “systematic evolution of ligands by exponential enrichment” (SELEX). The targets of nucleic acid aptamers range from small molecules, for example cocaine, proteins like VEGF (vascular endothelial growth factor), and even whole cells.

[0072] (II) Cancer Markers and Binding Domains Thereof. Cancer markers are markers preferentially expressed by cancer cells or tumors. A “cancer marker binding domain” refers to a binding domain that binds a cancer marker. In particular embodiments, a cancer marker includes a cancer antigen.

[0073] In particular embodiments, cancer markers are preferentially expressed by cancer cells. “Preferentially expressed” means that a cancer marker is found at higher levels on cancer cells as compared to other cell types. In some instances, a cancer marker is only expressed by the targeted cancer cell type. In other instances, the cancer marker is expressed on the targeted cancer cell type at least 25%, 35%, 45%, 55%, 65%, 75%, 85%, 95%, 96%, 97%, 98%, 99%, or 100% more than on non-targeted cells.

[0074] In particular embodiments, cancer markers are significantly expressed on cancer cells. In particular embodiments, significantly expressed means that the cancer cells express the cancer marker more often than they do not express the cancer marker. In some instances, all targeted cancer cells express the cancer marker. In other instances, at least 25%, 35%, 45%, 55%, 65%, 75%, 85%, 95%, 96%, 97%, 98%, 99%, or 100% of targeted cancer cells express the cancer marker.

[0075] The following table provides examples of particular cancers and cancer markers that can be targeted with ICEm.

Table 1. Exemplary Cancer Markers.

[0076] Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1) is an enzyme and transmembrane receptor that is part of the ROR family of proteins. Because receptor tyrosine kinases (RTKs) are key regulators of normal cellular processes, they are also involved in the development and progression of many types of cancer. In particular embodiments, ROR1 includes the sequence: MHRPRRRGTRPPLLALLAALLLAARGAAAQETELSVSAELVPTSSWNISSELNKDSYLTLDEPM NNITTSLGQTAELHCKVSGNPPPTIRWFKNDAPWQEPRRLSFRSTIYGSRLRIRNLDTTDTGYF QCVATNGKEVVSSTGVLFVKFGPPPTASPGYSDEYEEDGFCQPYRGIACARFIGNRTVYMESL HMQGEIENQITAAFTMIGTSSHLSDKCSQFAIPSLCHYAFPYCDETSSVPKPRDLCRDECEILEN VLCQTEYIFARSNPMILMRLKLPNCEDLPQPESPEAANCIRIGIPMADPINKNHKCYNSTGVDYR GTVSVTKSGRQCQPWNSQYPHTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKS DLCDIPACDSKDSKEKNKMEILYILVPSVAIPLAIALLFFFICVCRNNQKSSSAPVQRQPKHVRGQ NVEMSMLNAYKPKSKAKELPLSAVRFMEELGECAFGKIYKGHLYLPGMDHAQLVAIKTLKDYN NPQQWTEFQQEASLMAELHHPNIVCLLGAVTQEQPVCMLFEYINQGDLHEFLIMRSPHSDVGC SSDEDGTVKSSLDHGDFLHIAIQIAAGMEYLSSHFFVHKDLAARNILIGEQLHVKISDLGLSREIY SADYYRVQSKSLLPIRWMPPEAIMYGKFSSDSDIWSFGVVLWEIFSFGLQPYYGFSNQEVIEMV RKRQLLPCSEDCPPRMYSLMTECWNEIPSRRPRFKDIHVRLRSWEGLSSHTSSTTPSGGNAT TQTTSLSASPVSNLSNPRYPNYMFPSQGITPQGQIAGFIGPPIPQNQRFIPINGYPIPPGYAAFPA AHYQPTGPPRVIQHCPPPKSRSPSSASGSTSTGHVTSLPSSGSNQEANIPLLPHMSIPNHPGG MGITVFGNKSQKPYKIDSKQASLLGDANIHGHTESMISAEL (SEQ ID NO: 9).

[0077] Prostate specific membrane antigen (PSMA) is a protein widely expressed on the surface of prostate cancer cells. In particular embodiments, PSMA includes the sequence:

KSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSV ELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGD LVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAP GVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYY DAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLR GAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEF GLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEG KSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYH SVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMK HPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGL PDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAET LSEVA (SEQ ID NO: 1).

[0078] Prostate stem cell antigen (PSCA) is a membrane surface glycoprotein that may play a role in progenitor cell function. In particular embodiments, PSCA includes the sequence:

MKAVLLALLMAGLALQPGTALLCYSCKAQVSNEDCLQVENCTQLGEQCWTARIRAVGLLTVISK GCSLNCVDDSQDYYVGKKNITCCDTDLCNASGAHALQPAAAILALLPALGLLLWGPGQL (SEQ ID NO: 2).

[0079] Mesothelin is a glycosylphosphatidylinositol-anchored cell-surface protein that may function as a cell adhesion protein. This protein is overexpressed in epithelial mesotheliomas, ovarian cancers and in specific squamous cell carcinomas. In particular embodiments, mesothelin includes the sequence: MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVI-ANPPNISSLSPR QLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPLDLLLFLNP DAFSGPQACTHFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEADVRALGGLACD LPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTWSVSTMDALRGLLP VLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKTACPSGKKAREIDESLIFY KKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPE DIRKWNVTSLETLKALLEVNKGHEMSPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSP

EELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLK ALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLD TLGLGLQGGIPNGYLVLDLSVQEALSGTPCLLGPGPVLTVLALLLASTLA (SEQ ID NO: 3).

[0080] CD19 is a type-l transmembrane glycoprotein of 95 kDa that belongs to the immunoglobulin superfamily and is widely expressed on B cells throughout most stages of B-cell differentiation, though its expression is down-regulated during their terminal differentiation to plasma cells. In particular embodiments, CD19 includes the sequence:

MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLK LSLGLPGLGIHMRPLASWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRW NVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCVPPRDSLNQSLSQ DLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLL PRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILH LQRALVLRRKRKRMTDPTRRFFKVTPPPGSGPQNQYGNVLSLPTPTSGLGRAQRWAAGLGG

TAPSYGNPSSDVQADGALGSRSPPGVGPEEEEGEGYEEPDSEEDSEFYENDSNLGQDQLSQ DGSGYENPEDEPLGPEDEDSFSNAESYENEDEELTQPVARTMDFLSPHGSAWDPSREATSLG SQSYEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENMDNPDGPDPAWGGGGRMGTWSTR (SEQ ID NO: 4).

[0081] CD20 is B lymphocyte cell-surface molecule that is a 33-37 kDa non-glycosylated protein. CD20 is expressed on the surface of B-cells from the pre-B phase, the expression is lost in terminally differentiated plasma cells. In particular embodiments, CD20 includes the sequence: MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGAVQIMNGLF HIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIISGSLLAATEKNSRKCLVKGKMIMNSLSLFAAIS GMILSIMDILNIKISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGILSVM

LIFAFFQELVIAGIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEWGLTETSSQPKNEEDIEII PIQEEEEEETETNFPEPPQDQESSPIENDSSP (SEQ ID NO: 5).

[0082] CD33 is a myeloid cell surface antigen that is not expressed on blood stem cells or within the hematopoietic system, but it can be expressed on the surface of natural B-lymphocytes, activated T-lymphocytes, and natural killer (NK) cells. In particular embodiments, CD33 (full length) includes the sequence: MPLLLLLPLLWAGALAMDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFRE GAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTK YSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPR TTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETR AGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPKHQKKSKLHG PTETSSCSGAAPTVEMDEELHYASLNFHGMNPSKDTSTEYSEVRTQ (SEQ ID NO: 6).

[0083] Splice variants of CD33 can result in truncated forms of CD33. For example, lack of exon 2 (AE2) can result in a splice variant. In particular embodiments, CD33 (AE2 variant) includes the sequence: MPLLLLLPLLWADLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTT HSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAG VVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPKHQKKSKLHGPT ETSSCSGAAPTVEMDEELHYASLNFHGMNPSKDTSTEYSEVRTQ (SEQ ID NO: 7).

[0084] In particular embodiments, CD33 (with C-terminal truncation) includes the sequence: MPLLLLLPLLWAGALAM DPN FWLQVQESVTVQEGLCVLVPCTFFH PI PYYDKNSPVHGYWFRE GAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTK YSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPR TTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETR AGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPVR (SEQ ID NO: 8).

[0085] As will be understood by one of ordinary skill in the art, targeted cancer markers can lack signal peptides, such as the underlined segments of sequences underlined in SEQ ID NOs: 6-8 (representative CD33 antigens).

[0086] Wilms tumor protein (WT1) is a transcription factor that contains four zinc finger motifs at the C-terminus and a proline / glutamine-rich DNA-binding domain at the N-terminus. It has an essential role in the normal development of the urogenital system, and it is mutated in a subset of patients with Wilms' tumor, the gene's namesake. Multiple transcript variants, resulting from alternative splicing at two coding exons, have been well characterized. In particular embodiments, WT1 includes the sequence: IEGRHMRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCD FKDCERRFFRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFSCRWPS CQKKFARSDELVRHHNMHQRNMTKLQLAL (SEQ ID NO: 10). [0087] CD123, or the interleukin 3 receptor alpha chain (IL-3Ra), is a cytokine receptor that plays a role in production and function of hematopoietic cells. It is expressed on normal cells like monocytes, basophils, and plasmacytoid dendritic cells, but is also overexpressed in many hematologic malignancies, including acute myeloid leukemia (AML), acute lymphoblastic leukemia, and hairy cell leukemia. In particular embodiments, CD123 includes the sequence: MVLLWLTLLLIALPCLLQTKEDPNPPITNLRMKAKAQQLTWDLNRNVTDIECVKDADYSMPAVN NSYCQFGAISLCEVTNYTVRVANPPFSTWILFPENSGKPWAGAENLTCWIHDVDFLSCSWAVG PGAPADVQYDLYLNVANRRQQYECLHYKTDAQGTRIGCRFDDISRLSSGSQSSHILVRGRSAA FGIPCTDKFVVFSQIEILTPPNMTAKCNKTHSFMHWKMRSHFNRKFRYELQIQKRMQPVITEQV RDRTSFQLLNPGTYTVQIRARERVYEFLSAWSTPQRFECDQEEGANTRAWRTSLLIALGTLLAL VCVFVICRRYLVMQRLFPRIPHMKDPIGDSFQNDKLVVWEAGKAGLEECLVTEVQVVQKT (SEQ ID NO: 11).

[0088] In particular embodiments, the cancer marker binding domain binds RORI . Examples of ROR1 binding domains (i.e. , binding domains that bind ROR1) include a sequence selected from: QVKLVQSGGGLVQTGGSLRLSCAASEITFDMYSMGWYREAPGKARDAVASITNRGNTYYADS VKGRFTISRDNAKKTMYLQMNSLKPEDTAVYYCNVYRTGFSDYWGQGTQVTVSS (SEQ ID NO: 12);

ASVNQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKRA KSFSLRIKDLTVADSATYYCKAQSGMAISTGSGHGYNVVYDGAGTVLTVN (SEQ ID NO: 13); AKVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKRA KSFSLRIKDLTVADSATYYCKAQSGMAIDIGSGHGYNWYDGAGTVLTVN (SEQ ID NO: 14); TRVDQTPRTATKETGESLTINCWTGAKYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTM SFSLRIKDLTVADSATYYCKAYPWAMWGQWYDGAGTVLTVN (SEQ ID NO: 15); TRVDQTPRTATKETGESLTINCWTGAKYGLFATYWYRKNPGSSNQERISISGRYVESVNKRTM SFSLRIKDLTVADSATYYCKAVFMPQHWHPAAHWYDGAGTVLTVN (SEQ ID NO: 16); TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGA KSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVN (SEQ ID NO: 17); ASVNQTPRTATKETGESLTINCWTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTM SFSLRIKDLTVADSATYYCKAYPWGAGAPWLVQVVYDGAGTVLTVN (SEQ ID NO: 18);

TRVDQSPSSLSASVGDRVTITCVLTGANYGLASTYVVYRKNPGSSNKEQISISGRYSESVNKGT KSFTLTISSLQPEDSATYYCRAYPWGAGAPWLVQWYDGAGTKVEIK (SEQ ID NO: 19);

TRVDQSPSSLSASVGDRVTITCVLTGANYGLASTYWYRKNPGSSNQERISISGRYSESVNKRT MSFTLTISSLQPEDSATYYCRAYPWGAGAPWLVQVVYDGAGTKVEIK (SEQ ID NO: 20); TRVDQSPSSLSASVGDRVTITCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKG AKSFTLTISSLQPEDFATYYCKAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO: 21);

TRVDQSPSSLSASVGDRVTITCVLTDTSYGLYSTYVVYRKNPGSSNKEQISISGRYSESVNKGT KSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO: 22); and TRVDQSPSSLSASVGDRVTITCVLTDTSYGLYSTYVVYRKNPGTTDWERMSIGGRYSESVNKG AKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO: 23).

[0089] In particular embodiments, the cancer marker binding domain binds PSMA. In particular embodiments, the PSMA binding domain is a VHH. Examples of PSMA binding domains (i.e., binding domains that bind PSMA) include: a sequence having a CDR1 including the sequence GSTFSINA (SEQ ID NO: 151), a CDR2 including the sequence LSSGGSK (SEQ ID NO: 152), and a CDR3 including the sequence NAEIYYSDGVDDGYRGMDY (SEQ ID NO: 153); or a sequence having a CDR1 including the sequence GPPLSSYA (SEQ ID NO: 154), a CDR2 including the sequence ISWSGSNT (SEQ ID NO: 155), and a CDR3 including the sequence AADRRGGPLSDYEWEDEYAD (SEQ ID NO: 156).

[0090] In particular embodiments, the cancer marker binding domain binds mesothelin. Examples of mesothelin binding domains (i.e., binding domains that bind mesothelin) include a sequence having a CDR1 including the sequence GIDLSLYR (SEQ ID NO: 157) or GSIFGIRT (SEQ ID NO: 158), a CDR2 including the sequence ITDDGTS (SEQ ID NO: 159) or ITMDGRV (SEQ ID NO: 160), and a CDR3 including the sequence NAETPLSPVNY (SEQ ID NO: 161) or RYSGLTSREDY (SEQ ID NO: 162).

[0091] In particular embodiments, the cancer marker binding domain binds CD19. Examples of CD19 binding domains (i.e., binding domains that bind CD19) include: a sequence having a CDR1 including the sequence GNINSRNCMG (SEQ ID NO: 163) or RNCMG (SEQ ID NO: 164), a CDR2 including the sequence AIGQVTGRSYYVDSVKG (SEQ ID NO: 165), and a CDR3 including the sequence APGCLLSALRSADYRN (SEQ ID NO: 166); a sequence having a CDR1 including the sequence GNINSRNC (SEQ ID NO: 167), a CDR2 including the sequence IGQVTGRS (SEQ ID NO: 168), and a CDR3 including the sequence AAAPGCLLSALRSADYRN (SEQ ID NO: 169); or a sequence having a CDR1 including the sequence GDTLSNKWMG (SEQ ID NO: 170) or NKWMG (SEQ ID NO: 171), a CDR2 including the sequence TIRTDHAGTYADSVKG (SEQ ID NO: 172), and a CDR3 including the sequence SYSGATTFRY (SEQ ID NO: 173).

[0092] In particular embodiments, the cancer marker binding domain binds CD20. Examples of CD20 binding domains (i.e., binding domains that bind CD20) include a sequence having a CDR1 including the sequence GRTFSSYNMG (SEQ ID NO: 174) or SYNMG (SEQ ID NO: 175), a CDR2 including the sequence VVDWSGGSPYYADSVKG (SEQ ID NO: 176), and a CDR3 including the sequence RVQYGSSWSGDY (SEQ ID NO: 177);a CDR1 including the sequence GRTFSSYN (SEQ ID NO: 178), a CDR2 including the sequence VVDWSGGSP (SEQ ID NO: 179), and a CDR3 including the sequence VVDWSGGSP (SEQ ID NO: 179); and a CDR1 including the sequence GRTFSSYNMG (SEQ ID NO: 174) or SYNMG (SEQ ID NO: 175), a CDR2 including the sequence AISWSGGSPYYASSVRG (SEQ ID NO: 183) and a CDR3 including the sequence PIEYGSSWSADY (SEQ ID NO: 184).

[0093] In particular embodiments, the cancer marker binding domain binds CD33. Examples of CD33 binding domains (i.e., binding domains that bind CD33) include a sequence having a CDR1 including the sequence RSSGIDVMG (SEQ ID NO: 185), a CDR2 including the sequence EISGVGDTN (SEQ ID NO: 186), and a CDR3 including the sequence of HSFLDLVGA (SEQ ID NO: 187); a CDR1 including the sequence GSINSINVME (SEQ ID NO: 188), a CDR2 including the sequence GITSDGDTN (SEQ ID NO: 189), and a CDR3 including the sequence RDWGSLTDY (SEQ ID NO: 190); a CDR1 including the sequence GRTISDYVVG (SEQ ID NO: 191), a CDR2 including the sequence AISRYGTTY (SEQ ID NO: 192), and a CDR3 including the sequence LQNDVRNNHSPTSYDY (SEQ ID NO: 193).

[0094] In particular embodiments, the cancer marker binding domain binds CD123. Examples of CD123 binding domains (i.e., binding domains that bind CD123) include a sequence having a CDR1 including the sequence GGTFSSYGMA (SEQ ID NO: 194), a CDR2 including the sequence SNSWIAGSTY (SEQ ID NO: 195), and a CDR3 including the sequence DLLATADDEYDY (SEQ ID NO: 196); a CDR1 including the sequence GRTQSAVAMG (SEQ ID NO: 197), a CDR2 including the sequence AIRWSGGNTY (SEQ ID NO: 198), and a CDR3 including the sequence SMNHFGMYDY (SEQ ID NO: 199); or a CDR1 including the sequence GRAINTYAMG (SEQ ID NO: 200), a CDR2 including the sequence AISWNGGHTR (SEQ ID NO: 201), and a CDR3 including the sequence YSDYHRIATMEADADS (SEQ ID NO: 202).

[0095] In some cases, an ICEm can include more than one cancer marker binding domain and therefore can target more than one cancer marker. In particular embodiments, an ICEm targets cancer markers that are co-expressed in cancerous tissue. Targeted cancer marker epitopes that are co-expressed in cancerous tissues but not in non-cancerous tissues are different from one another. In particular embodiments, “different from” means that the targeted cancer marker epitopes are distinct from one another in sequence and/or structure. In particular embodiments, in addition to being different, targeted cancer marker epitopes are also non-overlapping. “Nonoverlapping” means that the binding of one binding domain in a group to an epitope is not decreased to a statistically-significant degree in a competitive binding assay by the presence of at least one other binding domain in the group. Non-overlapping epitopes may be epitopes on different molecules (e.g., ROR1 and CD33; CD3 and CD28) or may be non-overlapping epitopes located on the same molecule (e.g., non-overlapping ROR1 epitopes). Non-repetitive different epitopes on the same molecule exclude epitopes that are physically distinct in space from one another yet repetitive in sequence to each other. For example, MUC1 has a repetitive sequence, and the repeats within the sequence are not non-repetitive and different, as defined herein.

[0096] In particular embodiments, a targeted cancer marker can have high expression by a targeted cancer cell or tumor or low expression by a targeted cancer cell or tumor. In particular embodiments, high and low expression can be determined using flow cytometry or fluorescence- activated cell-sorting (FACs). As is understood by one of ordinary skill in the art of flow cytometry, “hi”, “Io”, “+” and refer to the intensity of a signal relative to negative or other populations. In particular embodiments, positive expression (+) means that the marker is detectable on a cell using flow cytometry. In particular embodiments, negative expression (-) means that the marker is not detectable using flow cytometry. In particular embodiments, “hi” means that the positive expression of a marker of interest is brighter as measured by fluorescence (using for example FACS) than other cells also positive for expression. In these embodiments, those of ordinary skill in the art recognize that brightness is based on a threshold of detection. Generally, one of skill in the art will analyze a negative control tube first, and set a gate (bitmap) around the population of interest by FSC and SSC and adjust the photomultiplier tube voltages and gains for fluorescence in the desired emission wavelengths, such that 97% of the cells appear unstained for the fluorescence marker with the negative control. Once these parameters are established, stained cells are analyzed and fluorescence recorded as relative to the unstained fluorescent cell population. In particular embodiments, and representative of a typical FACS plot, hi implies to the farthest right (x line) or highest top line (upper right or left) while Io implies within the left lower quadrant or in the middle between the right and left quadrant (but shifted relative to the negative population). In particular embodiments, "hi" refers to greater than 20-fold of +, greater than 30- fold of +, greater than 40-fold of +, greater than 50-fold of +, greater than 60-fold of +, greater than 70-fold of +, greater than 80-fold of +, greater than 90-fold of +, greater than 100-fold of +, or more of an increase in detectable fluorescence relative to + cells. Conversely, “Io” can refer to a reciprocal population of those defined as "hi".

[0097] The epitope is the part of an antigen to which a binding domain attaches itself.

[0098] (III) Immune Cell Activating Epitopes and Binding Domains Thereof. In particular embodiments, the ICEm include a binding domain that binds an ICAE. An “immune cell activating epitope binding domain” or “ICAE binding domain” refers to the binding domain of an ICEm that binds an ICAE. Immune cells that can be targeted for localized activation by ICEm of the current disclosure include, for example, T cells, natural killer (NK) cells, and macrophages. In particular embodiments, if the ICEm targets T cells, it can be considered a multi-specific T cell engager (MTE). In particular embodiments, the binding domain that targets an immune cell for localized activation is derived from an antibody that binds a protein that activates the immune cell. In particular embodiments, an ICAE includes a T cell activating epitope, NK cell activating epitope, or macrophage activating epitope.

[0099] T-cell activation, for example can be mediated by two distinct signals: those that initiate antigen-dependent primary activation and provide a T-cell receptor like signal (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). MTE disclosed herein can target any T cell activating epitopes that upon binding induce T-cell activation. Examples of such T cell activating epitopes are on T cell markers including CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, 4-1BB (CD 137), 0X40, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, and B7-H3. T cell suppressive receptors that can be blocked include 4-1 BB, PD-1, LAG3, TIM-3, BT LA, CT LA-4, and CD200. Binding domains that bind T-cell activating epitopes are known in the art.

[0100] CD3 is a primary signal transduction element of T cell receptors. CD3 is composed of a group of invariant proteins called gamma (y), delta (A), epsilon (Z), zeta (Z) and eta (H) chains. The y, A, and Z chains are structurally-related, each containing an Ig-like extracellular constant domain followed by a transmembrane region and a cytoplasmic domain of more than 40 amino acids. The Z and H chains have a distinctly different structure: both have a very short extracellular region of only 9 amino acids, a transmembrane region and a long cytoplasmic tail including 113 and 115 amino acids in the Z and H chains, respectively. The invariant protein chains in the CD3 complex associate to form noncovalent heterodimers of the Z chain with a y chain (Zy) or with a A chain (ZA) or of the Z and H chain (ZH), or a disulfide-linked homodimer of two Z chains (ZZ). 90% of the CD3 complex incorporate the ZZ homodimer.

[0101] The cytoplasmic regions of the CD3 chains include a motif designated the immunoreceptor tyrosine-based activation motif (ITAM). This motif is found in a number of other receptors including the I g-a/lg-p heterodimer of the B-cell receptor complex and Fc receptors for IgE and IgG. The ITAM sites associate with cytoplasmic tyrosine kinases and participate in signal transduction following TCR-mediated triggering. In CD3, the y, A and Z chains each contain a single copy of ITAM, whereas the Z and H chains harbor three ITAMs in their long cytoplasmic regions. Indeed, the Z and H chains have been ascribed a major role in T cell activation signal transduction pathways.

[0102] CD3 is expressed on all mature T cells. The MTE depicted in FIG. 2 includes an anti-CD3 binding domain. In particular embodiments, the anti-CD3 binding domain includes a VHH. In particular embodiments the anti-CD3 binding domain includes the sequence:

EVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMGWFRQAPGKEREFVAAIVWSGGNTYYE DSVKGRFTISRDNAKNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS (SEQ ID NO: 24).

[0103] In particular embodiments, the anti-CD3 binding domain includes the sequence:

EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATY

YADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTV SSGGGSGGGSGGGSGGGSQTWTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKP GQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVL (SEQ ID NO: 203);

MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTREGLTKTGYADSVKGR FAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 25);

MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTREGLTKTGYADSVKGR FAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 26);

MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTREGLTQTGYADSVKGR FAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 27);

MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTREGLTQTGYADSVKGR FAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 28);

MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTREGLPKTGYADSVKGR FAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 29);

MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTREGLPKTGYADSVKGR FAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 30);

MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTREGLPQTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 31);

MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTREGLPQTGYADSVKG RFAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 32);

MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTRDGLTKTGYADSVKGR

FAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 33);

MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTRDGLTKTGYADSVKGR

FAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 34);

MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTRDGLTQTGYADSVKG

RFAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 35);

MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTRDGLTQTGYADSVKG

RFAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 36);

MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTRDGLPKTGYADSVKGR

FAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 37);

MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTRDGLPKTGYADSVKGR

FAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 38);

MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTRDGLPQTGYADSVKG

RFAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 39);

MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTRDGLPQTGYADSVKG

RFAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 40);

MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTREGLTKTGYADSVKGR

FAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 41);

MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTREGLTKTGYADSVKGR

FAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 42);

MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTREGLTQTGYADSVKGR

FAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 43); MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTREGLTQTGYADSVKGR FAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 44);

MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTREGLPKTGYADSVKGR FAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 45);

MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTREGLPKTGYADSVKGR FAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 46);

MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTREGLPQTGYADSVKGR FAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 47);

MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTREGLPQTGYADSVKGR FAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 48);

MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTRDGLTKTGYADSVKGR FAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 49);

MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTRDGLTKTGYADSVKGR FAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 50);

MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTRDGLTQTGYADSVKGR FAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 51);

MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTRDGLTQTGYADSVKGR FAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 52);

MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTRDGLPKTGYADSVKGR FAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 53);

MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTRDGLPKTGYADSVKGR FAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 54);

MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTRDGLPQTGYADSVKGR FAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 55); or MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTRDGLPQTGYADSVKGR FAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 56).

[0104] In particular embodiments, the anti-CD3 binding domain is derived from a CD3 antibody such as the OKT3 antibody (the same as the one utilized in blinatumomab). The OKT3 antibody is described in detail in U.S. Patent No. 5,929,212. It includes a variable light chain including a CDRL1 sequence including SASSSVSYM N (SEQ ID NO: 57), a CDRL2 sequence including RWIYDTSKLAS (SEQ ID NO: 58), and a CDRL3 sequence including QQWSSNPFT (SEQ ID NO: 59). In particular embodiments, the anti-CD3 binding domain (i.e., binding domain that binds the ICAE CD3) is derived from an antibody including a variable heavy chain including a CDRH1 sequence including KASGYTFTRYTMH (SEQ ID NO: 60), a CDRH2 sequence including INPSRGYTNYNQKFKD (SEQ ID NO: 61), and a CDRH3 sequence including YYDDHYCLDY (SEQ ID NO: 62).

[0105] In particular embodiments, the anti-CD3 binding domain can include or be derived from an scFv derived from OKT3 which retains the capacity to bind CD3. This scFv includes the sequence: QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYN QKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSSGGG GSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYD TSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR (SEQ ID NO: 63).

[0106] In particular embodiments, the anti-CD3 binding domain is derived from an anti-CD3 binding domain including a variable light chain including a CDRL1 sequence including QSLVHNNGNTY (SEQ ID NO: 64), a CDRL2 sequence including KVS, and a CDRL3 sequence including GQGTQYPFT (SEQ ID NO: 65). In particular embodiments, the anti-CD3 binding domain is derived from a 20G6-F3 antibody including a variable heavy chain including a CDRH1 sequence including GFTFTKAW (SEQ ID NO: 66), a CDRH2 sequence including IKDKSNSYAT (SEQ ID NO: 67), and a CDRH3 sequence including RGVYYALSPFDY (SEQ ID NO: 68).

[0107] In particular embodiments, the anti-CD3 binding domain is derived from an anti-CD3 binding domain including a variable light chain including a CDRL1 sequence including QSLVHDNGNTY (SEQ ID NO: 69), a CDRL2 sequence including KVS, and a CDRL3 sequence including GQGTQYPFT (SEQ ID NO: 65). In particular embodiments, the anti-CD3 binding domain is derived from a 4B4-D7 antibody including a variable heavy chain including a CDRH1 sequence including GFTFSNAW (SEQ ID NO: 71), a CDRH2 sequence including IKARSNNYAT (SEQ ID NO: 72), and a CDRH3 sequence including RGTYYASKPFDY (SEQ ID NO: 73).

[0108] In particular embodiments, the anti-CD3 binding domain is derived from a 4E7-C9 antibody including a variable light chain including a CDRL1 sequence including QSLEHNNGNTY (SEQ ID NO: 74), a CDRL2 sequence including KVS, and a CDRL3 sequence including GQGTQYPFT (SEQ ID NO: 65). In particular embodiments, the anti-CD3 binding domain is derived from a 4E7- C9 antibody including a variable heavy chain including a CDRH1 sequence including GFTFSNAW (SEQ ID NO: 71), a CDRH2 sequence including IKDKSNNYAT (SEQ ID NO: 77), and a CDRH3 sequence including RYVHYGIGYAMDA (SEQ ID NO: 78).

[0109] In particular embodiments, the anti-CD3 binding domain is derived from a 18F5-H10 antibody including a variable light chain including a CDRL1 sequence including QSLVHTNGNTY (SEQ ID NO: 79), a CDRL2 sequence including KVS, and a CDRL3 sequence including GQGTHYPFT (SEQ ID NO: 80). In particular embodiments, the anti-CD3 binding domain is derived from a 18F5-H10 antibody including a variable heavy chain including a CDRH1 sequence including GFTFTNAW (SEQ ID NO: 81), a CDRH2 sequence including KDKSNNYAT (SEQ ID NO: 82), and a CDRH3 sequence including RYVHYRFAYALDA (SEQ ID NO: 83). T

[0110] Additional examples of anti-CD3 antibodies, binding domains, and CDRs can be found in WO2016/116626. TR66 may also be used.

[0111] CD28 is a surface glycoprotein present on 80% of peripheral T cells in humans, and is present on both resting and activated T cells. CD28 binds to B7-1 (CD80) and B7-2 (CD86) and is the most potent of the known co-stimulatory molecules (June et al., Immunol. Today 15:321 (1994); Linsley et al., Ann. Rev. Immunol. 11 :191 (1993)). In particular embodiments, the anti- CD28 binding domain is derived from a CD28 antibody. CD28 antibodies include CD80, CD86 or the 9D7 antibody. Additional antibodies that bind CD28 include 9.3, KOLT-2, 15E8, 248.23.2, and EX5.3D10.

[0112] Activated T-cells express 4-1 BB (CD137). T-cells can further be classified into helper cells (CD4+ T-cells) and cytotoxic T-cells (CTLs, CD8+ T-cells), which include cytolytic T-cells. T helper cells assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and activation of cytotoxic T-cells and macrophages, among other functions. These cells are also known as CD4+ T-cells because they express the CD4 protein on their surface. Helper T-cells become activated when they are presented with peptide antigens by MHC class II molecules that are expressed on the surface of antigen presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response. [0113] Particular embodiments can include activating CD4+ T cells by binding CD3, TLR2 or CD28 and/or by blocking the suppression of CD4 T cells by binding 4-1 BB, PD-1 , LAG3, TIM-3, BTLA, CTLA-4, CD200, and/or VISTA. In particular embodiments, the ICAE includes 4-1 BB, PD- 1 , l_AG3, TIM-3, BTLA, CTLA-4, CD200, or VISTA. In particular embodiments, the ICAE binding domain includes a binding domain that binds 4-1 BB, PD-1 , LAG3, TIM-3, BTLA, CTLA-4, CD200, or VISTA.

[0114] TLR2 (UniProt ID No. 060603) is involved in the innate immune response to bacterial lipoproteins and other microbial cell wall components. In particular embodiments, the anti-TLR2 binding domain is derived from an anti-TLR2 antibody. Commercially available anti-TLR2 antibodies include anti-hTLR2-lgA and mAb-hTLR2 (both available from Invivogen).

[0115] In particular embodiments, ICEm bind an epitope of co-stimulatory receptor 4- 1BB. 4-1 BB, also called CD137 or TNFSF9 (UniProt ID No. Q07011) is a T-cell co-stimulatory receptor. In particular embodiments, an anti-4-1 BB binding domain is derived from an anti-4-1 BB antibody including a variable light chain including a CDRL1 sequence including RASQSVS (SEQ ID NO: 84), a CDRL2 sequence including ASNRAT (SEQ ID NO: 85), and a CDRL3 sequence including QRSNWPPALT (SEQ ID NO: 86) and a variable heavy chain including a CDRH1 sequence including YYWS (SEQ ID NO: 87), a CDRH2 sequence including INH, and a CDRH3 sequence including YGPGNYDWYFDL (SEQ ID NO: 88).

[0116] Cytotoxic T-cells destroy tumor cells. These cells are also known as CD8+ T-cells because they express the CD8 glycoprotein at their surface. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of nearly every cell of the body. Particular embodiments can include activating CD8 T cells by binding CD3, CD28, or 4- 1 BB and/or by blocking the suppression of CD8 T cells by binding PD-1 , LAG3, TIM-3, or VISTA. [0117] Particular embodiments disclosed herein including binding domains that bind epitopes on CD8. In particular embodiments, the anti-CD8 binding domain is derived from the anti-OKT8 antibody.

[0118] In particular embodiments, natural killer cells (also known as NK cells, K cells, and killer cells) are targeted for localized activation. NK cells can induce apoptosis or cell lysis by releasing granules that disrupt cellular membranes, and can secrete cytokines to recruit other immune cells. [0119] Examples of activating proteins expressed on the surface of NK cells include NKG2D, CD8, CD16, KIR2DL4, KIR2DS1, KIR2DS2, KIR3DS1, NKG2C, NKG2E, NKG2D, and several members of the natural cytotoxicity receptor (NCR) family. Examples of NCRs that activate NK cells upon ligand binding include NKp30, NKp44, NKp46, NKp80, and DNAM-1.

[0120] Examples of commercially available antibodies that bind to an NK cell receptor and induce and/or enhance activation of NK cells include: 5C6 and 1D11 , which bind and activate NKG2D (available from BioLegend® San Diego, CA); mAb 33, which binds and activates KIR2DL4 (available from BioLegend®); P44-8, which binds and activates NKp44 (available from BioLegend®); SK1, which binds and activates CD8; and 3G8 which binds and activates CD16. [0121] In particular embodiments macrophages are targeted for localized activation. Macrophages are a type of leukocyte (or white blood cell) that can engulf and digest cells, cellular debris, and/or foreign substances in a process known as phagocytosis.

[0122] The ICEm can be designed to include a binding domain that binds to a molecule expressed on the surface of macrophages. Examples of activating proteins expressed on the surface of macrophages (and their precursors, monocytes) include CD11b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4/80, IFGR2 Toll-like receptors (TLRs) 1-9, IL-4Ra, and MARCO. Commercially available antibodies that bind to molecules expressed on the surface of macrophages include M1/70, which binds and activates CD11b (available from BioLegend®); KP1 , which binds and activates CD68 (available from ABCAM®, Cambridge, United Kingdom); and ab87099, which binds and activates CD163 (available from ABCAM®).

[0123] In particular embodiments, the ICEm includes a binding domain that binds an epitope of CD40. CD40 (or Tumor necrosis factor receptor superfamily member 5, UniProt ID No. P25942) is a receptor that can transduce activating signals in macrophages.

[0124] In particular embodiments, examples of inhibitory proteins expressed by macrophages (and their precursors, monocytes) include programmed cell death ligands 1 and 2 (PD-L1 and PD-L2) and galectin 9 (Gal-9).

[0125] In particular embodiments, the ICEm includes a binding domain that binds an epitope of Gal-9 (UniProt ID No. 000182) In particular embodiments, an anti-Gal-9 binding domain can be derived from an anti-Gal-9 antibody that blocks binding to TIM-3. An example of a commercially available anti-Gal-9 antibody that blocks TIM-3 binding is 9M1-3 (available from Biolegend).

[0126] (IV) Cancer Supporter and Binding Domains Thereof. In particular embodiments, an ICEm includes a binding domain that binds a cancer supporter (i.e. , cancer supporter binding domain). A cancer supporter is a molecule expressed by a cancer cell that renders the cancer cell (i) less susceptible to a treatment and/or (ii) less susceptible to detection or destruction by the immune system. A “cancer supporter binding domain” refers to the binding domain of an ICEm that binds a cancer supporter. In particular embodiments, the cancer supporter binding domain blocks and targets cancer supporters. In particular embodiments, the cancer supporter binding domain binds a molecule that that cancer cells upregulate or select for to promote their survival. In particular embodiments, the cancer supporter binding domain binds a molecule that down-regulates immune system activity against a cancer cell that expresses the cancer supporter. In particular embodiments, the cancer supporter binding domain binds a molecule that provides treatment resistance for a cancer cell that expresses the cancer supporter.

[0127] Cancer supporters can be selected by narrowing down a list of molecules such that they have the following properties: are induced by IFNy, are expressed on the cell surface, are expressed beyond hematopoietic lineages, are expressed on tumors, and provide selective advantage. Examples of a cancer supporter includes: PD-L1 (B5XDX7), ALCAM/CD166 (Uniprot ID: Q13740), C10orf54 (human homolog of mouse Vsir, a PD1 homolog) (Uniprot ID: Q2TA85), CEACAM1 (Uniprot ID: M0R109), CEACAM20 (Uniprot ID: Q6UY09), CMKLR1 (Uniprot ID: Q99788), HLA-E (Uniprot ID: P12747), IFITM1 (Uniprot ID: P13164), IFITM2 (Uniprot ID: Q01629), IFITM3 (Uniprot ID: Q01628), IL31RA (Uniprot ID: Q8NI17), LST1 (Uniprot ID: 000453), MEFV (Uniprot ID: 015553), OSMR (Uniprot ID: Q99650), PDL2 (Uniprot ID: Q9BQ51), PIM1 (Uniprot ID: P11309), PIM2 (Uniprot ID: Q9P1W9), PLAAT3 (Uniprot ID: P53816), PRAME (Uniprot ID: P78395), and SDCBP (Uniprot ID: 000560). In particular embodiments, the cancer supporter can include CD80/CD86.

[0128] In particular embodiments, cancer supporters are identified by treating healthy cells (PBMCs) and cancer cells (e.g., diffuse midline glioma (DMG) primary tumor cells) with an immune cell activation indicator; measuring gene expression in the treated healthy cells and treated cancer cells; and selecting a gene that is upregulated in the treated cancer cells and not in the treated healthy cells. In particular embodiments, the immune cell activation indicator includes IFNy, TNFa, IL-2, IL-7, or IL-15. In particular embodiments, the immune cell activation indicator includes supernatant from co-culture of activated T cells.

[0129] In particular embodiments, a cancer supporter includes: OCSTAMP, UNC5C, KCNF1 , TMEM45A, RXFP1 , MGAM2, PTGES, TMEM92, CLEC9A, CAV2, LAMA3, RIMS2, BCAR1 , PLEKHS1 , SSTR2, KCNE5, ITGA7, GPR31 , IL31 RA, SCUBE1 , HPN, GJA4, P2RY14, BPI, S1 PR3, FCGR1A, KREMEN1 , FLT1 , DCSTAMP, GRIN3A, MUC1 , DEFB1 , CALHM6, KCNJ10, CPNE6, FFAR2, SLC6A12, HCAR3, F3, XIRP1 , CLEC6A, FPR2, HCAR2, FRMD3, VAMP5, TMTC1 , CD274, NPFFR1 , GLDN, SMO, CD38, TNFSF10, GPR33, SLAMF7, NRN1 , CYSLTR2, ATP1 B2, PLSCR4, RRAD, KCNJ15, CCRL2, TMEM255A, CSPG4, FPR1 , JAG1 , CDCP1 , GPR146, MYOF, PDGFRA, MY01A, ABCC11 , LAG3, CAVIN1, PPFIA4, CR1 L, GPC4, P2RX7, RARRES3, PLEKHA6, TMEM132A, SMCO2, SMCO4, MMP25, KCNJ2, RALB, TIE1 , SCG3, PRRT2, SORT1 , VCAM1 , FAM241A, PTPRU, CLEC4E, NCF1 , SLC12A3, IL15RA, SECTM1 , LRRN2, OTOF, GEM, ENTHD1, OSBPL6, SCARF1 , SUCNR1 , SLC2A5, PSTPIP2, GPR84, GOLM1, GJB2, SLITRK4, SLC2A6, CLEC4D, SSPN, SORBS1 , GAREM1 , CLDN7, LRRC4, ASPHD2, STX11, EREG, RAPGEF2, MY01 B, CLEC5A, SYT17, LYPD5, PDCD1 LG2, PHLDA2, CTTN, SLC31A2, BEGAIN, RIPK2, CSF2RB, DGKG, FRRS1 , TNFSF13B, VRK2, D0CK7, DAPP1 , PRRG4, CACNA1A, ITGB7, PRAME, MFSD2A, CEACAM4, TNF, SLC1A5, CDK14, FAS, ATP1 B1, FFAR4, HVCN1, PROCR, CD80, IFITM1 , TENM4, PLAUR, NETO2, SCIMP, PAK1, CAMK2D, C19orf38, CD83, RHOU, CD40, PLEK, ITPRIPL2, TREML2, EPB41 L5, LILRA2, SLC1A4, LDLR, IFNGR2, SFT2D2, SLAMF8, TLR7, GPBAR1, SIRPB1 , SIRPB1, NYNRIN, C3orf14, TJP2, LMTK2, PAQR6, FZD2, DIXDC1 , STX1 B, CNIH4, SLC8A1 , CD33, TMEM131, NOD2, PDE4B, SH2B2, CLMP, SLC1A3, MCOLN2, IL12RB2, TLR1 , CSF1 , and TLR8 (see FIG. 16).

[0130] Examples of cancer supporters also include immune checkpoint molecules and ligands and efflux pumps.

[0131] As used herein, the terms “immune checkpoint,” “checkpoint pathway,” and “immune checkpoint pathway” refer to a pathway by which the binding of an immune checkpoint ligand to an immune checkpoint receptor modulates the amplitude and quality of the activation of immune cells (e.g., T cells, Jurkat cells, HuT-78, CEM, Molt-4, etc.).

[0132] As used herein “an immune checkpoint molecule” refers to at least the portion of an immune checkpoint molecule that is capable of binding a ligand thereof which modulates its activity. It is typically an immune checkpoint receptor. These immune checkpoint molecules are regulatory molecules that maintain immune homeostasis in physiological conditions. By sending T cells a series of co-stimulatory or co-inhibitory signals via receptors, immune checkpoints can both protect healthy tissues from adaptive immune response and activate lymphocytes to remove pathogens effectively. However, due to their mode of action, suppressive immune checkpoints serve as unwanted protection for cancer cells.

[0133] According to a specific embodiment, the immune checkpoint molecule is of an immune cell (e.g., PD-1) and the ligand is of a cancer cell (e.g., PD-L1).

[0134] As used herein, the term “ligand of an immune checkpoint molecule” or “immune checkpoint ligand” (“ICL”) refers to a ligand of an immune checkpoint receptor “Immune checkpoint ligands” are commonly surface-displayed proteins on antigen presenting cells (APCs) or tumor cells. Through an interaction with an immune-cell-displayed immune checkpoint receptor, an “immune checkpoint ligand” modulates the immune response of the immune cell (e.g., T cell) to the antigen presenting cell. Examples of immune checkpoint ligands that bind inhibitory immune checkpoint receptors include: PD-L1 , PD-L2, B7-H4, CD 155, galectin-9, and HVEM.

[0135] However, the terminology may be the other way around, as both ligand and receptor are typically membrane-anchored.

[0136] Examples of immune checkpoint molecules and their ligands that are contemplated according to some embodiments of the present disclousre are provided in Table 1.

[0137] Table 2. Examples of (negative) and stimulatory (positive) immune checkpoint ligandreceptor pairs with cellular distribution of these molecules under physiological conditions

(Marhelava et al., Cancers 2019, 11 , 1756).

[0138] Cancer supporters can include checkpoint molecules and checkpoint ligands described in

Table 2. In particular embodiments, the cancer supporter includes 2B4, A2aR, E37HI, B7H3, B7H4, BTLA, CCR7, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276, CTLA-4, DR3, GAL9, GITR, HAVCR2, HVEM, ID01 , ID02, ICOS, KIR, LAG3, LAIRI, LIGHT, MARCO, PD-1, PD-L1 , PD-L2, PD-L3, PD-L4, PS, OX-40, SLAM, TIGHT, TIGIT, TIM3, VISTA, or VTCNI.

[0139] Efflux pumps are membrane-based protein active transport channels found in the cytoplasmic membrane of living cells and are involved in removing endogenous wastes and xenobiotics out of the cell and protecting the cells from harmful effects of toxins and drugs. The overexpression of pumps leads to increased efflux of drugs/chemicals. Mammalian cell efflux pumps are monocarboxylate transporter (MCT), ATP-binding cassette transporters (ABC), peptide transporters (PEPTs), and Na+ phosphate transporters (NPTs). The ABC superfamily is one of the most prominent protein families containing 49 ABC genes in the human genome. The ABC proteins are classified into seven subfamilies; ABC-A to ABC-G. There are several ABC transporters that contribute to drug resistance by cancer cells including: M DR-associated protein (MRP1/ABCC1), breast cancer resistance protein (MXR or BCRP/ABCG2), and P-glycoprotein (MDR1/ABCB1). Additional ABC transporters include MRP/ABCC-2, 3, 4, 5, 6, or 7.

[0140] In particular embodiments, a cancer supporter includes an efflux pump (e.g., MRP1 , MXR, P-gp). In particular embodiments, a cancer supporter binding domain includes a binding domain that blocks or inhibits an efflux pump. In particular embodiments, a cancer supporter binding domain includes an anti-MRP1 binding domain, an anti-MXR binding domain, or an anti-P-gp binding domain.

[0141] In particular embodiments, the cancer supporter includes PD-L1. In particular embodiments, cancer supporter binding domain includes an anti-PD-L1 binding domain that binds to and inhibit PD-L1. PD-L1 (also known as CD274 or B7-H1, UniProt ID No. Q9NZQ7) can inhibit T-cell proliferation and cytokine production.

[0142] In particular embodiments, the anti-PD-L1 binding domain includes a miniprotein (i.e. , anti- PD-L1 miniprotein). In particular embodiments, the anti-PD-L1 miniprotein includes the sequence: EEDCKVHCVKEWMAGKACAERDKSYTIGRAHCSGQKFDVFKCLDHCAAP (SEQ ID NO: 89).

[0143] In particular embodiments, the anti-PD-L1 miniprotein includes the sequence: SEEDCKVHCVKEWMAGKACAERDKSYTIGRAHCSGQKFDVFKCLDHCAAPGS (SEQ ID NO: 204).

[0144] Additional examples of anti-PD-L1 miniproteins include:

EEDCKVHCVKEWAAYKACAERIKSDTTGQAHCSGQYFDFWKCVDHCAAP (SEQ ID NO: 90); EEDCKVHCVKEWAAYKACAERIKSYTIGRAHCSGQYFDVWKCLDHCAAP (SEQ ID NO: 91); EESCKPQCVKAWLEYQACAERVEKDESGEAHCTGQYFDYWHCVDKCAAK (SEQ ID NO: 92); EESCKPQCVKAWLEYQACAERVEKDESGEAHCTGQYFDLWGCVDKCVAP (SEQ ID NO: 93); ARTCESQSHRFKGPCVSDTNCASVCRTERFSGGHCRGFRRRCLCTKHC (SEQ ID NO: 94); ARTCESQSHRFKGPCVSDTMCASVCRTERFSGGHCRGFRRRCLCSKHC (SEQ ID NO: 95); EERCKPQCVKSLYEYEKCVKRVENDDTGHKHCTGQYFDYWSCIDKCVAS (SEQ ID NO: 96); and

EERCMPQCVKSLYEYEKCLKRVENDDTGHKHCTGHYFDYWSCIDKCVAS (SEQ ID NO: 97).

[0145] In particular embodiments, the anti-PD-L1 binding domain can be derived from an anti- PD-L1 antibody. Examples of a commercially available antibodies that block PD-L1 include nivolumab (Opdivo, Lawrence, NJ) and pembrolizumab (Keytruda, Rahway, NJ). An example of a neutralizing antibody that binds to and neutralizes PD-L1 is the monoclonal antibody 71213 (available from BPS Bioscience).

[0146] In particular embodiments, the cancer supporter includes an immune checkpoint molecule. In particular embodiments, the cancer supporter binding domain includes a binding domain that binds an epitope of programmed cell death protein 1 (PD-1) (i.e. , anti-PD-1 binding domain). PD- 1 , also called CD279 (UniProt ID No. Q15116) is an inhibitory cell surface receptor involved in regulating the T-cell immune response. In particular embodiments, an anti-PD-1 binding domain can include or be derived from an anti-PD-1 antibody. Anti-PD-1 antibodies include lambrolizumab (MK-3475, MERCK), nivolumab (BMS-936558, BRISTOL-MYERS SQUIBB), AMP-224 (MERCK), and pidilizumab (CT-011 , CURETECH LTD). These antibodies can be used to create PD-1 binding domain.

[0147] In certain examples, a cancer supporter binding domain includes an anti-PD-1 miniprotein (i.e., anti-PD-1 miniprotein). An anti-PD1 miniprotein includes PD-MP1 which is a hyperstable 40- residue miniprotein that specifically binds murine and human PD-1 at the PD-L1 interface with a Kd of 100 nM. PD-MP1 includes a sequence selected from: CLCWCARTKPFHRRYGKYLYGTRLQCKKWLSECAQQNPGARVNIQC (SEQ ID NO: 98);

GSAHIVMVDAYKPTKGDGGKGSDGEQKLISEEDLGKGSGGSSGCLCWCARTKPFHRRYGKYL YGTRLQCKKWLSECAQQNPGARVNIQC (SEQ ID NO: 99);

GAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISD GQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHILEGLNDIFEAQ KIEWHEGSGSGTKYELRRALEELEKALRELKKSLDELERSLEELEKNPSEDALVENNRLNVENN KIIVEVLRIIAEVLKIIAKSD (SEQ ID NO: 100); or GSAHIVMVDAYKPTKGDGGKGSDGEQKLISEEDLGKGSGGSSGCICWCTKTVPDGRRYWKY RYGNKLICKKWLSECQQKNPGAEINIQCMGSSHHHHHHSSGLVPRGSHMSGAMVDTLSGLSS EQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGK YTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHILEGLNDIFEAQKIEWHEGSGSGT KYELRRALEELEKALRELKKSLDELERSLEELEKNPSEDALVENNRLNVENNKIIVEVLRIIAEVLK IIAKSD (SEQ ID NO: 101).

[0148] Activated leukocyte cell adhesion molecule (ALCAM), also known as CD166, is a member of the immunoglobulin superfamily. In particular embodiments, a cancer supporter binding domain includes an anti-ALCAM binding domain. Anti-ALCAM binding domains can be derived from commercially available antibodies such as MAB656, eBioALC48 (eBioscienceTM, San Diego, CA), or 67768-1-lg.

[0149] C10orf54 is the human homolog of mouse VSIR which is a PD1 homolog. It is also referred to as V-domain Ig suppressor of T cell activation (VISTA) and is a type I transmembrane protein that functions as an immune checkpoint. In particular embodiments, a cancer supporter binding domain includes an anti-C10orf54 binding domain. Anti-C10orf54 binding domains can be derived from commercially available antibodies such as UMAB272, SAB4200644, or KVA12123.

[0150] Carcinoembryonic antigen-related cell adhesion molecule 1 (biliary glycoprotein) (CEACAM1) also known as CD66a is a human glycoprotein, and a member of the carcinoembryonic antigen (CEA) gene family. CEACAM1 functions as a MHC-class-l- independent inhibitory receptor on natural killer cells when ligated homophilically by CEACAM1 on target cells, such that overexpression of CEA CAM 1 on cancer cells might function as a means to avoid immune surveillance. In particular embodiments, a cancer supporter binding domain includes an anti-CEACAM1 binding domain. Anti-CEACAM1 binding domains can be derived from commercially available antibodies such as EPR26490-88, BLR032F, or MA5-23985.

[0151] Carcinoembryonic antigen-related cell adhesion molecule 20 (CEACAM20) is a carcinoembryonic antigen. It is found in various tissues and cells, including erythrocytes, endothelial cells, keratinocytes, and mammary epithelial cells. In particular embodiments, a cancer supporter binding domain includes an anti-CEACAM20 binding domain. Anti-CEACAM20 binding domains can be derived from commercially available antibodies such as HT-12D8, PA5- 103711 , or PA5-70882.

[0152] Chemokine like receptor 1 (CMKLR1) also known as ChemR23 (Chemerin Receptor 23) is expressed by circulating plasmocytoid dendritic cells in normal individuals and patients suffering from skin diseases, such as psoriasis and atopic dermatitis. In particular embodiments, a cancer supporter binding domain includes an anti-CMKLR1 binding domain. Anti-CMKLR1 binding domains can be derived from commercially available antibodies such as LS-B12924, 7H10L22, or PA5-50932.

[0153] Human leukocyte antigen E (HLA-E) is a protein that's part of the major histocompatibility complex (MHC) class I and is involved in the body's immune response. In particular embodiments, a cancer supporter binding domain includes an anti-HLA-E binding domain. Anti-HLA-E binding domains can be derived from commercially available antibodies such as EPR25300-104, MEM- E/02, or MEM-E/07.

[0154] Interferon-induced transmembrane protein (IFITM)1 is a part of the IFITM family which is involved in suppressing the early stage of viral replication. In particular embodiments, a cancer supporter binding domain includes an anti-l FITM 1 binding domain. Anti-IFITM1 binding domains can be derived from commercially available antibodies such as EPR22620-12, F-12, or 60074-1- IG.

[0155] In particular embodiments, a cancer supporter binding domain includes an anti-IFITM2 binding domain. Anti-l FITM2 binding domains can be derived from commercially available antibodies such as AF4834, 3D5F7, 66081-1-lg, CL488-66081 , or A-6.

[0156] In particular embodiments, a cancer supporter binding domain includes an anti-IFITM3 binding domain. Anti-l FITM3 binding domains can be derived from commercially available antibodies such as 11714-1-AP, PA5-11274, PA5-30382, or MA5-32798.

[0157] Interleukin 31 receptor A (IL31 RA) associates with OSMR to activate STAT3 signaling. IL31RA promotes basal-like breast cancer progression and metastasis. In particular embodiments, a cancer supporter binding domain includes an anti-l L31 RA binding domain. Anti- IL31RA binding domains can be derived from commercially available antibodies such as AF2769, ab113498, or AA 401-500.

[0158] Leukocyte Specific Transcript 1 (LST1) is a small adaptor protein expressed in leukocytes of myeloid lineage and may have a role in modulating immune responses. In particular embodiments, a cancer supporter binding domain includes an anti-LST1 binding domain. Anti- LST1 binding domains can be derived from commercially available antibodies such as 7E2, EPR27070-49, MDQ, or NBP1-45072.

[0159] Familial Mediterranean fever (MEFV) encodes a protein called pyrin which regulates inflammation. In particular embodiments, a cancer supporter binding domain includes an anti- MEFV binding domain. Anti-MEFV binding domains can be derived from commercially available antibodies such as ABIN2779421 , 24280-1-AP, or PA5-18410.

[0160] Oncostatin M receptor (OSMR) signaling reprograms fibroblasts to promote pancreatic cancer growth and metastasis. In particular embodiments, a cancer supporter binding domain includes an anti-OSMR binding domain. Anti-OSMR binding domains can be derived from commercially available antibodies such as EPR28222-64, EPR24786-50, AN-V2, or AA 503-749. [0161] Programmed cell death 1 ligand 2 (PD-L2 or PDL2) is a protein that regulates the adaptive immune response and plays a role in tumor progression. In particular embodiments, a cancer supporter binding domain includes an anti-PDL2 binding domain. Anti-PDL2 binding domains can be derived from commercially available antibodies such as B7-DC, CAL28, or TY25 (Ab21107, available from Abeam).

[0162] PIM1 (proto-oncogene serine/threonine kinase) is up-regulated by hypoxia in hepatocellular carcinoma and promotes tumor growth and metastasis by facilitating cancer cell glycolysis. In particular embodiments, a cancer supporter binding domain includes an anti-PIM1 binding domain. Anti-PIM1 binding domains can be derived from commercially available antibodies such as G-11, 12H8, #2907, or G.360.1.

[0163] PIM2 promotes hepatocellular carcinoma tumorigenesis and progression through activating NF-kappaB signaling pathway. In particular embodiments, a cancer supporter binding domain includes an anti-PIM2 binding domain. Anti-PIM2 binding domains can be derived from commercially available antibodies such as EPR6987, CHU61 B, 5H66L51 , 3V1S5, or JE55-57.

[0164] Phospholipase A And Acyltransferase 3 (PLAAT3) is involved in several processes including lipid metabolic processes and negative regulation of cell cycle. In particular embodiments, a cancer supporter binding domain includes an anti-PLAAT3 binding domain. Anti- PLAAT3 binding domains can be derived from commercially available antibodies such as HPA011749, ABIN335142, or 14G11.

[0165] Preferentially Expressed Antigen in Melanoma (PRAME) is a tumor-associated antigen expressed in many types of cancers. In particular embodiments, a cancer supporter binding domain includes an anti-PRAME binding domain. Anti-PRAME binding domains can be derived from commercially available antibodies such as EPR20330, E7I1 B, EP461 , or 11438-1-AP.

[0166] Syndecan binding protein (SDCBP) modulates tumor microenvironment, tumor progression and anti-PD1 efficacy in colorectal cancer. In particular embodiments, a cancer supporter binding domain includes an anti-SDCBP binding domain. Anti-SDCBP binding domains can be derived from commercially available antibodies such as HPA023840, 2C12, ABIN562830, or ABIN62664952.

[0167] A B7-H3 binding domain can be derived from, for example, an anti-B7-H3 antibody such as MGA271. A CTLA-4 binding domain may be derived from, for example, an anti-CTLA-4 antibody, such as ipilimumab (Bristol-Myers Squibb) or tremelimumab (PFIZER). A LAG3 binding domain may be derived from an anti-LAG3 antibody, such as IMP321 , a soluble Ig fusion protein. [0168] (V) Multimerization Domains and Linkers. An ICEm includes three or more binding domains disclosed herein. Binding domains can multimerize to form an ICEm using a multimerization domain or a linker.

[0169] A “multimerization domain” is a domain that causes two or more monomers (e.g., proteins) to interact with each other through covalent and/or non-covalent association(s). Multimerization domains are highly conserved protein sequences that can include different types of sequence motifs such as leucine zipper, helix loop-helix, ankyrin and PAS (Feuerstein et al, Proc. Natl. Acad. Sci. USA, 91 :10655-10659, 1994). Multimerization domains present in proteins can bind to form dimers, trimers, tetramers, pentamers, hexamers, heptamers, etc., depending on the number of units/monomers incorporated into the multimer, and/or homomultimers or heteromultimers, depending on whether the binding monomers are the same type or a different type (US Patent No. 10030065).

[0170] Dimerization domains can include protein sequence motifs such as coiled coils, acid patches, zinc fingers, calcium hands, a CH1-CL pair, an "interface" with an engineered "knob" and/or "protruberance" (US 5821333), leucine zippers (US 5932448), SH2 and SH3 (Vidal et al., Biochemistry, 43:7336-44, 2004), PTB (Zhou et al., Nature, 378:584-592, 1995), WW (Sudol Prog Biochys MoL Bio, 65:113-132, 1996), PDZ (Kim et al., Nature, 378: 85-88, 1995; Komau et al., Science, 269:1737-1740, 1995) and WD40 (Hu et al., J Biol Chem., 273:33489-33494, 1998). Additional examples of molecules that contain dimerization domains/motifs are receptor dimer pairs such as the interleukin-8 receptor (IL-8R), integrin heterodimers such as LFA-I and GPU Ib/ll la, dimeric ligand polypeptides such as nerve growth factor (NGF), neurotrophin-3 (NT- 3), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, PDGF members, and brain-derived neurotrophic factor (BDNF) (Arakawa et al., J Biol. Chem., 269:27833-27839, 1994; Radziejewski et al., Biochem, 32: 1350, 1993) and variants of some of these domains with modified affinities (PCT Publication No. WO 2012/001647).

[0171] In particular embodiments, the ICEm can be prepared using knobs-into holes techniques. Knobs-into-holes refers to forcing the pairing of two different binding domains (e.g., antibody heavy chains) by introducing mutations into the structure (e.g., in the CH3 domains) to modify the contact interface. For example, on one chain bulky amino acids are replaced by amino acids with short side chains to create a ‘hole’. Conversely, amino acids with large side chains were introduced into the other CH3 domain, to create a ‘knob’. By co-expressing these two heavy chains, high yields of heterodimer formation (‘knob-hole’) versus homodimer formation (‘holehole’ or ‘knob-knob’) is observed (Ridgway, J. B., Protein Eng. 9 (1996) 617-621 ; and WO 96/027011).

[0172] In particular embodiments, the ‘knob’ and/or the ‘hole’ may exist in the original polypeptide or may be introduced synthetically (e.g. by altering nucleic acid encoding the polypeptide). To synthetically introduce a knob and/or hole, the nucleic acid encoding the original amino acid residue (or other non-amino acid groups such as, for example carbohydrate groups) in the interface of the polypeptide is replaced with DNA encoding at least one import amino acid residue, wherein the interface refers to amino acid residues in contact between a first heavy chain constant region and one or more amino acid residues (or other non-amino acid groups) in a second heavy chain constant region.. The preferred import residues for the formation of a hole are amino acids with smaller side chain volumes than the original amino acid residue such as alanine (A), serine (S), threonine (T), valine (V), or glycine (G). The preferred import residues for the formation of a knob are amino acids with larger side chain volumes than the original amino acid residue such as tyrosine (Y), arginine (R), phenylalanine (F), or tryptophan (W). The percentage of heterodimer can be increased by remodeling the interaction surfaces of the two CH3 domains using a phage display approach and the introduction of a disulfide bridge to stabilize the heterodimers (Merchant A. M, et al., Nature Biotech 16 (1998) 677-681 ; Atwell, S., et al., J. Mol. Biol. 270 (1997) 26-35). [0173] In particular embodiments, the ICEm includes (i) a first polypeptide chain including a mutated human lgG1 Fc such that it has a knob mutation (referred to as lgG1 Fc Knob) and (ii) a second polypeptide chain including a mutated human lgG1 Fc such that it has a hole mutation (referred to as lgG1 Fc Hole).

[0174] In particular embodiments, the lgG1 Fc Knob includes the sequence: EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 125) with the mutations underlined and the lgG1 Fc Hole includes the sequence: EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 126) with the mutations underlined.

[0175] In particular embodiments, the lgG1 Fc Knob with the LALAPG Fc mutation includes the sequence: EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 205) and the lgG1 Fc Hole with the LALAPG Fc mutation includes the sequence: EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 206).

[0176] In particular embodiments, the sequence corresponding to a dimerization motif/domain includes the leucine zipper domain of Jun (US5932448; RIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMN (SEQ ID NO: 117)), the dimerization domain of Fos (US 5932448; LTDTLQAETDQLEDKKSALQTEIANLLKEKEKLEFILAA (SEQ ID NO: 118)), a consensus sequence for a WW motif (PCT Publication No. WO 1997/037223), the dimerization domain of the SH2B adapter protein from GenBank Accession no. AAF73912.1 (Nishi et al., Mol Cell Biol, 25: 2607-2621 , 2005;

WREFCESHARAAALDFARRFRLYLASHPQYAGPGAEAAFSRRFAELFLQHFEAEVARAS (SEQ ID NO: 119)), the SH3 domain of IB1 from GenBank Accession no. AAD22543.1 (Kristensen el al., EMBO J., 25: 785-797, 2006;

THRAIFRFVPRHEDELELEVDDPLLVELQAEDYWYEAYNMRTGARGVFPAYYAIE (SEQ ID NO: 76)), the PTB domain of human DOK-7 from GenBank Accession no. NP_005535.1 (Wagner et al., Cold Spring Harb Perspect Biol. 5: a008987, 2013;

LGEVHRFHVTVAPGTKLESGPATLHLCNDVLVLARDIPPAVTGQWKLSDLRRYGAVPSGFIFEG GTRCGYWAGVFFLSSAEGEQISFLFDCIVRGISPTKG (SEQ ID NO: 102)), the PDZ-like domain of SATB1 from UniProt Accession No. Q01826 (Galande et al., Mol Cell Biol. Aug; 21 : 5591- 5604, 2001;

DCKEEHAEFVLVRKDMLFNQLIEMALLSLGYSHSSAAQAKGLIQVGKWNPVPLSYVTDAPDAT VADMLQDVYHVVTLKIQLHSCPKLEDLPPEQWSHTTVRNALKDLLKDMNQSS (SEQ ID NO: 133)), the WD40 repeats of APAF from UniProt Accession No. 014727 (Jorgensen et al., 2009. PLOS One. 4(12):e8463;

CAPWPMVEKLIKQCLKENPQERPTSAQVFDILNSAELVCLTRRILLPKNVIVECMVATHHNSRN ASIWLGCGHTDRGQLSFLDLNTEGYTSEEVADSRILCLALVHLPVEKESWIVSGTQSGTLLVINT EDGKKRHTLEKMTDSVTCLYCNSFSKQSKQKNFLLVGTADGKLAIFEDKTVKLKGAAPLKILNIG NVSTPLMCLSESTNSTERNVMWGGCGSQLFSYAAFSDSNIITVVVDTALYIAKQNSPWEVWD KKTEKLCGLIDCVHFLREVMVKETKIFSFSNDFTIQKLIETRTNKESKHKMSYSGRVKTLCLQKN TALWIGTGGGHILLLDLSTRRLIRVIYNFCNSVRVMMTAQLGSLKNVMLVLGYNRKNTEGTQKQ KEIQSCLTVWDINLPHEVQNLEKHIEVRKELAEKMRRTSVE (SEQ ID NO: 134)), the PAS motif of the dioxin receptor from UniProt Accession No. I6L9E7 (Pongratz et al., Mol Cell Biol, 18:4079- 4088, 1998;

DQELKHLILEAADGFLFIVSCETGRVVYVSDSVTPVLNQQQSEWFGSTLYDQVHPDDVDKLRE QLSTSENALTGR (SEQ ID NO: 135)) and the EF hand motif of parvalbumin from UniProt Accession No. P20472 (Jamalian et al., Int J Proteomics, 2014: 153712, 2014; LSAKETKMLMAAGDKDGDGKIGVDEFSTLVAES (SEQ ID NO: 136)).

[0177] In particular embodiments, the dimerization domain can be a dimerization and docking domain (DDD) on one nanobody and an anchoring domain (AD) on another nanobody to facilitate a stably tethered structure. In particular embodiments, the DDD (DDD1 and DDD2) are derived from the regulatory subunits of a cAMP-dependent protein kinase (PKA), and the AD (AD1 and AD2) are derived from a specific region found in various A-kinase anchoring proteins (AKAPs) that mediates association with the R subunits of PKA. In particular embodiments, DDD1 includes the amino acid sequence: SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 143). In particular embodiments, DDD2 includes the amino acid sequence: CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 144). In particular embodiments, AD1 includes the amino acid sequence: QIEYLAKQIVDNAIQQA (SEQ ID NO: 145). In particular embodiments, AD2 includes the amino acid sequence: CGQIEYLAKQIVDNAIQQAGC (SEQ ID NO: 146). However, one skilled in the art will realize that other DDDs and ADs are known and can be used such as: the 4-helix bundle type DDD domains may be obtained from p53, DCoH (pterin 4 alpha carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor 1 alpha (TCF1)) and HNF-1 (hepatocyte nuclear factor 1). Other AD sequences of potential use may be found in Patent Publication No. US2003/0232420A1.

[0178] The X-type four-helix bundle dimerization motif that is a structural characteristic of the DDD (Newlon, et al. EMBO J. 2001 ; 20: 1651-1662; Newlon, et al. Nature Struct Biol. 1999; 3: 222-227) is found in other classes of proteins, such as the S100 proteins (for example, S100B and calcyclin), and the hepatocyte nuclear factor (HNF) family of transcriptional factors (for example, HNF-1 a and HNF-1 P). Over 300 proteins that are involved in either signal transduction or transcriptional activation also contain a module of 65-70 amino acids termed the sterile a motif (SAM) domain, which has a variation of the X-type four-helix bundle present on its dimerization interface. For S100B, this X-type four-helix bundle enables the binding of each dimer to two p53 peptides derived from the c-terminal regulatory domain (residues 367-388) with micromolar affinity (Rustandi, et al. Biochemistry. 1998; 37: 1951-1960). Similarly, the N-terminal dimerization domain of HNF- 1a (HNF-p1) was shown to associate with a dimer of DCoH (dimerization cofactor for HNF-1) via a dimer of HNF-p1 (Rose, et al. Nature Struct Biol. 2000; 7: 744-748). In alternative embodiments, these naturally occurring systems can also be used to provide stable multimeric structures with multiple functions or binding specificities. Other binding events such as those between an enzyme and its substrate/inhibitor, for example, cutinase and phosphonates (Hodneland, et al. Proc Natl Acd Sci USA. 2002; 99: 5048-5052), may also be utilized to generate the two associating components (the “docking” step), which are subsequently stabilized covalently (the “lock” step).

[0179] In particular embodiments, dimerization of nanobodies can be induced by a chemical inducer. This method of dimerization requires one nanobody to contain a chemical inducer of dimerization binding domain 1 (CBD1) and the second nanobody to contain the second chemical inducer of dimerization binding domain (CBD2), wherein CBD1 and CBD2 are capable of simultaneously binding to a chemical inducer of dimerization (CID). If the CID is rapamycin, CBD1 and CBD2 can be the rapamycin binding domain of FK-binding protein 12 (FKBP12) and the FKBP12-Rapamycin Binding (FRB) domain of mTOR. In particular embodiments, FKBP12 includes the sequence:

MGVQVETISPGDGRTFPKRGQTCWHYTGMLEDGKKFDSSRDRNPFKFMLGKQEVIRGWEEG VAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 120).

[0180] In particular embodiments, FRB includes the sequence:

MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRD LMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKLES (SEQ ID NO: 121). If the CID is FK506/cyclosporin fusion protein or a derivative thereof, CBD1 and CBD2 can be the FK506 (Tacrolimus) binding domain of FK-binding protein 12 (FKBP12) and the cyclosporin binding domain of cylcophilin A. If the CID is estrone/biotin fusion protein or a derivative thereof, CBD1 and CBD2 can be an oestrogen-binding domain (EBD) and a streptavidin binding domain. If the CID is dexamethasone/methotrexate fusion molecule or a derivative thereof, CBD1 and CBD2 can be a glucocorticoid-binding domain (GBD) and a dihydrofolate reductase (DHFR) binding domain. If the CID is O6-benzylguanine derivative/methotrexate fusion molecule or a derivative thereof, CBD1 and CBD2 can be an O6-alkylguanine-DNA alkyltransferase (AGT) binding domain and a dihydrofolate reductase (DHFR) binding domain. If the CID is RSL1 or a derivative thereof, CBD1 and CBD2 can be a retinoic acid receptor domain and an ecodysone receptor domain. If the CID is AP1903 or a derivative thereof, CBD1 and CBD2 can be the FK506 binding protein (FKBP12) binding domains including a F36V mutation. Use of the CID binding domains can also be used to alter the affinity to the CID. For instance, altering amino acids at positions 2095, 2098, and 2101 of FRB can alter binding to Rapamycin: KTW has high, KHF intermediate and PLW is low (Bayle et al, Chemistry & Biology 13, 99-107, January 2006).

[0181] In particular embodiments, nanobodies can multimerize using a transmembrane polypeptide derived from a FCERI chain. In particular embodiments, a nanobody can include a part of a FCERI alpha chain and another nanobody can include a part of an FCERI beta chain or variant thereof such that said FCERI chains spontaneously dimerize together to form a dimeric nanobody. In particular embodiments, nanobodies can include a part of a FCERI alpha chain and a part of a FCERI gamma chain or variant thereof such that said FCERI chains spontaneously trimerize together to form a trimeric nanobody, and in another embodiment the multi-chain nanobody can include a part of FCERI alpha chain, a part of FCERI beta chain and a part of FCERI gamma chain or variants thereof such that said FCERI chains spontaneously tetramerize together to form a tetrameric nanobody.

[0182] In particular embodiments, additional methods of causing dimerization can be utilized. Additional modifications to generate a dimerization domain in nanobody could include: replacing the C-terminus domain with murine counterparts; generating a second interchain disulfide bond in the C-terminus domain by introducing a second cysteine residue into both nanobodies; swapping interacting residues in each of the nanobodies in the C-terminus domains (“knob-in- hole”); and fusing the variable domains of the nanobodies directly to CD3 (CD3 fusion) (Schmitt et al., Hum. Gene Ther. 2009. 20:1240-1248).

[0183] In particular embodiments, an ICEm can be formed by linking binding domains (e.g., single-domain antibodies) together with linkers. In certain aspects, multimerization is achieved by linking single-domain antibodies in a fusion protein with protein linkers. Fusion proteins include different protein domains (e.g., single-domain antibodies) linked to each other directly or through intervening linker segments such that the function of each included domain is retained.

[0184] Commonly used flexible linkers include linker sequences with the amino acids glycine and serine (Gly-Ser linkers). In particular embodiments, the linker sequence includes sets of glycine and serine repeats such as from one to ten repeats of (GlyxSery)n, wherein x and y are independently an integer from 0 to 10 provided that x and y are not both 0 and wherein n is an integer of 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10). Particular examples include (Gly4Ser)n (SEQ ID NO: 103), (Gly3Ser)n(Gly4Ser)n (SEQ ID NO: 104), (Gly3Ser)n(Gly2Ser)n (SEQ ID NO: 105), and (Gly3Ser)n(Gly4Ser)1 (SEQ ID NO: 106). In particular embodiments, the linker is (Gly4Ser)4 (SEQ ID NO: 107), (Gly4Ser)3 (SEQ ID NO: 108), (Gly4Ser)2 (SEQ ID NO: 109), (Gly4Ser)1 (SEQ ID NO: 110), (Gly3Ser)2 (SEQ ID NO: 111), (Gly3Ser)1 (SEQ ID NO: 112), (Gly2Ser)2 (SEQ ID NO: 113) or (Gly2Ser)1, GGSGGGSGGSG (SEQ ID NO: 114), GGSGGGSGSG (SEQ ID NO: 115), or GGSGGGSG (SEQ ID NO: 116).

[0185] In some situations, flexible linkers may be incapable of maintaining a distance or positioning of binding domains needed for a particular use. In these instances, rigid or semi-rigid linkers may be useful. Examples of rigid or semi-rigid linkers include proline-rich linkers. In particular embodiments, a proline-rich linker is a peptide sequence having more proline residues than would be expected based on chance alone. In particular embodiments, a proline-rich linker is one having at least 30%, at least 35%, at least 36%, at least 39%, at least 40%, at least 48%, at least 50%, or at least 51 % proline residues. Particular examples of proline-rich linkers include fragments of proline-rich salivary proteins (PRPs).

[0186] Linkers can also include one or more antibody hinge regions and/or immunoglobulin heavy chain constant regions, such as CH3 alone or a CH2CH3 sequence. Additional examples of linkers can be found in Chen et al., Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357-1369. Linkers can be flexible, rigid, or semi-rigid, depending on the desired functional domain presentation to a target.

[0187] (VI) Variants & Nucleic Acid Sequences. Amino acid and nucleic acid sequence variations of the binding domains or ICEm disclosed herein are contemplated. Variations can include additions, deletions or substitutions of residues within the amino acid sequences. Variations of sequences disclosed herein include CDR variants, variant Fc regions, humanized antibodies [0188] In particular embodiments, variants of the sequences disclosed herein leave CDR sequences unchanged. In particular embodiments, variants of the sequences disclosed herein change the CDR sequences by less than 80%, less than 85%, less than 90%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or less than 100% compared to the sequence disclosed herein.

[0189] Variants of the sequences disclosed and referenced herein are also included. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity can be found using computer programs well known in the art, such as DNASTAR™ (Madison, Wisconsin) software. Preferably, amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.

[0190] In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. Co., p. 224). Naturally occurring amino acids are generally divided into conservative substitution families as follows: Group 1 : Alanine (Ala), Glycine (Gly), Serine (Ser), and Threonine (Thr); Group 2: (acidic): Aspartic acid (Asp), and Glutamic acid (Glu); Group 3: (acidic; also classified as polar, negatively charged residues and their amides): Asparagine (Asn), Glutamine (Gin), Asp, and Glu; Group 4: Gin and Asn; Group 5: (basic; also classified as polar, positively charged residues): Arginine (Arg), Lysine (Lys), and Histidine (His); Group 6 (large aliphatic, nonpolar residues): Isoleucine (lie), Leucine (Leu), Methionine (Met), Valine (Vai) and Cysteine (Cys); Group 7 (uncharged polar): Tyrosine (Tyr), Gly, Asn, Gin, Cys, Ser, and Thr; Group 8 (large aromatic residues): Phenylalanine (Phe), Tryptophan (Trp), and Tyr; Group 9 (nonpolar): Proline (Pro), Ala, Vai, Leu, lie, Phe, Met, and Trp; Group 11 (aliphatic): Gly, Ala, Vai, Leu, and lie; Group 10 (small aliphatic, nonpolar or slightly polar residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing): Met and Cys. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

[0191] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: lie (+4.5); Vai (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); Glutamate (-3.5); Gin (-3.5); aspartate (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).

[0192] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity.

[0193] As detailed in US 4,554,101 , the following hydrophilicity values have been assigned to amino acid residues: Arg (+3.0); Lys (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); Ser (+0.3); Asn (+0.2); Gin (+0.2); Gly (0); Thr (-0.4); Pro (-0.5+1); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Vai (-1.5); Leu (-1.8); lie (-1.8); Tyr (-2.3); Phe (-2.5); Trp (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0194] In particular embodiments, a binding domain of the present disclosure can be derived from or based on a binding domain of a known antibody (e.g., single domain antibody) and can include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the binding domain of the known antibody. An insertion, deletion or substitution may be anywhere in the VH region, including at the amino- or carboxy-terminus or both ends of this region, provided that each CDR includes zero changes or at most one, two, or three changes and provided a binding domain including the modified VH region can still specifically bind its target with an affinity similar to the wild type binding domain.

[0195] As outlined above, amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. As indicated elsewhere, variants of gene sequences can include codon optimized variants, sequence polymorphisms, splice variants, and/or mutations that do not affect the function of an encoded product to a statistically-significant degree.

[0196] Variants of the protein and nucleic acid sequences disclosed herein also include sequences with at least 70% sequence identity, 80% sequence identity, 85% sequence, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to the protein and nucleic acid sequences described or disclosed herein.

[0197] In particular embodiments, a binding domain includes or is a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to a known amino acid sequence of a heavy chain variable region (VH), or both, wherein each CDR includes zero changes or at most one, two, or three changes, from an antibody (e.g., single domain antibody) or fragment or derivative thereof that specifically binds to target of interest.

[0198] Variants also include nucleic acid molecules that hybridize under stringent hybridization conditions to a sequence disclosed herein and provide the same function as the reference sequence. Exemplary stringent hybridization conditions include an overnight incubation at 42 °C in a solution including 50% formamide, 5XSSC (750 mM NaCI, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5XDenhardt's solution, 10% dextran sulfate, and 20 pg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1XSSC at 50 °C. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, moderately high stringency conditions include an overnight incubation at 37°C in a solution including 6XSSPE (20XSSPE=3M NaCI; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 pg/ml salmon sperm blocking DNA; followed by washes at 50 °C with 1XSSPE, 0.1 % SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5XSSC). Variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

[0199] “% sequence identity” refers to a relationship between two or more sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between protein and nucleic acid sequences as determined by the match between strings of such sequences. "Identity" (often referred to as "similarity") can be readily calculated by known methods, including (but not limited to) those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple alignment of the sequences can also be performed using the Clustal method of alignment (Higgins and Sharp CABIOS, 5, 151-153 (1989) with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Relevant programs also include the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wsconsin); BLASTP, BLASTN, BLASTX (Altschul, et al., J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the PASTA program incorporating the Smith- Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. I nt. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N.Y.. Wthin the context of this disclosure it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the program referenced. "Default values" will mean any set of values or parameters, which originally load with the software when first initialized.

[0200] CDR sequences within binding domains can be based on LlamaMagic or based on other methods known in the art. For example, definitive delineation of a CDR and identification of residues including the binding site of a binding domain can be accomplished by solving the structure of the binding domain and/or solving the structure of the binding domain-epitope complex. In particular embodiments, this can be accomplished by methods such as X-ray crystallography. Alternatively, CDRs are determined by comparison to known nanobodies (linear sequence) and without resorting to solving a crystal structure.

[0201] In addition to LlamaMagic, CDR sets can be based on, for example, Kabat numbering (Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme)); Chothia (Al- Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme)), Martin (Abinandan et al., Mol Immunol. 45:3832-3839 (2008), “Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains”), Gelfand, Contact (MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745.” (Contact numbering scheme)), IMGT (Lefranc M P et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 January; 27(1):55-77 (“IMGT” numbering scheme)), AHo (Honegger A and Pluckthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun. 8; 309(3):657-70, (AHo numbering scheme)), North (North et al., J Mol Biol. 406(2) :228-256 (2011), “A new clustering of antibody CDR loop conformations”), or other numbering schemes.

[0202] Software programs and bioinformatic tools, such as ABodyBuilder and Paratome can also be used to determine CDR sequences.

[0203] In particular embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody, thereby generating an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., a human IgG 1 , lgG2, lgG3 or lgG4 Fc region) including an amino acid modification (e.g., a substitution) at one or more amino acid positions. An “Fc region variant” includes an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, e.g. from one to ten amino acid substitutions, and preferably from one to five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein will preferably possess at least 80% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, and most preferably at least 90% homology therewith, more preferably at least 95% homology therewith. Numerous Fc modifications are known in the art, and a representative sampling of such possible modifications are described elsewhere herein. [0204] Binding domains can be humanized. Humanized binding domains have lowered immunogenicity in humans and have a lower number of non-immunogenic epitopes compared to non-humanized binding domains.

[0205] A “humanized” antibody refers to a chimeric antibody including amino acid residues from non-human CDRs and amino acid residues from human FRs. In particular embodiments, a humanized antibody will include substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0206] Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633, 2008, and are further described, e.g., in Riechmann et al., Nature 332:323-329, 1988; Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033, 1989; U.S. Pat. Nos. 5,821 ,337, 7,527,791 , 6,982,321 , and 7,087,409; Kashmiri et al., Methods 36:25- 34, 2005 (describing SDR (a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498, 1991 (describing “resurfacing”); Dall'Acqua et al., Methods 36:43-60,2005 (describing “FR shuffling”); and Osbourn et al., Methods 36:61-68, 2005 and Klimka et al., Br. J. Cancer, 83:252-260, 2000 (describing the “guided selection” approach to FR shuffling). EP-B-0239400 provides additional description of “CDR-grafting”, in which one or more CDR sequences of a first antibody is/are placed within a framework of sequences not of that antibody, for instance of another antibody.

[0207] Human framework regions that may be used for humanization include: framework regions selected using the “best-fit” method (see, e.g., Sims et al. J. Immunol. 151 :2296, 1993); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al., Proc. Nati. Acad. Sci. USA, 89:4285, 1992; and Presta et al., J. Immunol., 151 :2623, 1993); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633, 2008); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684, 1997; and Rosok et al., J. Biol. Chem. 271 :22611-22618, 1996).

[0208] In particular embodiments, variants have the same or increased binding to a targeted antigen as the variant’s original reference sequence. "Bind" means that the binding domain associates with its target epitope with a dissociation constant (1(D) of 10'⁸ M or less, in particular embodiments of from 10^-5 M to 10¹³ M, in particular embodiments of from 10^-5 M to 10¹⁰ M, in particular embodiments of from 10^-5 M to 10^-7 M, in particular embodiments of from 10’⁸ M to 10’ ¹³ M, or in particular embodiments of from 10’⁹ M to 10^-13 M. The term can be further used to indicate that the binding molecule does not bind to other biomolecules present, (e.g., it binds to other biomolecules with a dissociation constant (K_D) of 10'⁴ M or more, in particular embodiments of from 10’⁴ M to 1 M). A targeted epitope is one that will be bound by its corresponding ICEm under relevant in vitro conditions and in in vivo conditions as described herein. In particular embodiments, relevant in vitro conditions for binding can include a buffered salt solution approximating physiological pH (7.4) at room temperature or 37°C.

[0209] (VII) Expression of Immune Cell Engaging Molecules. The present disclosure includes methods of producing the ICEm disclosed herein. In particular embodiments, the method includes vector construction and expression within a host. In particular embodiments, the method includes nucleic acid synthesis and codon optimization, vector construction, expression within a host cell, and purification.

[0210] Particular embodiments utilize genetic constructs (e.g., chimeric genes, expression cassettes, expression vectors, recombination vectors, etc.) including a nucleic acid sequence encoding the protein or proteins of interest (i.e., coding sequence) operatively linked to appropriate expression control sequences. These genetic constructs are not naturally-occurring DNA molecules and are useful for introducing DNA into host-cells to express selected proteins of interest.

[0211] Operatively linked refers to the linking of DNA sequences (including the order of the sequences, the orientation of the sequences, and the relative spacing of the various sequences) in such a manner that the encoded protein is expressed. Methods of operatively linking expression control sequences to coding sequences are well known in the art. See, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N. Y., 1982; and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N. Y., 1989.

[0212] Expression control sequences are nucleic acid (e.g., DNA) sequences involved in any way in the control of transcription or translation. Suitable expression control sequences and methods of making and using them are well known in the art. Expression control sequences generally include a promoter. The promoter may be inducible or constitutive. It may be naturally-occurring, may be composed of portions of various naturally-occurring promoters, or may be partially or totally synthetic. Guidance for the design of promoters is provided by studies of promoter structure, such as that of Harley and Reynolds, Nucleic Acids Res., 15, 2343-2361, 1987. Also, the location of the promoter relative to the transcription start may be optimized. See, e.g., Roberts et al., Proc. Natl. Acad. Sci. USA, 76:760-764, 1979.

[0213] The promoter may include, or be modified to include, one or more enhancer elements. In particular embodiments, the promoter will include a plurality of enhancer elements. Promoters including enhancer elements can provide for higher levels of transcription as compared to promoters that do not include them.

[0214] For efficient expression, the coding sequences can be operatively linked to a 3' untranslated sequence. In particular embodiments, the 3' untranslated sequence can include a transcription termination sequence and a polyadenylation sequence. The 3' untranslated region can be obtained, for example, from the flanking regions of genes.

[0215] In particular embodiments, a 5' untranslated leader sequence can also be employed. The 5' untranslated leader sequence is the portion of a nucleic acid sequence that extends from the 5' CAP site to the translation initiation codon.

[0216] In particular embodiments, the genetic construct includes sequence encoding linkers. Commonly used linkers are described elsewhere herein. In particular embodiments, the linker includes the sequence GS. In particular embodiments, the linker includes the sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 108).

[0217] In particular embodiments, the genetic construct includes a sequence encoding a signal peptide sequence. In particular embodiments, the signal peptide includes the sequence: METDTLLLWVLLLWVPGSTG (SEQ ID NO: 124).

[0218] In addition to the coding sequence and expression control sequence, genetic constructs can include a sequence encoding a tag. Example tags include tags can include, for example, His tag (HHHHHH (SEQ ID NO: 123)), Flag tag (DYKDDDD (SEQ ID NO: 207), Xpress tag (DLYDDDDK (SEQ ID NO: 208)), Avi tag (GLNDIFEAQKIEWHE (SEQ ID NO: 122)), Calmodulin binding peptide (CBP) tag (KRRWKKNFIAVSAANRFKKISSSGAL (SEQ ID NO: 209)), Polyglutamate tag (EEEEEE (SEQ ID NO: 210)), HA tag (YPYDVPDYA (SEQ ID NO: 211)), Myc tag (EQKLISEEDL (SEQ ID NO: 212)), Strep tag (WRHPQFGG (SEQ ID NO: 213)), STREP® tag II (WSHPQFEK (SEQ ID NO: 214); IBA Institutfur Bioanalytik, Germany; see, e g., US 7,981,632), Softag 1 (SLAELLNAGLGGS (SEQ ID NO: 215)), Softag 3 (TQDPSRVG (SEQ ID NO: 216)), and V5 tag (GKPIPNPLLGLDST (SEQ ID NO: 217)). In particular embodiments, the tag includes the sequence AWSHPQFEK (SEQ ID NO: 218). [0219] In particular embodiments, a “hisavi” tag can be added to the N-terminus or C-terminus of a gene by the addition of nucleotides coding for the Avitag amino acid sequence (SEQ ID NO: 122), as well as the 6xhistidine tag coding sequence (SEQ ID NO: 123). The Avitag avidity tag can be biotinylated by a biotin ligase to allow for biotin-avidin or biotin-streptavidin based interactions for protein purification, as well as for immunobiology (such as immunoblotting or immunofluorescence) using anti-biotin antibodies. The 6xhistidine tag allows for protein purification using Ni-2+ affinity chromatography.

[0220] In particular embodiments, the genetic construct can include a selectable marker. A selectable marker includes any marker that allows for the selection of cells that have been successfully transduced. In particular embodiments, a selectable marker includes a transduction marker or a selection cassette.

[0221] Transduction markers may be selected from at least one of a truncated CD19 (tCD19; see Budde et al., Blood 122: 1660, 2013); a truncated human EGFR (tEGFR or EGFRt; see Wang et al., Blood 118: 1255, 2011); an ECD of human CD34; and/or RQR8 which combines target epitopes from CD34 (see Fehse et al, Mol. Therapy 1(5 Pt 1); 448-456, 2000) and CD20 antigens (see Philip et al, Blood 124: 1277-1278). In particular embodiments, cells are genetically modified to express EGFRt.

[0222] In particular embodiments, a selection cassette provides for positive selection or negative selection of a desired cell population. Negative selection is when several cell types are removed, leaving the cell type of interest. Positive selection involves targeting the desired cell population to retain desired cells.

[0223] A selection cassette can encode proteins that (a) confer resistance to antibiotics or other toxins, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli. Any number of selection systems may be used to recover transformed cells. In particular embodiments, a positive selection cassette includes resistance genes to neomycin, hygromycin, ampicillin, puromycin, phleomycin, zeomycin, blasticidin, or viomycin. In particular embodiments, a selection cassette includes the DHFR (dihydrofolate reductase) gene or DHFR double mutant (DHFRdm) gene providing resistance to methotrexate (MTX), the MGMT P140K gene responsible for the resistance to O⁶BG/BCNU, the HPRT (Hypoxanthine phosphoribosyl transferase) gene responsible for the transformation of specific bases present in the HAT selection medium (aminopterin, hypoxanthine, thymidine) or other genes for detoxification with respect to some drugs. In particular embodiments, the selection agent includes neomycin, hygromycin, puromycin, phleomycin, zeomycin, blasticidin, viomycin, ampicillin, O⁶BG/BCNU, MTX, tetracycline, aminopterin, hypoxanthine, thymidine kinase, DHFR, Gin synthetase, or ADA.

[0224] In particular embodiments, negative selection cassettes include a gene for transformation of a substrate present in the culture medium into a toxic substance for the cell that expresses the gene. These molecules include detoxification genes of diptheria toxin (DTA) (Yagi et al., Anal Biochem. 214(1):77-86, 1993; Yanagawa et al., Transgenic Res. 8(3):215-221 , 1999), the kinase thymidine gene of the Herpes virus (HSV TK) sensitive to the presence of ganciclovir or FIAU. The HPRT gene may also be used as a negative selection by addition of 6-thioguanine (6TG) into the medium, and for all positive and negative selections, a poly A transcription termination sequence from different origins, the most classical being derived from SV40 poly A, or a eukaryotic gene poly A (bovine growth hormone, rabbit -globin, etc.).

[0225] In particular embodiments, genetic constructs can include a polynucleotide that encodes a self-cleaving polypeptide, wherein the polynucleotide encoding the self-cleaving polypeptide is located between the polynucleotide encoding the coding sequence and a polynucleotide encoding a selectable marker. Exemplary self-cleaving polypeptides include 2A peptide from porcine teschovirus-1 (P2A), Thosea asigna virus (T2A), equine rhinitis A virus (E2A), foot-and-mouth disease virus (F2A). Further exemplary nucleic acid and amino acid sequences of 2A peptides are set forth in, for example, Kim et al. (PLOS One 6:e18556 (2011).

[0226] Nucleic acid sequences encoding proteins disclosed herein can be derived by those of ordinary skill in the art. Nucleic acid sequences can also include one or more of various sequence polymorphisms, mutations, and/or sequence variants. In particular embodiments, the sequence polymorphisms, mutations, and/or sequence variants do not affect the function of the encoded protein. The sequences can also include degenerate codons of a native sequence or sequences that may be introduced to provide codon preference.

[0227] In some aspects, the genetic constructs can be introduced by transfection, a technique that involves introduction of genetic constructs into the nucleus of eukaryotic cells. In some aspects, the proteins can be synthesized by transient transfection (DNA does not integrate with the genome of the eukaryotic cells, but the genes are expressed for 24-96 hours). Various methods can be used to introduce the genetic constructs into the host-cells, and transfection can be achieved by chemical-based means including by the calcium phosphate, by dendrimers, by liposomes, and by the use of cationic polymers. Non-chemical methods of transfection include electroporation, sono-poration, optical transfection, protoplast fusion, impalefection, and hydrodynamic delivery. In some embodiments, transfection can be achieved by particle-based methods including gene gun where the genetic construct is coupled to a nanoparticle of an inert solid which is then "shot" directly into the target-cell's nucleus. Other particle-based transfection methods include magnet assisted transfection and impalefection.

[0228] Any cell suitable for expression of a genetic construct can be used as a host cell. For example, the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as mammalian cells. Examples of bacterial host cells include Escherichia coli, Streptomyces, or Salmonella typhimurium. Mammalian host cells can include HEK293 cells or CHO cells.

[0229] Once expressed, ICEm and/or binding domains can be purified according to standard procedures of the art, including high-performance liquid chromatography (HPLC) purification, column chromatography, gel electrophoresis and the like (see generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0230] (VIII) Modifications to Provide Administration Benefits. In particular embodiments, the ICEm can be modified to produce an administration benefit. Exemplary administration benefits can include (1) reduced susceptibility to proteolysis, (2) reduced susceptibility to oxidation, (3) altered binding affinity for forming protein complexes, (4) altered binding affinities, (5) reduced immunogenicity; and/or (6) modified (extended or shortened) half-life. While the disclosure below describes these modifications in terms of their application to antibodies, the modifications can also be applied to the ICEm format. In particular embodiments, the ICEm are modified at the nucleic acid level or at the protein level. In particular embodiments, modifications include Fc silencing mutations. Fc silencing provides several benefits including biodistribution of the ICEm, lowers toxicity, and prevents exhaustion of immune cells in the periphery.

[0231] In particular embodiments, modified ICEm include those wherein one or more amino acids have been replaced with a different amino acid, a non-amino acid component, or where the amino acid has been conjugated to a functional group or a functional group has been otherwise associated with an amino acid. The modified amino acid may be, e.g., a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety, or an amino acid conjugated to an organic derivatizing agent. Amino acid(s) can be modified, for example, co-translationally or post- translationally during recombinant production (e.g., N-linked glycosylation at N-X-S/T motifs during expression in mammalian cells) or modified by synthetic means. The modified amino acid can be within the sequence or at the terminal end of a sequence. Modifications also include nitrited constructs. Nucleic acid(s) can be modified to result in an amino acid mutation.

[0232] In particular embodiments, variants include glycosylation variants wherein the number and/or type of glycosylation site has been altered compared to the amino acid sequences of a reference sequence. In particular embodiments, glycosylation variants include a greater or a lesser number of N-linked glycosylation sites than the reference sequence. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X can be any amino acid residue except proline. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions which eliminate this sequence will remove an existing N-linked carbohydrate chain. Also provided is a rearrangement of N-linked carbohydrate chains wherein one or more N-linked glycosylation sites (e.g., those that are naturally occurring) are eliminated and one or more new N-linked sites are created. Additional variants include cysteine variants wherein one or more cysteine residues are deleted from or substituted for another amino acid (e.g., serine) as compared to the reference sequence. These cysteine variants can be useful when antibodies must be refolded into a biologically active conformation such as after the isolation of insoluble inclusion bodies. These cysteine variants generally have fewer cysteine residues than the reference sequence, and typically have an even number to minimize interactions resulting from unpaired cysteines.

[0233] In particular embodiments, the ICEm can be fused or coupled to an Fc polypeptide that includes amino acid alterations that extend the in vivo half-life of an ICEm that contains the altered Fc polypeptide as compared to the half-life of a similar ICEm containing the same Fc polypeptide without the amino acid alterations. In particular embodiments, the Fc polypeptide includes a substitution at positions CH2 4, CH2 5, or both. In general, the amino acid at positions 4 and 5 of CH2 of the wild-type lgG1 and lgG3 is a leucine ("L"). In particular embodiments, the antibody includes an amino acid at position CH2 4, CH2 5, or both, that is not an L. In particular embodiments, an antibody includes an alanine ("A") at position CH2 4, or CH2 5, or both. In particular embodiments, the antibody includes both, a CH2 L4A and a CH2 L5A substitution. In one aspect, the substitutions are L234A and L235A (LALA). Such antibodies are referred to herein as a "LALA" variant. Interestingly, a "LALA" mutation in the Fc moiety does not only result in a lack of contribution of the respective antibody in antibody-dependent enhancement (ADE), but also blocks ADE.

[0234] In particular embodiments, the Fc polypeptide includes a substitution at position L234A, L235A and P329G in an Fc region derived from a human lgG1 Fc region. Such mutations are referred to as “LALAPG” mutations. In particular embodiments, the LALAPG mutation contributes to Fc silencing.

[0235] In another aspect, the substitutions are L234A, L235A and D265A in an Fc region derived from a human lgG1 Fc region. This mutation is referred to as a “LALA-DA” mutation.

[0236] In particular embodiments, Fc polypeptide amino acid alterations can include M252Y, S254T, T256E, M428L, and/or N434S and can be used together, separately or in any combination. For example, M428L/N434S is a pair of mutations that increase the half-life of antibodies in serum, as described in Zalevsky et al., Nature Biotechnology 28, 157-159, 2010. Other alterations that can be helpful are described in US Patent No. 7,083,784, US Patent No. 7,670,600, US Publication No. 2010/0234575, PCT/US2012/070146, and Zwolak, Scientific Reports 7: 15521 , 2017. In particular embodiments, any substitution at one of the following amino acid positions in an Fc polypeptide can be considered an Fc alteration that extends half-life: 250, 251 , 252, 259, 307, 308, 332, 378, 380, 428, 430, 434, 436. Each of these alterations or combinations of these alterations can be used to extend the half-life of ICEm described herein.

[0237] In particular embodiments, an lgG4 Fc region is mutated to form the lgG4_S228P Fc region. lgG4 antibodies can undergo a process called Fab arm exchange which results in functionally monovalent, bispecific antibodies with unknown specificity and thus potentially reduced therapeutic efficacy. Mutating the wildtype lgG4 serine at position 228 within the corehinge region to a proline creates the lgG4_S228P mutant. In particular embodiments, the lgG4_S228P mutant prevents Fab arm exchange.

[0238] In particular embodiments, it may be desirable to create cysteine engineered ICEm in which one or more residues of an ICEm are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the ICEm. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the ICEm and may be used to conjugate the ICEm to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further below. In particular embodiments, residue 5400 (EU numbering) of the heavy chain Fc region is selected. Cysteine engineered antibodies may be generated as described, e.g., in U.S. Pat. No. 7,521 ,541.

[0239] In particular embodiments, Fc modifications include hulgG4 ProAlaAla, hulgG2m4, and/or hulgG2sigma mutations. In particular embodiments, one or several amino acids at the amino or carboxy terminus of the light and/or heavy chain, such as the C-terminal lysine of the heavy chain, may be missing or derivatized in a proportion or all of the molecules. Substitutions can be made in the constant regions to reduce or increase effector function such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al., US Patent No. 5,624,821 ; Tso et al., US Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to prolong half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004). For additional information regarding Fc mutations that create administration benefits, see Saunders, Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life, Frontiers in Immunology (2019) Vol. 10, Article 1296. [0240] PEGylation particularly is a process by which polyethylene glycol (PEG) polymer chains are covalently conjugated to other molecules such as proteins. Several methods of PEGylating proteins have been reported in the literature. For example, N-hydroxy succinimide (NHS)-PEG was used to PEGylate the free amine groups of lysine residues and N-terminus of proteins; PEGs bearing aldehyde groups have been used to PEGylate the amino-termini of proteins in the presence of a reducing reagent; PEGs with maleimide functional groups have been used for selectively PEGylating the free thiol groups of cysteine residues in proteins; and site-specific PEGylation of acetyl-phenylalanine residues can be performed.

[0241] Covalent attachment of proteins to PEG has proven to be a useful method to increase the half-lives of proteins in the body (Abuchowski, A. et al., Cancer Biochem. Biophys. ,1984, 7:175- 186; Hershfield, M. S. et al., N. Engl. J. Medicine, 1987, 316:589-596; and Meyers, F. J. et al., Clin. Pharmacol. Then, 49:307-313, 1991). The attachment of PEG to proteins not only protects the molecules against enzymatic degradation, but also reduces their clearance rate from the body. The size of PEG attached to a protein has significant impact on the half-life of the protein. The ability of PEGylation to decrease clearance is generally not a function of how many PEG groups are attached to the protein, but the overall molecular weight of the altered protein. Usually the larger the PEG is, the longer the in vivo half-life of the attached protein. In addition, PEGylation can also decrease protein aggregation (Suzuki et al., Biochem. Bioph. Acta 788:248, 1984), alter protein immunogenicity (Abuchowski et al., J. Biol. Chem. 252: 3582, 1977), and increase protein solubility as described, for example, in PCT Publication No. WO 92/16221).

[0242] Several sizes of PEGs are commercially available (Nektar Advanced PEGylation Catalog 2005-2006; and NOF DDS Catalogue Ver 7.1), which are suitable for producing proteins with targeted circulating half-lives. A variety of active PEGs have been used including mPEG succinimidyl succinate, mPEG succinimidyl carbonate, and PEG aldehydes, such as mPEG- propionaldehyde.

[0243] Variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody or ICEm may be from 1 % to 80%, from 1 % to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., W02000/61739; WO 2001/29246; W02002/031140; US2002/0164328;

W02003/085119; W02003/084570; US2003/0115614; US2003/0157108; US2004/0093621 ; US2004/0110704; US2004/0132140; US2004/0110282; US2004/0109865; W02005/035586; W02005/035778; W02005/053742; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); and Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Led 3 CHO cells deficient in protein fucosylation (Ripka etal. Arch. Biochem. Biophys. 249:533-545, 1986, and knockout cell lines, such as alpha- 1 ,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614, 2004; Kanda et al., Biotechnol. Bioeng., 94(4):680-688, 2006; and W02003/085107).

[0244] (IX) Compositions for Administration. Any of the ICEm described herein in any exemplary format can be formulated alone or in combination into compositions for administration to subjects. Additionally, nucleic acids encoding the ICEm can also be formulated into compositions for administration (e.g., nucleic acids encapsulated within nanoparticles (e.g., liposomes or polymer- based nanoparticles) and/or as part of a vector delivery system (e.g., a viral vector or plasmid). ICEm and/or nucleic acids encoding ICEm are collectively referred to herein as “active ingredients”.

[0245] Salts and/or pro-drugs of active ingredients can also be used.

[0246] A pharmaceutically acceptable salt includes any salt that retains the activity of the active ingredient and is acceptable for pharmaceutical use. A pharmaceutically acceptable salt also refers to any salt which may form in vivo as a result of administration of an acid, another salt, or a prodrug which is converted into an acid or salt.

[0247] Suitable pharmaceutically acceptable acid addition salts can be prepared from an inorganic acid or an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Appropriate organic acids can be selected from aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids.

[0248] Suitable pharmaceutically acceptable base addition salts include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N,N'-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N- methylglucamine, lysine, arginine and procaine.

[0249] A prodrug includes an active ingredient which is converted to a therapeutically active compound after administration, such as by cleavage of an active ingredient or by hydrolysis of a biologically labile group.

[0250] In particular embodiments, the compositions include active ingredients of at least 0.1% w/v or w/w of the composition; at least 1% w/v or w/w of composition; at least 10% w/v or w/w of composition; at least 20% w/v or w/w of composition; at least 30% w/v or w/w of composition; at least 40% w/v or w/w of composition; at least 50% w/v or w/w of composition; at least 60% w/v or w/w of composition; at least 70% w/v or w/w of composition; at least 80% w/v or w/w of composition; at least 90% w/v or w/w of composition; at least 95% w/v or w/w of composition; or at least 99% w/v or w/w of composition.

[0251] Exemplary generally used pharmaceutically acceptable carriers include any and all absorption delaying agents, antioxidants, binders, buffering agents, bulking agents or fillers, chelating agents, coatings, disintegration agents, dispersion media, gels, isotonic agents, lubricants, preservatives, salts, solvents or co-solvents, stabilizers, surfactants, and/or delivery vehicles.

[0252] Exemplary antioxidants include ascorbic acid, methionine, and vitamin E.

[0253] Exemplary buffering agents include citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and/or trimethylamine salts.

[0254] An exemplary chelating agent is EDTA (ethylene-diamine-tetra-acetic acid).

[0255] Exemplary isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.

[0256] Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0257] Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the active ingredient or helps to prevent denaturation or adherence to the container wall. Typical stabilizers can include polyhydric sugar alcohols; amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, and cyclitols, such as inositol; PEG; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium th ioglycol ate, thioglycerol, a- monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (i.e., <10 residues); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides such as raffinose, and polysaccharides such as dextran. Stabilizers are typically present in the range of from 0.1 to 10,000 parts by weight based on therapeutic weight.

[0258] The compositions disclosed herein can be formulated for administration by, for example, injection, inhalation, infusion, perfusion, lavage, or ingestion. The compositions disclosed herein can further be formulated for intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral and/or subcutaneous administration and more particularly by intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, intrathecal, intratumoral, intramuscular, intravesicular, and/or subcutaneous injection. A delivery vehicle refers to any method, apparatus, or system used to administer or introduce an active ingredient. Examples of delivery vehicles include syringes, needles, catheters, infusion pumps, transdermal patches, inhalers, or oral dosage forms.

[0259] For injection, compositions can be formulated as aqueous solutions, such as in buffers including Hanks' solution, Ringer's solution, or physiological saline. The aqueous solutions can include formulatory agents such as suspending, stabilizing, and/or dispersing agents. Alternatively, the composition can be in lyophilized and/or powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0260] For oral administration, the compositions can be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like. For oral solid compositions such as powders, capsules and tablets, suitable excipients include binders (gum tragacanth, acacia, cornstarch, gelatin), fillers such as sugars, e.g., lactose, sucrose, mannitol and sorbitol; dicalcium phosphate, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate; cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxy-methylcellulose, and/or polyvinylpyrrolidone (PVP); granulating agents; and binding agents. If desired, disintegrating agents can be added, such as corn starch, potato starch, alginic acid, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. If desired, solid dosage forms can be sugar-coated or enteric-coated using standard techniques. Flavoring agents, such as peppermint, oil of Wintergreen, cherry flavoring, orange flavoring, etc. can also be used.

[0261] Compositions can be formulated as an aerosol. In particular embodiments, the aerosol is provided as part of an anhydrous, liquid or dry powder inhaler. Aerosol sprays from pressurized packs or nebulizers can also be used with a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, a dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of gelatin for use in an inhaler or insufflator may also be formulated including a powder mix of active ingredient and a suitable powder base such as lactose or starch.

[0262] Compositions can also be formulated as depot preparations. Depot preparations can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salts.

[0263] Additionally, compositions can be formulated as sustained-release systems utilizing semipermeable matrices of solid polymers including at least one active ingredient. Various sustained-release materials have been established and are well known by those of ordinary skill in the art. Sustained-release systems may, depending on their chemical nature, release one or more active ingredients following administration for a few weeks up to over 100 days. Depot preparations can be administered by injection; parenteral injection; instillation; or implantation into soft tissues, a body cavity, or occasionally into a blood vessel with injection through fine needles. [0264] Depot compositions can include a variety of bioerodible polymers including poly(lactide), poly(glycolide), poly(caprolactone) and poly(lactide)-co(glycolide) (PLG) of desirable lactide:glycolide ratios, average molecular weights, polydispersities, and terminal group chemistries. Blending different polymer types in different ratios using various grades can result in characteristics that borrow from each of the contributing polymers.

[0265] The use of different solvents (for example, dichloromethane, chloroform, ethyl acetate, triacetin, N-methyl pyrrolidone, tetrahydrofuran, phenol, or combinations thereof) can alter microparticle size and structure in order to modulate release characteristics. Other useful solvents include water, ethanol, dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), acetone, methanol, isopropyl alcohol (IPA), ethyl benzoate, and benzyl benzoate.

[0266] Exemplary release modifiers can include surfactants, detergents, internal phase viscosity enhancers, complexing agents, surface active molecules, co-solvents, chelators, stabilizers, derivatives of cellulose, (hydroxypropyl)methyl cellulose (HPMC), HPMC acetate, cellulose acetate, pluronics (e.g., F68/F127), polysorbates, Span® (Croda Americas, Wilmington, Delaware), poly(vinyl alcohol) (PVA), Brij® (Croda Americas, Wilmington, Delaware), sucrose acetate isobutyrate (SAIB), salts, and buffers. [0267] Excipients that partition into the external phase boundary of nanoparticles or microparticles such as surfactants including polysorbates, dioctylsulfosuccinates, poloxamers, PVA, can also alter properties including particle stability and erosion rates, hydration and channel structure, interfacial transport, and kinetics in a favorable manner.

[0268] Additional processing of the disclosed sustained release depot formulations can utilize stabilizing excipients including mannitol, sucrose, trehalose, and glycine with other components such as polysorbates, PVAs, and dioctylsulfosuccinates in buffers such as Tris, citrate, or histidine. A freeze-dry cycle can also be used to produce very low moisture powders that reconstitute to similar size and performance characteristics of the original suspension.

[0269] Any composition disclosed herein can advantageously include any other pharmaceutically acceptable carriers which include those that do not produce significantly adverse, allergic, or other untoward reactions that outweigh the benefit of administration. Exemplary pharmaceutically acceptable carriers are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Moreover, compositions can be prepared to meet sterility, pyrogenicity, general safety, and purity standards as required by U.S. FDA Office of Biological Standards and/or other relevant foreign regulatory agencies.

[0270] In particular embodiments, compositions include immunogenic compositions. An immunogenic composition refers to a composition that stimulates an immune response in a subject. The immune response can be, for example, a T-cell response. A T-cell response can be detected, for example, by measuring production of cytokines, such as interferon-gamma (IFN-y), tumor necrosis factor-alpha (TNF-a), interleukin (IL)-2, IL-4, IL-10, and IL-17. In particular embodiments, a T-cell response can be detected by measuring production of granzyme B and/or perforin.

[0271] In particular embodiments, compositions include therapeutic compositions. A therapeutic composition refers to a composition that treats a subject. In particular embodiments, efficacy of a treatment can be detected by a reduction in a subject’s disease (e.g., cancer) or symptoms as described elsewhere herein.

[0272] (X) Kits. Also disclosed herein are kits including at least one ICEm, sequences encoding at least one ICEm, at least one binding domain, sequence encoding at least one binding domain, and/or compositions disclosed herein. Kits may be formed with components to practice, for example, the methods described herein. The kit may include material(s), which may be desirable from a user standpoint, such as a buffer(s), a diluent(s), a standard(s), and/or other material useful in sample processing, washing, or conducting any other step of the method described herein. In particular embodiments, a kit includes a pharmaceutically acceptable carrier and/or a delivery vehicle.

[0273] In particular embodiments, kits can include one or more containers including one or more ICEm or compositions disclosed herein. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use, or sale for human administration. In particular embodiments, ICEm within kits are chosen based on assessment of a particular subject’s anticipated disease course. In particular embodiments, ICEm within kits are updated for a particular subject based on on-going assessments of the subject’s current disease status.

[0274] The kit according to the present disclosure may also include instructions for carrying out the method. Instructions included in the kit of the present disclosure may be affixed to packaging material or may be included as a package insert. While instructions are typically written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term “instructions” can include the address of an internet site which provides instructions.

[0275] (XI) Methods of Use. Methods disclosed herein include treating subjects (e.g., humans, veterinary animals (dogs, cats, reptiles, birds) livestock (e.g., horses, cattle, goats, pigs, chickens) and research animals (e.g., monkeys, rats, mice, fish) with compositions disclosed herein. Treating subjects includes delivering therapeutically effective amounts. Therapeutically effective amounts include those that provide effective amounts, prophylactic treatments and/or therapeutic treatments.

[0276] An “effective amount” is the amount of a composition necessary to result in a desired physiological change in the subject. For example, an effective amount can provide an immunogenic effect. Effective amounts are often administered for research purposes. Effective amounts disclosed herein can cause a statistically-significant effect in an in vitro assay, an animal model or clinical study relevant to the assessment of a cancer’s development or progression. An immunogenic composition can be provided in an effective amount, wherein the effective amount stimulates an immune response.

[0277] A "prophylactic treatment" includes a treatment administered to a subject who does not display signs or symptoms of a cancer or displays only early signs or symptoms of a cancer such that treatment is administered for the purpose of diminishing or decreasing the risk of developing the cancer further. Thus, a prophylactic treatment functions as a preventative treatment against a cancer. In particular embodiments, prophylactic treatments reduce, delay, or prevent metastasis from a primary cancer tumor site.

[0278] A "therapeutic treatment" includes a treatment administered to a subject who displays symptoms or signs of a cancer and is administered to the subject for the purpose of diminishing or eliminating those signs or symptoms of the cancer. The therapeutic treatment can reduce, control, or eliminate the presence or activity of the cancer and/or reduce control or eliminate side effects of the cancer.

[0279] Function as an effective amount, prophylactic treatment, or therapeutic treatment are not mutually exclusive, and in particular embodiments, administered dosages may accomplish more than one treatment type.

[0280] In particular embodiments, therapeutically effective amounts provide anti-cancer effects. Anti-cancer effects include a decrease in the number of cancer cells, decrease in the number of metastases, a decrease in tumor volume, an increase in life expectancy, induced chemo- or radiosensitivity in cancer cells, inhibited angiogenesis near cancer cells, inhibited cancer cell proliferation, inhibited tumor growth, prevented or reduced metastases, prolonged subject life, reduced cancer-associated pain, and/or reduced relapse or re-occurrence of cancer following treatment.

[0281] A "tumor" is a swelling or lesion formed by an abnormal growth of cells (called neoplastic cells or tumor cells). A "tumor cell" is an abnormal cell that grows by a rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that initiated the new growth cease. Tumors show partial or complete lack of structural organization and functional coordination with the normal tissue, and usually form a distinct mass of tissue, which may be benign, pre-malignant or malignant.

[0282] The disclosed ICEm provide a versatile platform that can be utilized to target a large variety of cancers, such as adrenal cancers, bladder cancers, blood cancers (e.g., leukemias, lymphomas, myelomas), bone cancers, brain cancers, breast cancers, carcinoma, cervical cancers, colon cancers, colorectal cancers, corpus uterine cancers, ENT (ear, nose, and throat) cancers, endometrial cancers, esophageal cancers, gastrointestinal cancers, head and neck cancers, intestinal cancers, kidney cancers, larynx cancers, liver cancers, lymph node cancers, lung cancers (e.g., mesothelioma), nasopharynx cancers, neuroblastomas, oral cancers, ovarian cancers, pancreatic cancers, penile cancers, pharynx cancers, prostate cancers, rectal cancers, sarcoma, seminomas, skin cancers (e.g., melanomas), stomach cancers, teratomas, testicular cancers, thyroid cancers, uterine cancers, vaginal cancers, vascular tumors, and metastases thereof. [0283] For administration, therapeutically effective amounts (also referred to herein as doses) can be initially estimated based on results from in vitro assays and/or animal model studies. Such information can be used to more accurately determine useful doses in subjects of interest. The actual dose amount administered to a particular subject can be determined by a physician, veterinarian or researcher taking into account parameters such as physical and physiological factors including target, body weight, severity of condition, type of cancer, stage of cancer, previous or concurrent therapeutic interventions, idiopathy of the subject and route of administration.

[0284] Useful doses can range from 0.1 to 5 pg/kg or from 0.5 to 1 pg /kg. In other examples, a dose can include 1 pg /kg, 15 pg /kg, 30 pg /kg, 50 pg/kg, 55 pg/kg, 70 pg/kg, 90 pg/kg, 150 pg/kg, 350 pg/kg, 500 pg/kg, 750 pg/kg, 1000 pg/kg, 0.1 to 5 mg/kg or from 0.5 to 1 mg/kg. In other examples, a dose can include 1 mg/kg, 10 mg/kg, 30 mg/kg, 50 mg/kg, 70 mg/kg, 100 mg/kg, 300 mg/kg, 500 mg/kg, 700 mg/kg, 1000 mg/kg or more.

[0285] Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or yearly). In particular embodiments, the treatment protocol may be dictated by a clinical trial protocol or an FDA- approved treatment protocol.

[0286] In particular embodiments, therapeutically effective amounts are administered at a time interval to reduce or eliminate cancer recurrence without causing autoimmune toxicity.

[0287] The pharmaceutical compositions described herein can be administered by, for example, injection, inhalation, infusion, perfusion, lavage, or ingestion. Routes of administration can include intravenous, intradermal, intraarterial, intraparenteral, intranasal, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral, subcutaneous, and/or sublingual administration and more particularly by intravenous, intradermal, intraarterial, intraparenteral, intranasal, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral, subcutaneous, and/or sublingual injection.

[0288] In particular embodiments, ICEm can be administered through a pump such as a programmable pump (e.g., an insulin pump). In particular embodiments, staged administration of different ICEm can be achieved using, for example, a programmed pump. In particular embodiments, ICEm have a short half-life (e.g., short in vivo half-life) such that the ICEm are administered using continuous infusion with a pump. In particular embodiments, any ICEm with an in vivo half-life of less than 5 hours can be administered through continuous infusion. In contrast, antibodies can have in vivo half-lives of several weeks due to their larger size and Fc portion, and bi-specific formats that contain an Fc portion can similarly have extended in vivo halflives.

[0289] A benefit of using an ICEm is that it can be used during the course of treatment. Nevertheless, in alternative embodiments, the administration of ICEm can evolve during the course of a subject’s treatment. In particular embodiments, a method of treating a subject in need thereof can include: administering a first ICEm to a subject at a first point in time, wherein the first ICEm targets a first cancer marker; monitoring for changes in cancer marker expression of the subject; and administering a second ICEm to the subject at a second point in time. In particular embodiments, the second ICEm can include a binding domain that binds a second cancer marker or can include a binding domain that includes a cancer supporter. In particular embodiments, the first ICEm includes a cancer marker binding domain and an ICAE binding domain and the second ICEm includes a cancer supporter binding domain and an ICAE binding domain. In particular embodiments, the majority of cancer marker expression changes from a first cancer marker at a first time point to a second cancer marker at a second time point. Cancer marker expression often changes during the course of cancer. For example, Her2, the molecular target of the cancer drug trastuzumab, can become down-regulated during treatment, leading to treatment resistance (Shi et al. Breast Cancer Research 2014 16: R33). In particular embodiments, a subject being treated with an ICEm that targets Her2 can be monitored for Her2 downregulation, and if Her-2 expression decreases. Examples of cancer markers that become down-regulated during the course of treatment include Her2, epidermal growth factor receptor (EGFR), prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), and CD19. A benefit of using an ICEm is that even though the cancer marker may become downregulated, the ICEm targets cancer supporters on the cancer which may become upregulated and turns the cancer supporters into a therapeutic target.

[0290] In particular embodiments, during the course of treatment, a first ICEm can be replaced with a second ICEm (or third or fourth, etc). In particular embodiments, a different ICEm includes a different cancer marker binding domain, ICAE binding domain, and/or cancer supporter binding domain. In particular embodiments, a different ICEm can add, remove, or replace a cancer marker binding domain, ICAE binding domain, and/or cancer supporter binding domain.

[0291] In particular embodiments, a subject can be monitored for immune suppression in the tumor microenvironment and/or T-cell suppression. Immune suppression in the microenvironment and/or T-cell suppression can be monitored, for example, by measuring cytokine levels and/or the number of T-cells in a sample derived from the patient.

[0292] In particular embodiments, methods disclosed herein include activating immune cells in the tumor microenvironment. In particular embodiments, activating immune cells in the tumor microenvironment includes reducing or reversing T cell suppression in the tumor microenvironment. T cell suppression can refer to a reduction in T cell activation, such as can be caused by regulatory T cells. Methods to measure T cell suppression can be found, for example in McMurchy & Levings (European Journal of Immunology 42(1): 27-34). In one example, T cells in a tumor microenvironment might, during the treatment with for example an ICEm with an ICAE binding domain that binds CD28, reduce expression of CD28. To reduce or reverse this T cell suppression in the tumor microenvironment, the treatment can be changed such that the treatment includes an ICEm with an ICAE binding domain that binds an ICAE that is not CD28.

[0293] The present disclosure also provides methods and kits for designing an ICEm for treatment of a subject with a cancer. In particular embodiments, the method includes obtaining a sample of the cancer; determining a cancer marker expressed on a portion of the cancer cells; selecting a cancer marker binding domain that binds the cancer marker; selecting an ICAE binding domain that binds and activates immune cells; and selecting a cancer supporter binding domain that binds a cancer supporter. In particular embodiments, the method further includes constructing a nucleic acid molecule or molecules that express an ICEm with the selected binding domains. In particular embodiments, the method further includes expressing the ICEm. In particular embodiments, the cancer marker is preferentially expressed on cancer cells as opposed to healthy cells. In particular embodiments, the method to select a cancer supporter includes selecting a molecule that cancer cells upregulate or select for to promote their survival. In particular embodiments, the method to select a cancer supporter includes selecting a molecule that down-regulates immune system activity against a cancer cell that expresses the cancer supporter. In particular embodiments, the method to select a cancer supporter includes selecting a molecule that provides treatment resistance for a cancer cell that expresses the cancer supporter. In particular embodiments, the method to select a cancer supporter includes selecting from a list of molecules having the following properties: altered by exposure to a molecule released in response to immune cell activation (e.g., IFNy) or therapeutics; expressed on the cell surface; expressed outside the hematopoietic lineage; expressed on tumors or tumor cells; and/or provides a selective benefit to the tumor or tumor cell.

[0294] The design considerations will include considerations for the number of binding domains to include for each of the cancer marker binding domain, ICAE binding domain, and cancer supporter binding domain. The design consideration will include considerations for the relative placement of each binding domain, Fc mutations, multimerization domains, tags, signal peptides, and linkers. Kits including nucleic acids, vectors, and cells to perform the method are provided.

[0295] The Exemplary Embodiments and Example below are included to demonstrate particular embodiments of the disclosure. Those of ordinary skill in the art should recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the disclosure. [0296] (XII) Examples and Exemplary Embodiments. In particular embodiments, an ICEm includes a first polypeptide sequence including a first multimerization domain and a second polypeptide sequence including a second multimerization domain. In particular embodiments, an ICEm includes a first polypeptide sequence including a knob and a second polypeptide sequence including a hole.

[0297] In particular embodiments, MTE1 includes and MTE 1 Knob and an MTE 1 Hole. In particular embodiments, the MTE 1 Knob includes a cancer marker binding domain (CM BD)- linker-IgG 1 Fc Knob-linker-cancer supporter binding domain (CS BD) including the sequence: QVKLVQSGGGLVQTGGSLRLSCAASEITFDMYSMGWYREAPGKARDAVASITNRGNTYYADS VKGRFTISRDNAKKTMYLQMNSLKPEDTAVYYCNVYRTGFSDYWGQGTQVTVSSGSEPKSSD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSSEEDCKVH CVKEWMAGKACAERDKSYTIGRAHCSGQKFDVFKCLDHCAAP (SEQ ID NO: 127).

[0298] In particular embodiments, the MTE 1 Knob includes a signal peptide (SP)-CM BD-linker- IgG 1 Fc Knob-linker-CS BD-linker-His tag including the sequence: METDTLLLWVLLLWVPGSTGQVKLVQSGGGLVQTGGSLRLSCAASEITFDMYSMGWYREAPG KARDAVASITNRGNTYYADSVKGRFTISRDNAKKTMYLQMNSLKPEDTAVYYCNVYRTGFSDY WGQGTQVTVSSGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGS GGGGSGGGGSSEEDCKVHCVKEWMAGKACAERDKSYTIGRAHCSGQKFDVFKCLDHCAAP GSHHHHHH (SEQ ID NO: 128).

[0299] In particular embodiments, the MTE 1 Hole includes a CM BD-linker-lgG1 Fc Hole-linker- immune cell activating epitope binding domain (ICAE BD) including the sequence: QVKLVQSGGGLVQTGGSLRLSCAASEITFDMYSMGWYREAPGKARDAVASITNRGNTYYADS VKGRFTISRDNAKKTMYLQMNSLKPEDTAVYYCNVYRTGFSDYWGQGTQVTVSSGSEPKSSD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSEVQLVESG GGPVQAGGSLRLSCAASGRTYRGYSMGWFRQAPGKEREFVAAIVWSGGNTYYEDSVKGRFT ISRDNAKNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS (SEQ ID NO: 129).

[0300] In particular embodiments, the MTE 1 Hole includes a SP-CM BD-linker-lgG1 Fc Hole- linker-ICAE BD-linker-tag including the sequence:

METDTLLLWVLLLWVPGSTGQVKLVQSGGGLVQTGGSLRLSCAASEITFDMYSMGWYREAPG KARDAVASITNRGNTYYADSVKGRFTISRDNAKKTMYLQMNSLKPEDTAVYYCNVYRTGFSDY WGQGTQVTVSSGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSG GGGSGGGGSEVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMGWFRQAPGKEREFVAAI VWSGGNTYYEDSVKGRFTISRDNAKNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYW GQGTLVTVSSGSAWSHPQFEK (SEQ ID NO: 130).

[0301] In particular embodiments, MTE 2 includes an MTE 2 Knob and an MTE 2 Hole. In particular embodiments, the MTE 2 Knob includes a CM BD-linker-lgG1 Fc Knob including the sequence:

QVKLVQSGGGLVQTGGSLRLSCAASEITFDMYSMGWYREAPGKARDAVASITNRGNTYYADS VKGRFTISRDNAKKTMYLQMNSLKPEDTAVYYCNVYRTGFSDYWGQGTQVTVSSGSEPKSSD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 131).

[0302] In particular embodiments, the MTE 2 Knob includes a SP-CM BD-linker-lgG1 Fc Knob- linker-His tag including the sequence:

METDTLLLWVLLLWVPGSTGQVKLVQSGGGLVQTGGSLRLSCAASEITFDMYSMGWYREAPG KARDAVASITNRGNTYYADSVKGRFTISRDNAKKTMYLQMNSLKPEDTAVYYCNVYRTGFSDY WGQGTQVTVSSGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSHHH HHH (SEQ ID NO: 132).

[0303] In particular embodiments, the MTE 2 Hole includes a CM BD-linker-lgG1 Fc Hole-linker- ICAE BD including the sequence:

QVKLVQSGGGLVQTGGSLRLSCAASEITFDMYSMGWYREAPGKARDAVASITNRGNTYYADS VKGRFTISRDNAKKTMYLQMNSLKPEDTAVYYCNVYRTGFSDYWGQGTQVTVSSGSEPKSSD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSEVQLVESG GGPVQAGGSLRLSCAASGRTYRGYSMGWFRQAPGKEREFVAAIVWSGGNTYYEDSVKGRFT ISRDNAKNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS (SEQ ID NO: 129).

[0304] In particular embodiments, the MTE 2 Hole includes a SP-CM BD-linker-lgG1 Fc Hole- linker-ICAE BD-linker-tag including the sequence:

[0305] In particular embodiments, MTE 3 includes an MTE 3 Knob and an MTE 3 Hole. In particular embodiments, the MTE 3 Knob includes a CM BD-linker-lgG1 Fc Knob-linker-CS BD including the sequence:

QVKLVQSGGGLVQTGGSLRLSCAASEITFDMYSMGWYREAPGKARDAVASITNRGNTYYADS VKGRFTISRDNAKKTMYLQMNSLKPEDTAVYYCNVYRTGFSDYWGQGTQVTVSSGSEPKSSD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSSEEDCKVH CVKEWMAGKACAERDKSYTIGRAHCSGQKFDVFKCLDHCAAP (SEQ ID NO: 127).

[0306] In particular embodiments, the MTE 3 Knob includes a SP-CM BD-linker-lgG1 Fc Knob- linker-CS BD-linker-His tag including the sequence:

METDTLLLWVLLLWVPGSTGQVKLVQSGGGLVQTGGSLRLSCAASEITFDMYSMGWYREAPG KARDAVASITNRGNTYYADSVKGRFTISRDNAKKTMYLQMNSLKPEDTAVYYCNVYRTGFSDY

WGQGTQVTVSSGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVD

VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGS GGGGSGGGGSSEEDCKVHCVKEWMAGKACAERDKSYTIGRAHCSGQKFDVFKCLDHCAAP GSHHHHHH (SEQ ID NO: 128).

[0307] In particular embodiments, the MTE 3 Hole includes a CM BD-linker-lgG1 Fc Hole including the sequence:

QVKLVQSGGGLVQTGGSLRLSCAASEITFDMYSMGWYREAPGKARDAVASITNRGNTYYADS VKGRFTISRDNAKKTMYLQMNSLKPEDTAVYYCNVYRTGFSDYWGQGTQVTVSSGSEPKSSD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV

HNAKTKPREEQYASTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ

VYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 137).

[0308] In particular embodiments, the MTE 3 Hole includes a SP-CM BD-linker-lgG1 Fc Hole- linker-tag including the sequence:

METDTLLLWVLLLWVPGSTGQVKLVQSGGGLVQTGGSLRLSCAASEITFDMYSMGWYREAPG KARDAVASITNRGNTYYADSVKGRFTISRDNAKKTMYLQMNSLKPEDTAVYYCNVYRTGFSDY WGQGTQVTVSSGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSAWSH PQFEK (SEQ ID NO: 138).

[0309] In particular embodiments, MTE 4 includes an MTE 4 Knob and an MTE 4 Hole. In particular embodiments, the MTE 4 Knob includes an I gG 1 Fc Knob-linker-CS BD including the sequence:

EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSS EEDCKVHCVKEWMAGKACAERDKSYTIGRAHCSGQKFDVFKCLDHCAAP (SEQ ID NO: 139). [0310] In particular embodiments, the MTE 4 Knob includes a SP-lgG1 Fc Knob-linker-CS BD- linker-His tag including the sequence:

METDTLLLWVLLLWVPGSTGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GGGGSGGGGSGGGGSSEEDCKVHCVKEWMAGKACAERDKSYTIGRAHCSGQKFDVFKCLD HCAAPGSHHHHHH (SEQ ID NO: 140).

[0311] In particular embodiments, the MTE 4 Hole includes an lgG1 Fc Hole-linker-ICAE BD including the sequence:

EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSEV QLVESGGGPVQAGGSLRLSCAASGRTYRGYSMGWFRQAPGKEREFVAAIVWSGGNTYYEDS VKGRFTISRDNAKNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS (SEQ ID NO: 141).

[0312] In particular embodiments, the MTE 4 Hole includes a SP-lgG1 Fc Hole-linker-ICAE BD- linker-tag including the sequence:

METDTLLLWVLLLWVPGSTGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GGGGSGGGGSGGGGSEVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMGWFRQAPGKE REFVAAIVWSGGNTYYEDSVKGRFTISRDNAKNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAG QYDYWGQGTLVTVSSGSAWSHPQFEK (SEQ ID NO: 142).

[0313] In particular embodiments, MTE 5 includes an MTE 5 Knob and an MTE 5 Hole. In particular embodiments, the MTE 5 Knob includes a CM BD-linker-lgG1 Fc Knob including the sequence:

[0314] In particular embodiments, the MTE 5 Knob includes a SP-CM BD-linker-lgG1 Fc Knob- linker-His tag including the sequence:

[0315] In particular embodiments, the MTE 5 Hole includes a CM BD-linker-lgG1 Fc Hole including the sequence:

HNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ

[0316] In particular embodiments, the MTE 5 Hole includes a SP-CM BD-linker-lgG1 Fc Hole- linker-tag including the sequence:

[0317] In particular embodiments, an ICEm includes a: CM BD-multimerization domain (MD)1- CS BD and CM BD-MD2-ICAE BD; CM BD-MD1-ICAE BD and CM BD-MD2-CS BD; CM BD- MD1-CM BD and ICAE BD-MD2-CS BD; CM BD-MD1-CM BD and CS BD-MD2-ICAE BD; CM BD-MD1-ICAE BD and CS BD-MD2-CM BD; CM BD-MD1-CS BD and ICAE BD-MD2-CM BD; ICAE BD-MD1-CM BD and CM BD-MD2-CS BD; CM BD-MD1-CS BD and MD2-ICAE BD; CM BD-MD1-ICAE BD and MD2-CS BD; CM BD-MD1 and ICAE BD-MD2-CS BD; CM BD-MD1 and CS BD-MD2-ICAE BD; CM BD-MD1-ICAE BD and CS BD-MD2; MD1-CS BD and ICAE BD-MD2- CM BD; ICAE BD-MD1 and CM BD-MD2-CS BD; CM BD-MD1-CS BD and CS BD-MD2-ICAE BD; CM BD-MD1-ICAE BD and CP-BD-MD2-CS BD; CM BD-MD1-CS BD and ICAE BD-MD2- CS BD; CM BD-MD1-ICAE BD and CS BD-MD2-ICAE BD; CM BD-MD1-ICAE BD and CS BD- MD2-CS BD; CP-BD-MD1-CS BD and ICAE BD-MD2-CM BD; CS BD-MD1-CM BD and CS BD- MD2-ICAE BD; CM BD-MD1-ICAE BD and CM BD-MD2; CM BD-MD1 and CM BD-MD2-ICAE BD; CM BD-MD1-CM BD and ICAE BD-MD2; ICAE BD-MD1-CM BD and CM BD-MD2; CM BD- MD1 and CM BD-MD2-CS BD; CM BD-MD1-CS BD and CM BD-MD2; CM BD-MD1-CM BD and CS BD-MD2; CM BD-MD1-CM BD and MD2-CS BD; MD1-ICAE BD and MD2-CS BD; CS BD- MD1-ICAE BD and MD2; ICAE BD-MD1 and MD2-CS BD; MD1-ICAE BD and CS BD-MD2; CM BD-MD1 and CM BD-MD2; and CM BD-MD1-CM BD and MD2. In particular embodiments, the ICEm further includes signal peptides, linkers, and/or tags.

[0318] Additional examples of binding domains that can be used with embodiments disclosed here are described in WO2022115719.

[0319] Exemplary Embodiments.

1. A molecule including a first polypeptide including a sequence as set forth in SEQ ID NO: 127 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 127; and a second polypeptide including a sequence as set forth in SEQ ID NO: 129 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 129; a first polypeptide including the sequence as set forth in SEQ ID NO: 128 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 128; and a second polypeptide including the sequence as set forth in SEQ ID NO: 130 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 130; or a first polypeptide including the sequence as set forth in SEQ ID NO: 150 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 150; and a second polypeptide including the sequence as set forth in SEQ ID NO: 149 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 149.

2. A molecule including a first polypeptide including a first ROR1 binding domain, a CD3 binding domain, and a first multimerization domain, and a second polypeptide including a second ROR1 binding domain, a PD-L1 binding domain, and a second multimerization domain, wherein the first multimerization domain and the second multimerization domain link the first polypeptide to the second polypeptide.

3. A molecule including a first polypeptide including a first PD-L1 binding domain, a CD3 binding domain, and a first multimerization domain, and a second polypeptide including a second PD-L1 binding domain, a ROR1 binding domain, and a second multimerization domain, wherein the first multimerization domain and the second multimerization domain link the first polypeptide to the second polypeptide.

4. A molecule including a first polypeptide including a first CD3 binding domain, a ROR1 binding domain, and a first multimerization domain, and a second polypeptide including a second CD3 binding domain, a PD-L1 binding domain, and a second multimerization domain, wherein the first multimerization domain and the second multimerization domain link the first polypeptide to the second polypeptide.

5. A molecule including: a cancer marker binding domain, an immune cell activating epitope binding domain, and a cancer supporter binding domain.

6. The molecule of embodiment 5, wherein the cancer marker binding domain binds receptor tyrosine kinase like orphan receptor 1 (ROR1), prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), mesothelin, CD19, CD20, CD33, Wilms tumor protein (WT1), CD123, CD22, CD133, B-cell maturation antigen (BCMA), human epidermal growth factor receptor 2 (HER2), carbonic Anhydrase IX (CAIX), carcinoembryonic antigen (CEA), or GD2.

7. The molecule of embodiments 5 or 6, wherein the cancer marker binding domain binds ROR1 , PSMA, PSCA, mesothelin, CD19, CD20, CD33, WT1 , or CD123.

8. The molecule of any of embodiments 5-7, wherein the cancer marker binding domain includes a ROR1 binding domain.

9. The molecule of any of embodiments 5-8, wherein the cancer marker includes ROR1 and the cancer marker binding domain includes a sequence having SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, or SEQ ID NO: 23 or at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, or SEQ ID NO: 23.

10. The molecule of any of embodiments 5-9, wherein the immune cell activating epitope includes an epitope on a T cell, an NK cell, or a macrophage.

11. The molecule of any of embodiments 5-10, wherein the immune cell activating epitope includes CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, CD83, 4-1 BB, 0X40, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, or B7-H3.

12. The molecule of any of embodiments 5-11, wherein the immune cell activating epitope includes CD3, CD28, 4-1 BB, or CD8.

13. The molecule of any of embodiments 5-11, wherein the immune cell activating epitope includes NKG2D, CD8, CD16, KIR2DL4, KIR2DS1 , KIR2DS2, KIR3DS1 , NKG2C, NKG2E, or NKG2D.

14. The molecule of any of embodiments 5-11, wherein the immune cell activating epitope includes CD11b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4/80, IFGR2 Toll-like receptors (TLRs) 1-9, IL-4Ra, or MARCO.

15. The molecule of any of embodiments 5-12, wherein the immune cell activating epitope binding domain binds and activates a T cell.

16. The molecule of any of embodiments 5-15, wherein the immune cell activating epitope binding domain binds CD3.

17. The molecule of any of embodiments 5-16, wherein the immune cell activating epitope binding domain binds CD3 and includes a sequence as set forth in SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 63 or a sequence having at least 95% sequence identity to SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 63.

18. The molecule of any of embodiments 5-17, wherein the cancer supporter binding domain binds a molecule that cancer cells upregulate or select for to promote their survival.

19. The molecule of any of embodiments 5-18, wherein the cancer supporter binding domain binds a molecule that down-regulates immune system activity against a cancer cell that expresses the cancer supporter.

20. The molecule of any of embodiments 5-19, wherein the cancer supporter binding domain binds a molecule that provides treatment resistance for a cancer cell that expresses the cancer supporter.

21. The molecule of any of embodiments 5-20, wherein the cancer supporter includes PD-L1 , ALCAM/CD166, C10orf54, CEACAM1 , CEACAM20, CMKLR1, HLA-E, IFITM1 , IFITM2, IFITM3, IL31RA, LST1 , MEFV, OSMR, PDL2, PIM1 , PIM2, PLAAT3, PRAME, or SDCBP.

22. The molecule of any of embodiments 5-21 , wherein the cancer supporter includes OCSTAMP, UNC5C, KCNF1 , TMEM45A, RXFP1 , MGAM2, PTGES, TMEM92, CLEC9A, CAV2, LAMA3, RIMS2, BCAR1, PLEKHS1 , SSTR2, KCNE5, ITGA7, GPR31 , IL31RA, SCUBE1, HPN, GJA4, P2RY14, BPI, S1PR3, FCGR1A, KREMEN1 , FLT1 , DCSTAMP, GRIN3A, MUC1, DEFB1 , CALHM6, KCNJ10, CPNE6, FFAR2, SLC6A12, HCAR3, F3, XIRP1 , CLEC6A, FPR2, HCAR2, FRMD3, VAMP5, TMTC1 , CD274, NPFFR1 , GLDN, SMO, CD38, TNFSF10, GPR33, SLAMF7, NRN1 , CYSLTR2, ATP1 B2, PLSCR4, RRAD, KCNJ15, CCRL2, TMEM255A, CSPG4, FPR1 , JAG1 , CDCP1 , GPR146, MYOF, PDGFRA, MYO1A, ABCC11 , LAG3, CAVIN1 , PPFIA4, CR1 L, GPC4, P2RX7, RARRES3, PLEKHA6, TMEM132A, SMCO2, SMCO4, MMP25, KCNJ2, RALB, TIE1 , SCG3, PRRT2, SORT1, VCAM1 , FAM241A, PTPRU, CLEC4E, NCF1, SLC12A3, IL15RA, SECTM1 , LRRN2, OTOF, GEM, ENTHD1 , OSBPL6, SCARF1 , SUCNR1 , SLC2A5, PSTPIP2, GPR84, GOLM1 , GJB2, SLITRK4, SLC2A6, CLEC4D, SSPN, SORBS1 , GAREM1 , CLDN7, LRRC4, ASPHD2, STX11 , EREG, RAPGEF2, MYO1 B, CLEC5A, SYT17, LYPD5, PDCD1 LG2, PHLDA2, CTTN, SLC31A2, BEGAIN, RIPK2, CSF2RB, DGKG, FRRS1 , TNFSF13B, VRK2, DOCK7, DAPP1 , PRRG4, CACNA1A, ITGB7, PRAME, MFSD2A, CEACAM4, TNF, SLC1A5, CDK14, FAS, ATP1 B1 , FFAR4, HVCN1 , PROCR, CD80, IFITM1 , TENM4, PLAUR, NETO2, SCIMP, PAK1 , CAMK2D, C19orf38, CD83, RHOU, CD40, PLEK, ITPRIPL2, TREML2, EPB41 L5, LILRA2, SLC1A4, LDLR, IFNGR2, SFT2D2, SLAMF8, TLR7, GPBAR1 , SIRPB1 , SIRPB1, NYNRIN, C3orf14, TJP2, LMTK2, PAQR6, FZD2, DIXDC1 , STX1 B, CNIH4, SLC8A1 , CD33, TMEM131 , NOD2, PDE4B, SH2B2, CLMP, SLC1A3, MCOLN2, IL12RB2, TLR1 , CSF1 , or TLR8.

23. The molecule of any of embodiments 5-22, wherein the cancer supporter is an immune checkpoint molecule, immune checkpoint ligand, or a drug efflux pump. 24. The molecule of any of embodiments 5-23, wherein the cancer supporter includes PD-L1 , 2B4, A2aR, B7HI, B7H3, B7H4, BTLA, CCR7, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276, CTLA-4, DR3, GAL9, GITR, HAVCR2, HVEM, ID01 , ID02, ICOS, KIR, LAG3, LAIRI, LIGHT, MARCO, PD-1 , PD-L2, PD-L3, PD-L4, PS, OX-40, SLAM, TIGHT, TIGIT, TIM3, VISTA, or VTCNI.

25. The molecule of any of embodiments 5-24, wherein the cancer supporter includes PD-L1.

26. The molecule of any of embodiments 5-25, wherein the cancer supporter includes PD-L1 and the cancer supporter binding domain includes a sequence as set forth in SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97 or a sequence having at least 95% sequence identity to SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97.

27. The molecule of embodiment 23, wherein the immune checkpoint molecule includes CD27, CD28, CD137, CEACAM1 , CTLA4, GITR, ICOS, LAG3, PD-1 , OX-40, TIGIT, TIM3, or VISTA.

28. The molecule of embodiment 23, wherein the immune checkpoint ligand includes CD70, CD80, CD86, CD137L, CD155/CD112, CEACAM1 , ICOSLG, Galectin 9, GITRL, lectins, OC40L, PD-L1 , PD-L2, or VSIG-3.

29. The molecule of embodiment 23, wherein the drug efflux pump includes P-glycoprotein (P-gP or MDR1/ABCB1), monocarboxylate transporter (MCT), ATP-binding cassette transporters (ABC), peptide transporters (PEPTs), Na+ phosphate transporters (NPTs), MDR-associated protein (MRP1/ABCC1), breast cancer resistance protein (MXR or BCRP/ABCG2), MRP/ABCC- 2, MRP/ABCC-3, MRP/ABCC-4, MRP/ABCC-5, MRP/ABCC-6, or MRP/ABCC-7.

30. The molecule of any of embodiments 5-29, including a first polypeptide including a first multimerization domain and a second polypeptide including a second multimerization domain wherein the first multimerization domain and the second multimerization domain link the first polypeptide to the second polypeptide.

31. The molecule of embodiment 30, wherein first polypeptide includes the cancer marker binding domain and the immune cell activating epitope binding domain and the second polypeptide includes the cancer supporter binding domain.

32. The molecule of embodiment 31 , wherein the first polypeptide includes a ROR1 binding domain, an lgG1 Fc knob, and a CD3 binding domain, and the second polypeptide includes a PDL1 binding domain and an IgG 1 Fc hole.

33. The molecule of embodiments 31 or 32, wherein the second polypeptide further includes a second cancer marker binding domain, a second immune cell activating epitope binding domain, or a second cancer supporter binding domain.

34. The molecule of embodiment 30, wherein the first polypeptide includes the cancer marker binding domain and the cancer supporter binding domain and the second polypeptide includes the immune cell activating epitope binding domain.

35. The molecule of embodiment 34, wherein the first polypeptide includes a ROR1 binding domain, an I gG1 Fc knob, and a PDL1 binding domain, and the second polypeptide includes a CD3 binding domain and an IgG 1 Fc hole; or the first polypeptide includes a ROR1 binding domain, an IgG 1 Fc hole, and a PDL1 binding domain, and the second polypeptide includes a CD3 binding domain and an IgG 1 Fc knob.

36. The molecule of embodiments 34 or 35, wherein the second polypeptide further includes a second cancer marker binding domain, a second immune cell activating epitope binding domain, or a second cancer supporter binding domain.

37. The molecule of embodiment 30, wherein the first polypeptide includes the immune cell activating epitope binding domain and the cancer supporter binding domain and the second polypeptide includes the cancer marker binding domain.

38. The molecule of embodiment 37, wherein the first polypeptide includes a PDL1 binding domain, an lgG1 Fc knob, and a CD3 binding domain, and the second polypeptide includes a ROR1 binding domain and an lgG1 Fc hole.

39. The molecule of any of embodiments 37 or 38, wherein the second polypeptide further includes a second cancer marker binding domain, a second immune cell activating epitope binding domain, or a second cancer supporter binding domain.

40. The molecule of any of embodiments 5-39, wherein at least one of the cancer marker binding domain, the immune cell activating epitope biding domain, and the cancer supporter binding domain include an antibody or a peptide.

41. The molecule of embodiment 40, wherein the antibody includes an immunoglobulin G (IgG), a Fab fragment, an Fv fragment, a single chain variable fragments (scFv), a single domain antibody (sdAb), a camelid heavy chain antibody, an immunoglobulin new antigen receptor (IgNAR), or a picobody.

42. The molecule of embodiment 41 , wherein the peptide includes a miniprotein or a peptide aptamer.

43. The molecule of embodiments 41 or 42, wherein the cancer marker binding domain includes an sdAb and the immune cell activating epitope binding domain includes an sdAb.

44. The molecule of any of embodiments 41-43, wherein the cancer marker binding domain includes an sdAb, the immune cell activating epitope binding domain includes an sdAb, and the cancer supporter binding domain includes a miniprotein.

45. The molecule of any of embodiments 30-44, wherein the first polypeptide further includes a linker, a signal peptide, and/or a tag.

46. The molecule of any of embodiments 30-45, wherein the second polypeptide further includes a linker, a signal peptide, and/or a tag.

47. The molecule of any of embodiments 5-46, including Fc silencing mutations.

48. The molecule of embodiment 47, wherein the Fc silencing mutations include aglycosylation mutations or LALAPG mutations.

49. The molecule of any of embodiments 5-48, wherein the molecule includes: a first polypeptide encoded by the sequence as set forth in SEQ ID NO: 219 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 219; and a second polypeptide encoded by the sequence as set forth in SEQ ID NO: 220 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 220; or a first polypeptide encoded by the sequence as set forth in SEQ ID NO: 225 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 225; and a second polypeptide encoded by the sequence as set forth in SEQ ID NO: 226 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 226.

50. A nucleotide encoding the molecule of any of embodiments 5-49.

51. A nucleotide including: the sequence as set forth in SEQ ID NO: 219 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 219; the sequence as set forth in SEQ ID NO: 220 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 220; the sequence as set forth in SEQ ID NO: 225 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 225; or the sequence as set forth in SEQ ID NO: 226 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 226

52. A kit including: a cancer marker binding domain and/or a nucleic acid encoding the cancer marker binding domain, an immune cell activating epitope binding domain and/or a nucleic acid encoding the immune cell activating epitope binding domain, and a cancer supporter binding domain and/or a nucleic acid encoding the cancer supporter binding domain.

53. The kit of embodiment 52, wherein the cancer marker binding domain binds receptor tyrosine kinase like orphan receptor 1 (ROR1), prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), mesothelin, CD19, CD20, CD33, Wilms tumor protein (WT1), CD123, CD22, CD133, B-cell maturation antigen (BCMA), human epidermal growth factor receptor 2 (HER2), carbonic Anhydrase IX (CAIX), carcinoembryonic antigen (CEA) or GD2.

54. The kit of embodiments 52 or 53, wherein the cancer marker binding domain binds ROR1 , PSMA, PSCA, mesothelin, CD19, CD20, CD33, WT1, or CD123.

55. The kit of any of embodiments 52-54, wherein the cancer marker binding domain includes a ROR1 binding domain.

56. The kit of any of embodiments 52-55, wherein the cancer marker includes ROR1 and the cancer marker binding domain includes a sequence having SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23 or at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, or SEQ ID NO: 23.

57. The kit of any of embodiments 52-56, wherein the immune cell activating epitope includes an epitope on a T cell, an NK cell, or a macrophage.

58. The kit of any of embodiments 52-57, wherein the immune cell activating epitope includes CD3, CD2, CD4, CD7, CD8, CD27, CD28, CD30, CD40, CD83, 4-1 BB, 0X40, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, or B7-H3.

59. The kit of any of embodiments 52-58, wherein the immune cell activating epitope includes CD3, CD28, 4-1 BB, or CD8.

60. The kit of any of embodiments 52-57, wherein the immune cell activating epitope includes NKG2D, CD8, CD16, KIR2DL4, KIR2DS1, KIR2DS2, KIR3DS1, NKG2C, NKG2E, or NKG2D.

61. The kit of any of embodiments 52-57, wherein the immune cell activating epitope includes CD11b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4/80, IFGR2 Toll-like receptors (TLRs) 1-9, IL-4RO, or MARCO.

62. The kit of any of embodiments 52-57, wherein the immune cell activating epitope binding domain binds and activates a T cell.

63. The kit of any of embodiments 52-62, wherein the immune cell activating epitope binding domain binds CD3.

64. The kit of any of embodiments 52-63, wherein the immune cell activating epitope binding domain binds CD3 and includes a sequence as set forth in SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 63 or a sequence having at least 95% sequence identity to SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 63.

65. The kit of any of embodiments 52-64, wherein the cancer supporter binding domain binds a molecule that cancer cells upregulate or select for to promote their survival.

66. The kit of any of embodiments 52-65, wherein the cancer supporter binding domain binds a molecule that down-regulates immune system activity against a cancer cell that expresses the cancer supporter.

67. The kit of any of embodiments 52-66, wherein the cancer supporter binding domain binds a molecule that provides treatment resistance for a cancer cell that expresses the cancer supporter.

68. The kit of any of embodiments 52-67, wherein the cancer supporter includes PD-L1 , ALCAM/CD166, C10orf54, CEACAM1 , CEACAM20, CMKLR1, HLA-E, IFITM1 , IFITM2, IFITM3, IL31RA, LST1 , MEFV, OSMR, PDL2, PIM1 , PIM2, PLAAT3, PRAME, or SDCBP.

69. The kit of any of embodiments 52-68, wherein the cancer supporter includes OCSTAMP, UNC5C, KCNF1, TMEM45A, RXFP1 , MGAM2, PTGES, TMEM92, CLEC9A, CAV2, LAMA3, RIMS2, BCAR1, PLEKHS1, SSTR2, KCNE5, ITGA7, GPR31 , IL31 RA, SCUBE1 , HPN, GJA4, P2RY14, BPI, S1PR3, FCGR1A, KREMEN1, FLT1, DCSTAMP, GRIN3A, MUC1 , DEFB1 , CALHM6, KCNJ10, CPNE6, FFAR2, SLC6A12, HCAR3, F3, XIRP1 , CLEC6A, FPR2, HCAR2, FRMD3, VAMP5, TMTC1 , CD274, NPFFR1 , GLDN, SMO, CD38, TNFSF10, GPR33, SLAMF7, NRN1 , CYSLTR2, ATP1 B2, PLSCR4, RRAD, KCNJ15, CCRL2, TMEM255A, CSPG4, FPR1 , JAG1 , CDCP1 , GPR146, MYOF, PDGFRA, MYO1A, ABCC11 , LAG3, CAVIN1 , PPFIA4, CR1 L, GPC4, P2RX7, RARRES3, PLEKHA6, TMEM132A, SMCO2, SMCO4, MMP25, KCNJ2, RALB, TIE1 , SCG3, PRRT2, S0RT1, VCAM1 , FAM241A, PTPRU, CLEC4E, NCF1, SLC12A3, IL15RA, SECTM1 , LRRN2, OTOF, GEM, ENTHD1 , OSBPL6, SCARF1 , SUCNR1 , SLC2A5, PSTPIP2, GPR84, GOLM1 , GJB2, SLITRK4, SLC2A6, CLEC4D, SSPN, SORBS1 , GAREM1 , CLDN7, LRRC4, ASPHD2, STX11 , EREG, RAPGEF2, MYO1 B, CLEC5A, SYT17, LYPD5, PDCD1 LG2, PHLDA2, CTTN, SLC31A2, BEGAIN, RIPK2, CSF2RB, DGKG, FRRS1 , TNFSF13B, VRK2, DOCK7, DAPP1 , PRRG4, CACNA1A, ITGB7, PRAME, MFSD2A, CEACAM4, TNF, SLC1A5, CDK14, FAS, ATP1 B1 , FFAR4, HVCN1 , PROCR, CD80, IFITM1 , TENM4, PLAUR, NETO2, SCIMP, PAK1 , CAMK2D, C19orf38, CD83, RHOU, CD40, PLEK, ITPRIPL2, TREML2, EPB41 L5, LILRA2, SLC1A4, LDLR, IFNGR2, SFT2D2, SLAMF8, TLR7, GPBAR1 , SIRPB1 , SIRPB1 , NYNRIN, C3orf14, TJP2, LMTK2, PAQR6, FZD2, DIXDC1 , STX1 B, CNIH4, SLC8A1 , CD33, TMEM131 , NOD2, PDE4B, SH2B2, CLMP, SLC1A3, MCOLN2, IL12RB2, TLR1 , CSF1 , or TLR8.

70. The kit of any of embodiments 52-69, wherein the cancer supporter is an immune checkpoint molecule, immune checkpoint ligand, or a drug efflux pump.

71. The kit of any of embodiments 52-70, wherein the cancer supporter includes PD-L1 , 2B4, A2aR, B7HI, B7H3, B7H4, BTLA, CCR7, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276, CTLA-4, DR3, GAL9, GITR, HAVCR2, HVEM, ID01 , ID02, ICOS, KIR, LAG3, LAIRI, LIGHT, MARCO, PD-1 , PD-L2, PD-L3, PD-L4, PS, OX-40, SL M, TIGHT, TIGIT, TIM3, VISTA, or VTCNI.

72. The kit of any of embodiments 52-71 , wherein the cancer supporter includes PD-L1.

73. The kit of any of embodiments 52-72, wherein the cancer supporter includes PD-L1 and the cancer supporter binding domain includes a sequence as set forth in SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97 or a sequence having at least 95% sequence identity to SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97.

74. The kit of embodiment 70, wherein the immune checkpoint molecule includes CD27, CD28, CD137, CEACAM1 , CTLA4, GITR, ICOS, LAG3, PD-1, OX-40, TIGIT, TIM3, or VISTA.

75. The kit of embodiment 70, wherein the immune checkpoint ligand includes CD70, CD80, CD86, CD137L, CD155/CD112, CEACAM1 , ICOSLG, Galectin 9, GITRL, lectins, OC40L, PD-L1 , PD-L2, or VSIG-3.

76. The kit of embodiment 70, wherein the drug efflux pump includes P-glycoprotein (P-gP or MDR1/ABCB1), monocarboxylate transporter (MCT), ATP-binding cassette transporters (ABC), peptide transporters (PEPTs), Na+ phosphate transporters (NPTs), M DR-associated protein (MRP1/ABCC1), breast cancer resistance protein (MXR or BCRP/ABCG2), MRP/ABCC-2, MRP/ABCC-3, MRP/ABCC-4, MRP/ABCC-5, MRP/ABCC-6, or MRP/ABCC-7.

77. The kit of any of embodiments 52-76, further including a multimerization domain.

78. The kit of any of embodiments 52-77, including a first polypeptide including a first multimerization domain and a second polypeptide including a second multimerization domain wherein the first multimerization domain and the second multimerization domain link the first polypeptide to the second polypeptide.

79. The kit of embodiment 78, wherein first polypeptide includes the cancer marker binding domain and the immune cell activating epitope binding domain and the second polypeptide includes the cancer supporter binding domain.

80. The kit of embodiment 79, wherein the first polypeptide includes a ROR1 binding domain, an I gG 1 Fc knob, and a CD3 binding domain, and the second polypeptide includes a PDL1 binding domain and an IgG 1 Fc hole.

81. The kit of embodiments 79 or 80, wherein the second polypeptide further includes a second cancer marker binding domain, a second immune cell activating epitope binding domain, or a second cancer supporter binding domain.

82. The kit of embodiment 78, wherein first polypeptide includes the cancer marker binding domain and the cancer supporter binding domain and the second polypeptide includes the immune cell activating epitope binding domain.

83. The kit of embodiment 82, wherein the first polypeptide includes a ROR1 binding domain, an I gG1 Fc knob, and a PDL1 binding domain, and the second polypeptide includes a CD3 binding domain and an IgG 1 Fc hole; or the first polypeptide includes a ROR1 binding domain, an IgG 1 Fc hole, and a PDL1 binding domain, and the second polypeptide includes a CD3 binding domain and an IgG 1 Fc knob.

84. The kit of embodiments 82 or 83, wherein the second polypeptide further includes a second cancer marker binding domain, a second immune cell activating epitope binding domain, or a second cancer supporter binding domain.

85. The kit of embodiment 78, wherein first polypeptide includes the immune cell activating epitope binding domain and the cancer supporter binding domain and the second polypeptide includes the cancer marker binding domain.

86. The kit of embodiment 85, wherein the first polypeptide includes a PDL1 binding domain, an lgG1 Fc knob, and a CD3 binding domain, and the second polypeptide includes a ROR1 binding domain and an lgG1 Fc hole.

87. The kit of embodiments 85 or 86, wherein the second polypeptide further includes a second cancer marker binding domain, a second immune cell activating epitope binding domain, or a second cancer supporter binding domain.

88. The kit of any of embodiments 52-87 including a first polypeptide including a first cancer marker binding domain, a first multimerization domain, and a first immune cell activating epitope binding domain, a second polypeptide including a second cancer marker binding domain and a second multimerization domain wherein the first multimerization domain and the second multimerization domain link the first polypeptide to the second polypeptide, a third polypeptide including a third multimerization domain and a second immune cell activating epitope binding domain, and a fourth polypeptide including a fourth multimerization domain and a cancer supporter binding domain wherein the third multimerization domain and the fourth multimerization domain link the third polypeptide to the fourth polypeptide.

89. The kit of any of embodiments 52-87 including a first polypeptide including a first cancer supporter binding domain, a first multimerization domain, and a first immune cell activating epitope binding domain, a second polypeptide including a second cancer supporter binding domain and a second multimerization domain wherein the first multimerization domain and the second multimerization domain link the first polypeptide to the second polypeptide, a third polypeptide including a third multimerization domain and a second immune cell activating epitope binding domain, and a fourth polypeptide including a fourth multimerization domain and a cancer marker binding domain wherein the third multimerization domain and the fourth multimerization domain link the third polypeptide to the fourth polypeptide.

90. The kit of any of embodiments 52-89, wherein at least one of the cancer marker binding domain, the immune cell activating epitope biding domain, and the cancer supporter binding domain include an antibody or a peptide.

91. The kit of embodiment 90, wherein the antibody includes an immunoglobulin G (IgG), a Fab fragment, an Fv fragment, a single chain variable fragments (scFv), a single domain antibody (sdAb), a camelid heavy chain antibody, an immunoglobulin new antigen receptor (IgNAR), or a picobody.

92. The kit of embodiment 90, wherein the peptide includes a miniprotein or a peptide aptamer.

93. The kit of any of embodiments 90-92, wherein the cancer marker binding domain includes an sdAb and the immune cell activating epitope binding domain includes an sdAb. 94. The kit of any of embodiments 90-93, wherein the cancer marker binding domain includes an sdAb, the immune cell activating epitope binding domain includes an sdAb, and the cancer supporter binding domain includes a miniprotein.

95. The kit of any of embodiments 78-94, wherein the first polypeptide further includes a linker, a signal peptide, and/or a tag.

96. The kit of any of embodiments 78-95, wherein the second polypeptide further includes a linker, a signal peptide, and/or a tag.

97. The kit of any of embodiments 52-96, further including a pharmaceutically acceptable carrier.

98. The kit of any of embodiments 52-97, further including a delivery vehicle.

99. The kit of embodiment 98, wherein the delivery vehicle includes a syringe.

100. A composition including a molecule of any of embodiments 5-49 and a pharmaceutically acceptable carrier.

101. A method of killing cancer antigen-positive and cancer antigen-negative cancer cells within a tumor, the method including administering a therapeutically effective amount of a composition of embodiment 100 to the tumor for a therapeutically effective duration, thereby killing cancer antigen-positive and cancer antigen-negative cancer cells within the tumor.

102. The method of embodiment 101 , wherein the tumor is within a subject.

103. The method of embodiments 101 or 102, wherein the method reduces or reverses checkpoint inhibition at the tumor.

104. The method of any of embodiments 101-103, wherein cancer marker-positive cancer cells include less than 50%, less than 40%, less than 30%, less than 20%, less, less than 10%, or less than 5% of the total cancer cells

105. The method of any of embodiments 101-104, wherein the administering is intratumoral, intravenous, intradermal, intraarterial, intranodal, intravesicular, intrathecal, intraperitoneal, intraparenteral, intranasal, intralesional, intramuscular, or subcutaneous administering.

106. The method of any of embodiments 101-105, wherein the administering is intratumoral administering.

107. The method of any of embodiments 101-106, further including identifying a cancer marker expressed by the tumor.

108. The method of embodiment 107, further including selecting a molecule of any of embodiments 5-49, wherein the cancer marker binding domain binds the identified cancer marker.

109. The method of embodiments 107 or 108, wherein the identified cancer marker includes receptor tyrosine kinase like orphan receptor 1 (ROR1), prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), mesothelin, CD19, CD20, CD33, Wilms tumor protein (WT1), CD123, CD22, CD133, B-cell maturation antigen (BCMA), human epidermal growth factor receptor 2 (HER2), carbonic Anhydrase IX (CAIX), carcinoembryonic antigen (CEA), or GD2.

110. The method of any of embodiments 107-109, wherein the identified cancer marker includes ROR1, PSMA, PSCA, mesothelin, CD19, CD20, CD33, WT1, or CD123.

111. The method of any of embodiments 107-110, wherein the identified cancer marker includes a ROR1 binding domain.

112. The method of any of embodiments 107-111, wherein the identified cancer marker includes ROR1 and the cancer marker binding domain includes a sequence having SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, or SEQ ID NO: 23 or at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, or SEQ ID NO: 23.

113. The method of any of embodiments 101-112, further including selecting an immune cell to activate.

114. The method of embodiment 113, further including selecting a molecule of any of embodiments 5-49, wherein the immune cell activating epitope binding domain binds an immune cell activating epitope on the selected immune cell.

115. The method of embodiments 113 or 114, wherein the selected immune cell is a T cell, an NK cell, or a macrophage.

116. The method of any of embodiments 113-115, wherein the selected immune cell is a T cell, an NK cell, or a macrophage and the immune cell activating epitope includes CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, CD83, 4-1 BB, 0X40, lymphocyte function- associated antigen-1 (LFA-1), LIGHT, NKG2C, or B7-H3.

117. The method of any of embodiments 113-116, wherein the selected immune cell is a T cell and the immune cell activating epitope includes CD3, CD28, 4-1 BB, or CD8.

118. The method of any of embodiments 113-116, wherein the selected immune cell is an NK cell and the immune cell activating epitope includes NKG2D, CD8, CD16, KIR2DL4, KIR2DS1 , KIR2DS2, KIR3DS1, NKG2C, NKG2E, or NKG2D.

119. The method of any of embodiments 113-116, wherein the selected immune cell is a macrophage and the immune cell activating epitope includes CD11b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4/80, IFGR2 Toll-like receptors (TLRs) 1-9, IL-4Ra, or MARCO.

120. The method of any of embodiments 113-117, wherein the selected immune cell is a T cell, and the immune cell activating epitope binding domain binds CD3 and includes a sequence as set forth in SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 63 or a sequence having at least 95% sequence identity to SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 63.

121. The method of any of embodiments 101-120, further including identifying a cancer supporter that is or will be expressed by the tumor.

122. The method of embodiment 121 , further including selecting a molecule of any of embodiments 5-49, wherein the cancer supporter binding domain binds the identified cancer supporter.

123. The method of embodiment 122, wherein the cancer supporter binding domain binds a molecule that cancer cells upregulate or select for to promote their survival.

124. The method of embodiments 122 or 123, wherein the cancer supporter binding domain binds a molecule that down-regulates immune system activity against a cancer cell that expresses the cancer supporter.

125. The method of any of embodiments 122-124, wherein the cancer supporter binding domain binds a molecule that provides treatment resistance for a cancer cell that expresses the cancer supporter.

126. The method of any of embodiments 121-125, wherein the cancer supporter includes PD- L1 , ALCAM/CD166, C10orf54, CEACAM1 , CEACAM20, CMKLR1, HLA-E, IFITM1, IFITM2, IFITM3, IL31 RA, LST1 , MEFV, OSMR, PDL2, PIM1 , PIM2, PLAAT3, PRAME, or SDCBP.

127. The method of any of embodiments 121-126, wherein the cancer supporter includes OCSTAMP, UNC5C, KCNF1 , TMEM45A, RXFP1 , MGAM2, PTGES, TMEM92, CLEC9A, CAV2, LAMA3, RIMS2, BCAR1 , PLEKHS1 , SSTR2, KCNE5, ITGA7, GPR31 , IL31RA, SCUBE1 , HPN, GJA4, P2RY14, BPI, S1PR3, FCGR1A, KREMEN1 , FLT1 , DCSTAMP, GRIN3A, MUC1, DEFB1 , CALHM6, KCNJ10, CPNE6, FFAR2, SLC6A12, HCAR3, F3, XIRP1, CLEC6A, FPR2, HCAR2, FRMD3, VAMP5, TMTC1 , CD274, NPFFR1 , GLDN, SMO, CD38, TNFSF10, GPR33, SLAMF7, NRN1 , CYSLTR2, ATP1 B2, PLSCR4, RRAD, KCNJ15, CCRL2, TMEM255A, CSPG4, FPR1 , JAG1 , CDCP1 , GPR146, MYOF, PDGFRA, MYO1A, ABCC11 , LAG3, CAVIN1 , PPFIA4, CR1 L, GPC4, P2RX7, RARRES3, PLEKHA6, TMEM132A, SMCO2, SMCO4, MMP25, KCNJ2, RALB, TIE1 , SCG3, PRRT2, SORT1, VCAM1 , FAM241A, PTPRU, CLEC4E, NCF1, SLC12A3, IL15RA, SECTM1 , LRRN2, OTOF, GEM, ENTHD1 , OSBPL6, SCARF1 , SUCNR1 , SLC2A5, PSTPIP2, GPR84, GOLM1 , GJB2, SLITRK4, SLC2A6, CLEC4D, SSPN, SORBS1 , GAREM1 , CLDN7, LRRC4, ASPHD2, STX11 , EREG, RAPGEF2, MYO1 B, CLEC5A, SYT17, LYPD5, PDCD1 LG2, PHLDA2, CTTN, SLC31A2, BEGAIN, RIPK2, CSF2RB, DGKG, FRRS1 , TNFSF13B, VRK2, DOCK7, DAPP1 , PRRG4, CACNA1A, ITGB7, PRAME, MFSD2A, CEACAM4, TNF, SLC1A5, CDK14, FAS, ATP1 B1 , FFAR4, HVCN1 , PROCR, CD80, IFITM1 , TENM4, PLAUR, NETO2, SCIMP, PAK1 , CAMK2D, C19orf38, CD83, RHOU, CD40, PLEK, ITPRIPL2, TREML2, EPB41 L5, LILRA2, SLC1A4, LDLR, IFNGR2, SFT2D2, SLAMF8, TLR7, GPBAR1 , SIRPB1 , SIRPB1 , NYNRIN, C3orf14, TJP2, LMTK2, PAQR6, FZD2, DIXDC1 , STX1 B, CNIH4, SLC8A1 , CD33, TMEM131 , NOD2, PDE4B, SH2B2, CLMP, SLC1A3, MCOLN2, IL12RB2, TLR1 , CSF1 , or TLR8.

128. The method of any of embodiments 121-127, wherein the cancer supporter is an immune checkpoint molecule, immune checkpoint ligand, or a drug efflux pump.

129. The method of any of embodiments 121-128, wherein the cancer supporter includes PD- L1 , 2B4, A2aR, B7HI, B7H3, B7H4, BTLA, CCR7, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276, CTLA-4, DR3, GAL9, GITR, HAVCR2, HVEM, ID01 , ID02, ICOS, KIR, LAG3, LAIRI, LIGHT, MARCO, PD-1 , PD-L2, PD-L3, PD-L4, PS, OX-40, SLAM, TIGHT, TIGIT, TIM3, VISTA, or VTCNI.

130. The method of any of embodiments 121-129, wherein the cancer supporter includes PD- L1.

131. The method of any of embodiments 121-130, wherein the cancer supporter includes PD- L1 and the cancer supporter binding domain includes a sequence as set forth in SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97 or a sequence having at least 95% sequence identity to SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97.

132. The method of embodiment 128, wherein the immune checkpoint molecule includes CD27, CD28, CD137, CEACAM1 , CTLA4, GITR, ICOS, LAG3, PD-1 , OX-40, TIGIT, TIM3, or VISTA. 133. The method of embodiment 128, wherein the immune checkpoint ligand includes CD70, CD80, CD86, CD137L, CD155/CD112, CEACAM1 , ICOSLG, Galectin 9, GITRL, lectins, OC40L, PD-L1 , PD-L2, or VSIG-3.

134. The method of embodiment 128, wherein the drug efflux pump includes P-glycoprotein (P-gP or MDR1/ABCB1), monocarboxylate transporter (MCT), ATP-binding cassette transporters (ABC), peptide transporters (PEPTs), Na+ phosphate transporters (NPTs), MDR-associated protein (MRP1/ABCC1), breast cancer resistance protein (MXR or BCRP/ABCG2), MRP/ABCC- 2, MRP/ABCC-3, MRP/ABCC-4, MRP/ABCC-5, MRP/ABCC-6, or MRP/ABCC-7.

135. A method of selecting cancer supporter therapeutic targets, the method including: exposing healthy cells and cancer cells to an immune cell activation indicator; measuring gene expression in the healthy cells and the cancer cells after the exposing; and creating a set of genes with upregulated expression in the cancer cells but not the healthy cells after the exposing; and selecting from the set of genes those that encode a cell-surface expressed protein, thereby selecting cancer supporter therapeutic targets.

136. The method of embodiment 135, wherein the healthy cells include peripheral blood mononuclear cells (PBMCs).

137. The method of embodiments 135 or 136, wherein the cancer cells include diffuse midline glioma (DMG) primary tumor cells.

138. The method of any of embodiments 135-137, wherein the immune cell activation indicator includes I FNy, TNFa, IL-2, IL-7, or IL-15.

139. The method of any of embodiments 135-138, wherein the immune cell activation indicator includes supernatant from co-culture of activated T cells.

140. A method of designing a multi-specific binding molecule, the method including: selecting a cancer marker binding domain; selecting an immune cell activating epitope binding domain; exposing healthy cells and cancer cells to an immune cell activation indicator; measuring gene expression in the healthy cells and the cancer cells after the exposing; and creating a set of genes with upregulated expression in the cancer cells but not the healthy cells after the exposing; selecting a gene from the set of genes that encodes a cell-surface expressed protein, and selecting a binding domain that binds the cell-surface expressed protein thereby designing a multi-specific molecule.

141. The method of embodiment 140, wherein the healthy cells include peripheral blood mononuclear cells (PBMCs).

142. The method of embodiments 140 or 141 , wherein the cancer cells include diffuse midline glioma (DMG) primary tumor cells.

143. The method of any of embodiments 140-142, wherein the immune cell activation indicator includes I FNy, TNFa, IL-2, IL-7, or IL-15.

144. The method of any of embodiments 140-143, wherein the immune cell activation indicator includes supernatant from co-culture of activated T cells.

145. The method of any of embodiments 140-144, further including selecting a multimerization domain.

146. The method of embodiment 145, wherein the multimerization domain includes an lgG1 Fc knob and lgG1 Fc hole.

[0320] (XIII) Closing Paragraphs. The nucleic acid and amino acid sequences provided herein are shown using letter abbreviations for nucleotide bases and amino acid residues, as defined in 37 C.F.R. §1.831-1.835 and set forth in WIPO Standard ST.26 (implemented on July 1, 2022). Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included in embodiments where it would be appropriate.

[0321] Sequence information provided by public databases can be used to identify additional gene and protein sequences that can be used with the systems and methods disclosed herein. [0322] In particular embodiments, the term can be further used to indicate that the binding domain. A cognate binding molecule or targeted epitope is one that will be bound by its corresponding binding domain (e.g., binding domain in an ICEm) under relevant in vitro conditions and in in vivo conditions as described herein. Unless otherwise indicated, the practice of the present disclosure can employ conventional techniques of immunology, molecular biology, cell biology and recombinant DNA. These methods are described in the following publications. See, e.g., Sambrook, et al. Molecular Cloning: A Laboratory Manual, 2nd Edition (1989); F. M. Ausubel, et al. eds., Current Protocols in Molecular Biology, (1987); the series Methods IN Enzymology (Academic Press, Inc.); M. MacPherson, et al., PCR: A Practical Approach, IRL Press at Oxford University Press (1991); MacPherson et al., eds. PCR 2: Practical Approach, (1995); Harlow and Lane, eds. Antibodies, A Laboratory Manual, (1988); and R. I. Freshney, ed. Animal Cell Culture (1987).

[0323] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment. A material effect would cause a statistically significant increase in reduction in cancer cell killing, as described herein.

[0324] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11 % of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.

[0325] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0326] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0327] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0328] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0329] Furthermore, numerous references have been made to patents, printed publications, journal articles and other written text throughout this specification (referenced materials herein). Each of the referenced materials are individually incorporated herein by reference in their entirety for their referenced teaching.

[0330] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

[0331] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and/or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0332] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the examples or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3rd Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Eds. Attwood T et al., Oxford University Press, Oxford, 2006).

Claims

CLAIMS What is claimed is:

1. A molecule comprising a first polypeptide comprising a sequence as set forth in SEQ ID NO: 127 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 127; and a second polypeptide comprising a sequence as set forth in SEQ ID NO: 129 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 129; a first polypeptide comprising the sequence as set forth in SEQ ID NO: 128 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 128; and a second polypeptide comprising the sequence as set forth in SEQ ID NO: 130 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 130; or a first polypeptide comprising the sequence as set forth in SEQ ID NO: 150 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 150; and a second polypeptide comprising the sequence as set forth in SEQ ID NO: 149 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 149.

2. A molecule comprising a first polypeptide comprising a first ROR1 binding domain, a CD3 binding domain, and a first multimerization domain, and a second polypeptide comprising a second ROR1 binding domain, a PD-L1 binding domain, and a second multimerization domain, wherein the first multimerization domain and the second multimerization domain link the first polypeptide to the second polypeptide.

3. A molecule comprising a first polypeptide comprising a first PD-L1 binding domain, a CD3 binding domain, and a first multimerization domain, and a second polypeptide comprising a second PD-L1 binding domain, a ROR1 binding domain, and a second multimerization domain, wherein the first multimerization domain and the second multimerization domain link the first polypeptide to the second polypeptide.

4. A molecule comprising a first polypeptide comprising a first CD3 binding domain, a ROR1 binding domain, and a first multimerization domain, and a second polypeptide comprising a second CD3 binding domain, a PD-L1 binding domain, and a second multimerization domain, wherein the first multimerization domain and the second multimerization domain link the first polypeptide to the second polypeptide.

5. A molecule comprising: a cancer marker binding domain, an immune cell activating epitope binding domain, and a cancer supporter binding domain.

6. The molecule of claim 5, wherein the cancer marker binding domain binds receptor tyrosine kinase like orphan receptor 1 (ROR1), prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), mesothelin, CD19, CD20, CD33, Wilms tumor protein (WT1), CD123, CD22, CD133, B-cell maturation antigen (BCMA), human epidermal growth factor receptor 2 (HER2), carbonic Anhydrase IX (CAIX), carcinoembryonic antigen (CEA), or GD2.

7. The molecule of claim 5, wherein the cancer marker binding domain binds ROR1 , PSMA, PSCA, mesothelin, CD19, CD20, CD33, WT1, or CD123.

8. The molecule of claim 5, wherein the cancer marker binding domain comprises a ROR1 binding domain.

9. The molecule of claim 5, wherein the cancer marker comprises ROR1 and the cancer marker binding domain comprises a sequence having SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, or SEQ ID NO: 23 or at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, or SEQ ID NO: 23.

10. The molecule of claim 5, wherein the immune cell activating epitope comprises an epitope on a T cell, an NK cell, or a macrophage.

11. The molecule of claim 5, wherein the immune cell activating epitope comprises CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, CD83, 4-1 BB, 0X40, lymphocyte function- associated antigen-1 (LFA-1), LIGHT, NKG2C, or B7-H3.

12. The molecule of claim 5, wherein the immune cell activating epitope comprises CD3, CD28, 4-1 BB, or CD8.

13. The molecule of claim 5, wherein the immune cell activating epitope comprises NKG2D, CD8, CD16, KIR2DL4, KIR2DS1 , KIR2DS2, KIR3DS1 , NKG2C, NKG2E, or NKG2D.

14. The molecule of claim 5, wherein the immune cell activating epitope comprises CD11b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4/80, IFGR2 Toll-like receptors (TLRs) 1-9, IL-4Ra, or MARCO.

15. The molecule of claim 5, wherein the immune cell activating epitope binding domain binds and activates a T cell.

16. The molecule of claim 5, wherein the immune cell activating epitope binding domain binds CD3.

17. The molecule of claim 5, wherein the immune cell activating epitope binding domain binds CD3 and comprises a sequence as set forth in SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 63 or a sequence having at least 95% sequence identity to SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 63.

18. The molecule of claim 5, wherein the cancer supporter binding domain binds a molecule that cancer cells upregulate or select for to promote their survival.

19. The molecule of claim 5, wherein the cancer supporter binding domain binds a molecule that down-regulates immune system activity against a cancer cell that expresses the cancer supporter.

20. The molecule of claim 5, wherein the cancer supporter binding domain binds a molecule that provides treatment resistance for a cancer cell that expresses the cancer supporter.

21. The molecule of claim 5, wherein the cancer supporter comprises PD-L1 , ALCAM/CD166, C10orf54, CEACAM1 , CEACAM20, CMKLR1 , HLA-E, IFITM1 , IFITM2, IFITM3, IL31 RA, LST1 , MEFV, OSMR, PDL2, PIM1 , PIM2, PLAAT3, PRAME, or SDCBP.

22. The molecule of claim 5, wherein the cancer supporter comprises OCSTAMP, UNC5C, KCNF1 , TMEM45A, RXFP1 , MGAM2, PTGES, TMEM92, CLEC9A, CAV2, LAMA3, RIMS2, BCAR1, PLEKHS1 , SSTR2, KCNE5, ITGA7, GPR31 , IL31RA, SCUBE1 , HPN, GJA4, P2RY14, BPI, S1 PR3, FCGR1A, KREMEN1 , FLT1, DCSTAMP, GRIN3A, MUC1 , DEFB1 , CALHM6, KCNJ10, CPNE6, FFAR2, SLC6A12, HCAR3, F3, XIRP1 , CLEC6A, FPR2, HCAR2, FRMD3, VAMP5, TMTC1 , CD274, NPFFR1 , GLDN, SMO, CD38, TNFSF10, GPR33, SLAMF7, NRN1 , CYSLTR2, ATP1 B2, PLSCR4, RRAD, KCNJ15, CCRL2, TMEM255A, CSPG4, FPR1 , JAG1 , CDCP1, GPR146, MYOF, PDGFRA, MYO1A, ABCC11 , LAG3, CAVIN1 , PPFIA4, CR1L, GPC4, P2RX7, RARRES3, PLEKHA6, TMEM132A, SMCO2, SMCO4, MMP25, KCNJ2, RALB, TIE1, SCG3, PRRT2, SORT1 , VCAM1 , FAM241A, PTPRU, CLEC4E, NCF1 , SLC12A3, IL15RA, SECTM1 , LRRN2, OTOF, GEM, ENTHD1 , OSBPL6, SCARF1 , SUCNR1 , SLC2A5, PSTPIP2, GPR84, GOLM1 , GJB2, SLITRK4, SLC2A6, CLEC4D, SSPN, SORBS1 , GAREM1 , CLDN7, LRRC4, ASPHD2, STX11 , EREG, RAPGEF2, MYO1 B, CLEC5A, SYT17, LYPD5, PDCD1 LG2, PHLDA2, CTTN, SLC31A2, BEGAIN, RIPK2, CSF2RB, DGKG, FRRS1 , TNFSF13B, VRK2, DOCK7, DAPP1 , PRRG4, CACNA1A, ITGB7, PRAME, MFSD2A, CEACAM4, TNF, SLC1A5, CDK14, FAS, ATP1 B1 , FFAR4, HVCN1 , PROCR, CD80, IFITM1 , TENM4, PLAUR, NETO2, SCIMP, PAK1 , CAMK2D, C19orf38, CD83, RHOU, CD40, PLEK, ITPRIPL2, TREML2, EPB41 L5, LILRA2, SLC1A4, LDLR, IFNGR2, SFT2D2, SLAMF8, TLR7, GPBAR1 , SIRPB1 , SIRPB1 , NYNRIN, C3orf14, TJP2, LMTK2, PAQR6, FZD2, DIXDC1 , STX1 B, CNIH4, SLC8A1 , CD33, TMEM131 , NOD2, PDE4B, SH2B2, CLMP, SLC1A3, MCOLN2, IL12RB2, TLR1 , CSF1 , or TLR8.

23. The molecule of claim 5, wherein the cancer supporter is an immune checkpoint molecule, immune checkpoint ligand, or a drug efflux pump.

24. The molecule of claim 5, wherein the cancer supporter comprises PD-L1 , 2B4, A2aR, B7HI, B7H3, B7H4, BTLA, CCR7, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276, CTLA-4, DR3, GAL9, GITR, HAVCR2, HVEM, ID01 , ID02, ICOS, KIR, LAG3, LAIRI, LIGHT, MARCO, PD-1 , PD-L2, PD-L3, PD-L4, PS, OX-40, SLAM, TIGHT, TIGIT, TIM3, VISTA, or TCNI.

25. The molecule of claim 5, wherein the cancer supporter comprises PD-L1.

26. The molecule of claim 5, wherein the cancer supporter comprises PD-L1 and the cancer supporter binding domain comprises a sequence as set forth in SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97 or a sequence having at least 95% sequence identity to SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97.

27. The molecule of claim 23, wherein the immune checkpoint molecule comprises CD27, CD28, CD137, CEACAM1 , CTLA4, GITR, ICOS, LAG3, PD-1, OX-40, TIGIT, TIM3, or VISTA.

28. The molecule of claim 23, wherein the immune checkpoint ligand comprises CD70, CD80, CD86, CD137L, CD155/CD112, CEACAM1 , ICOSLG, Galectin 9, GITRL, lectins, OC40L, PD-L1 , PD-L2, or VSIG-3.

29. The molecule of claim 23, wherein the drug efflux pump comprises P-glycoprotein (P-gP or MDR1/ABCB1), monocarboxylate transporter (MCT), ATP-binding cassette transporters (ABC), peptide transporters (PEPTs), Na+ phosphate transporters (NPTs), MDR-associated protein (MRP1/ABCC1), breast cancer resistance protein (MXR or BCRP/ABCG2), MRP/ABCC- 2, MRP/ABCC-3, MRP/ABCC-4, MRP/ABCC-5, MRP/ABCC-6, or MRP/ABCC-7.

30. The molecule of claim 5, comprising a first polypeptide comprising a first multimerization domain and a second polypeptide comprising a second multimerization domain wherein the first multimerization domain and the second multimerization domain link the first polypeptide to the second polypeptide.

31. The molecule of claim 30, wherein first polypeptide comprises the cancer marker binding domain and the immune cell activating epitope binding domain and the second polypeptide comprises the cancer supporter binding domain.

32. The molecule of claim 31 , wherein the first polypeptide comprises a ROR1 binding domain, an lgG1 Fc knob, and a CD3 binding domain, and the second polypeptide comprises a PDL1 binding domain and an IgG 1 Fc hole.

33. The molecule of claim 31 , wherein the second polypeptide further comprises a second cancer marker binding domain, a second immune cell activating epitope binding domain, or a second cancer supporter binding domain.

34. The molecule of claim 30, wherein the first polypeptide comprises the cancer marker binding domain and the cancer supporter binding domain and the second polypeptide comprises the immune cell activating epitope binding domain.

35. The molecule of claim 34, wherein the first polypeptide comprises a ROR1 binding domain, an I gG 1 Fc knob, and a PDL1 binding domain, and the second polypeptide comprises a CD3 binding domain and an lgG1 Fc hole; or the first polypeptide comprises a ROR1 binding domain, an I gG 1 Fc hole, and a PDL1 binding domain, and the second polypeptide comprises a CD3 binding domain and an IgG 1 Fc knob.

36. The molecule of claim 34, wherein the second polypeptide further comprises a second cancer marker binding domain, a second immune cell activating epitope binding domain, or a second cancer supporter binding domain.

37. The molecule of claim 30, wherein the first polypeptide comprises the immune cell activating epitope binding domain and the cancer supporter binding domain and the second polypeptide comprises the cancer marker binding domain.

38. The molecule of claim 37, wherein the first polypeptide comprises a PDL1 binding domain, an lgG1 Fc knob, and a CD3 binding domain, and the second polypeptide comprises a ROR1 binding domain and an lgG1 Fc hole.

39. The molecule of claim 37, wherein the second polypeptide further comprises a second cancer marker binding domain, a second immune cell activating epitope binding domain, or a second cancer supporter binding domain.

40. The molecule of claim 5, wherein at least one of the cancer marker binding domain, the immune cell activating epitope biding domain, and the cancer supporter binding domain comprise an antibody or a peptide.

41. The molecule of claim 40, wherein the antibody comprises an immunoglobulin G (IgG), a Fab fragment, an Fv fragment, a single chain variable fragments (scFv), a single domain antibody (sdAb), a camelid heavy chain antibody, an immunoglobulin new antigen receptor (IgNAR), or a picobody.

42. The molecule of claim 41 , wherein the peptide comprises a miniprotein or a peptide aptamer.

43. The molecule of claim 41 , wherein the cancer marker binding domain comprises an sdAb and the immune cell activating epitope binding domain comprises an sdAb.

44. The molecule of claim 41 , wherein the cancer marker binding domain comprises an sdAb, the immune cell activating epitope binding domain comprises an sdAb, and the cancer supporter binding domain comprises a miniprotein.

45. The molecule of claim 30, wherein the first polypeptide further comprises a linker, a signal peptide, and/or a tag.

46. The molecule of claim 30, wherein the second polypeptide further comprises a linker, a signal peptide, and/or a tag.

47. The molecule of claim 5, comprising Fc silencing mutations.

48. The molecule of claim 47, wherein the Fc silencing mutations comprise aglycosylation mutations or LALAPG mutations.

49. The molecule of claim 5, wherein the molecule comprises: a first polypeptide encoded by the sequence as set forth in SEQ ID NO: 219 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 219; and a second polypeptide encoded by the sequence as set forth in SEQ ID NO: 220 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 220; or a first polypeptide encoded by the sequence as set forth in SEQ ID NO: 225 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 225; and a second polypeptide encoded by the sequence as set forth in SEQ ID NO: 226 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 226.

50. A nucleotide encoding the molecule of claim 5.

51. A nucleotide comprising: the sequence as set forth in SEQ ID NO: 219 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 219; the sequence as set forth in SEQ ID NO: 220 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 220; the sequence as set forth in SEQ ID NO: 225 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 225; or the sequence as set forth in SEQ ID NO: 226 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 226

52. A kit comprising: a cancer marker binding domain and/or a nucleic acid encoding the cancer marker binding domain, an immune cell activating epitope binding domain and/or a nucleic acid encoding the immune cell activating epitope binding domain, and a cancer supporter binding domain and/or a nucleic acid encoding the cancer supporter binding domain.

53. The kit of claim 52, wherein the cancer marker binding domain binds receptor tyrosine kinase like orphan receptor 1 (ROR1), prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), mesothelin, CD19, CD20, CD33, Wilms tumor protein (WT1), CD123, CD22, CD133, B-cell maturation antigen (BCMA), human epidermal growth factor receptor 2 (HER2), carbonic Anhydrase IX (CAIX), carcinoembryonic antigen (CEA) or GD2.

54. The kit of claim 52, wherein the cancer marker binding domain binds ROR1 , PSMA, PSCA, mesothelin, CD19, CD20, CD33, WT1 , or CD123.

55. The kit of claim 52, wherein the cancer marker binding domain comprises a ROR1 binding domain.

56. The kit of claim 52, wherein the cancer marker comprises ROR1 and the cancer marker binding domain comprises a sequence having SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, or SEQ ID NO: 23 or at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, or SEQ ID NO: 23.

57. The kit of claim 52, wherein the immune cell activating epitope comprises an epitope on a T cell, an NK cell, or a macrophage.

58. The kit of claim 52, wherein the immune cell activating epitope comprises CD3, CD2, CD4, CD7, CD8, CD27, CD28, CD30, CD40, CD83, 4-1 BB, 0X40, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, or B7-H3.

59. The kit of claim 52, wherein the immune cell activating epitope comprises CD3, CD28, 4- 1 BB, or CD8.

60. The kit of claim 52, wherein the immune cell activating epitope comprises NKG2D, CD8, CD16, KIR2DL4, KIR2DS1 , KIR2DS2, KIR3DS1, NKG2C, NKG2E, or NKG2D.

61. The kit of claim 52, wherein the immune cell activating epitope comprises CD11 b, CD11 c, CD64, CD68, CD119, CD163, CD206, CD209, F4/80, IFGR2 Toll-like receptors (TLRs) 1-9, IL- 4Ro, or MARCO.

62. The kit of claim 52, wherein the immune cell activating epitope binding domain binds and activates a T cell.

63. The kit of claim 52, wherein the immune cell activating epitope binding domain binds CD3.

64. The kit of claim 52, wherein the immune cell activating epitope binding domain binds CD3 and comprises a sequence as set forth in SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 63 or a sequence having at least 95% sequence identity to SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 63.

65. The kit of claim 52, wherein the cancer supporter binding domain binds a molecule that cancer cells upregulate or select for to promote their survival.

66. The kit of claim 52, wherein the cancer supporter binding domain binds a molecule that down-regulates immune system activity against a cancer cell that expresses the cancer supporter.

67. The kit of claim 52, wherein the cancer supporter binding domain binds a molecule that provides treatment resistance for a cancer cell that expresses the cancer supporter.

68. The kit of claim 52, wherein the cancer supporter comprises PD-L1 , ALCAM/CD166, C10orf54, CEACAM1, CEACAM20, CMKLR1, HLA-E, IFITM1, IFITM2, IFITM3, IL31RA, LST1, MEFV, OSMR, PDL2, PIM1, PIM2, PLAAT3, PRAME, or SDCBP.

69. The kit of claim 52, wherein the cancer supporter comprises OCSTAMP, UNC5C, KCNF1, TMEM45A, RXFP1, MGAM2, PTGES, TMEM92, CLEC9A, CAV2, LAMA3, RIMS2, BCAR1, PLEKHS1, SSTR2, KCNE5, ITGA7, GPR31, IL31RA, SCUBE1, HPN, GJA4, P2RY14, BPI, S1PR3, FCGR1A, KREMEN1, FLT1, DCSTAMP, GRIN3A, MUC1, DEFB1, CALHM6, KCNJ10, CPNE6, FFAR2, SLC6A12, HCAR3, F3, XIRP1, CLEC6A, FPR2, HCAR2, FRMD3, VAMP5, TMTC1, CD274, NPFFR1, GLDN, SMO, CD38, TNFSF10, GPR33, SLAMF7, NRN1, CYSLTR2, ATP1B2, PLSCR4, RRAD, KCNJ15, CCRL2, TMEM255A, CSPG4, FPR1, JAG1, CDCP1, GPR146, MYOF, PDGFRA, MYO1A, ABCC11, LAG3, CAVIN1, PPFIA4, CR1L, GPC4, P2RX7, RARRES3, PLEKHA6, TMEM132A, SMCO2, SMCO4, MMP25, KCNJ2, RALB, TIE1, SCG3, PRRT2, SORT1, VCAM1, FAM241A, PTPRU, CLEC4E, NCF1, SLC12A3, IL15RA, SECTM1, LRRN2, OTOF, GEM, ENTHD1, OSBPL6, SCARF1, SUCNR1, SLC2A5, PSTPIP2, GPR84, GOLM1, GJB2, SLITRK4, SLC2A6, CLEC4D, SSPN, SORBS1, GAREM1, CLDN7, LRRC4, ASPHD2, STX11, EREG, RAPGEF2, MYO1B, CLEC5A, SYT17, LYPD5, PDCD1LG2, PHLDA2, CTTN, SLC31A2, BEGAIN, RIPK2, CSF2RB, DGKG, FRRS1, TNFSF13B, VRK2, DOCK7, DAPP1, PRRG4, CACNA1A, ITGB7, PRAME, MFSD2A, CEACAM4, TNF, SLC1A5, CDK14, FAS, ATP1B1, FFAR4, HVCN1, PROCR, CD80, IFITM1, TENM4, PLAUR, NETO2, SCIMP, PAK1, CAMK2D, C19orf38, CD83, RHOU, CD40, PLEK, ITPRIPL2, TREML2, EPB41L5, LILRA2, SLC1A4, LDLR, IFNGR2, SFT2D2, SLAMF8, TLR7, GPBAR1, SIRPB1, SIRPB1, NYNRIN, C3orf14, TJP2, LMTK2, PAQR6, FZD2, DIXDC1, STX1B, CNIH4, SLC8A1, CD33, TMEM131, NOD2, PDE4B, SH2B2, CLMP, SLC1A3, MCOLN2, IL12RB2, TLR1, CSF1, orTLR8.

70. The kit of claim 52, wherein the cancer supporter is an immune checkpoint molecule, immune checkpoint ligand, or a drug efflux pump.

71. The kit of claim 52, wherein the cancer supporter comprises PD-L1, 2B4, A2aR, B7HI, B7H3, B7H4, BTLA, CCR7, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276, CTLA-4, DR3, GAL9, GITR, HAVCR2, HVEM, ID01, ID02, ICOS, KIR, LAG3, LAIRI, LIGHT, MARCO, PD-1, PD-L2, PD-L3, PD-L4, PS, OX-40, SLAM, TIGHT, TIGIT, TIM3, VISTA, or TCNI.

72. The kit of claim 52, wherein the cancer supporter comprises PD-L1.

73. The kit of claim 52, wherein the cancer supporter comprises PD-L1 and the cancer supporter binding domain comprises a sequence as set forth in SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97 or a sequence having at least 95% sequence identity to SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97.

74. The kit of claim 70, wherein the immune checkpoint molecule comprises CD27, CD28, CD137, CEACAM1 , CTLA4, GITR, ICOS, LAG3, PD-1, OX-40, TIGIT, TIM3, or VISTA.

75. The kit of claim 70, wherein the immune checkpoint ligand comprises CD70, CD80, CD86, CD137L, CD155/CD112, CEACAM1 , ICOSLG, Galectin 9, GITRL, lectins, OC40L, PD-L1 , PD- L2, or SIG-3.

76. The kit of claim 70, wherein the drug efflux pump comprises P-glycoprotein (P-gP or MDR1/ABCB1), monocarboxylate transporter (MCT), ATP-binding cassette transporters (ABC), peptide transporters (PEPTs), Na+ phosphate transporters (NPTs), M DR-associated protein (MRP1/ABCC1), breast cancer resistance protein (MXR or BCRP/ABCG2), MRP/ABCC-2, MRP/ABCC-3, MRP/ABCC-4, MRP/ABCC-5, MRP/ABCC-6, or MRP/ABCC-7.

77. The kit of claim 52, further comprising a multimerization domain.

78. The kit of claim 52, comprising a first polypeptide comprising a first multimerization domain and a second polypeptide comprising a second multimerization domain wherein the first multimerization domain and the second multimerization domain link the first polypeptide to the second polypeptide.

79. The kit of claim 78, wherein first polypeptide comprises the cancer marker binding domain and the immune cell activating epitope binding domain and the second polypeptide comprises the cancer supporter binding domain.

80. The kit of claim 79, wherein the first polypeptide comprises a ROR1 binding domain, an IgG 1 Fc knob, and a CD3 binding domain, and the second polypeptide comprises a PDL1 binding domain and an IgG 1 Fc hole.

81. The kit of claim 79, wherein the second polypeptide further comprises a second cancer marker binding domain, a second immune cell activating epitope binding domain, or a second cancer supporter binding domain.

82. The kit of claim 78, wherein first polypeptide comprises the cancer marker binding domain and the cancer supporter binding domain and the second polypeptide comprises the immune cell activating epitope binding domain.

83. The kit of claim 82, wherein the first polypeptide comprises a ROR1 binding domain, an I gG1 Fc knob, and a PDL1 binding domain, and the second polypeptide comprises a CD3 binding domain and an I gG 1 Fc hole; or the first polypeptide comprises a ROR1 binding domain, an I gG 1 Fc hole, and a PDL1 binding domain, and the second polypeptide comprises a CD3 binding domain and an IgG 1 Fc knob.

84. The kit of claim 82, wherein the second polypeptide further comprises a second cancer marker binding domain, a second immune cell activating epitope binding domain, or a second cancer supporter binding domain.

85. The kit of claim 78, wherein first polypeptide comprises the immune cell activating epitope binding domain and the cancer supporter binding domain and the second polypeptide comprises the cancer marker binding domain.

86. The kit of claim 85, wherein the first polypeptide comprises a PDL1 binding domain, an IgG 1 Fc knob, and a CD3 binding domain, and the second polypeptide comprises a ROR1 binding domain and an IgG 1 Fc hole.

87. The kit of claim 85, wherein the second polypeptide further comprises a second cancer marker binding domain, a second immune cell activating epitope binding domain, or a second cancer supporter binding domain.

88. The kit of claim 52 comprising a first polypeptide comprising a first cancer marker binding domain, a first multimerization domain, and a first immune cell activating epitope binding domain, a second polypeptide comprising a second cancer marker binding domain and a second multimerization domain wherein the first multimerization domain and the second multimerization domain link the first polypeptide to the second polypeptide, a third polypeptide comprising a third multimerization domain and a second immune cell activating epitope binding domain, and a fourth polypeptide comprising a fourth multimerization domain and a cancer supporter binding domain wherein the third multimerization domain and the fourth multimerization domain link the third polypeptide to the fourth polypeptide.

89. The kit of claim 52 comprising a first polypeptide comprising a first cancer supporter binding domain, a first multimerization domain, and a first immune cell activating epitope binding domain, a second polypeptide comprising a second cancer supporter binding domain and a second multimerization domain wherein the first multimerization domain and the second multimerization domain link the first polypeptide to the second polypeptide, a third polypeptide comprising a third multimerization domain and a second immune cell activating epitope binding domain, and a fourth polypeptide comprising a fourth multimerization domain and a cancer marker binding domain wherein the third multimerization domain and the fourth multimerization domain link the third polypeptide to the fourth polypeptide.

Ill

90. The kit of claim 52, wherein at least one of the cancer marker binding domain, the immune cell activating epitope biding domain, and the cancer supporter binding domain comprise an antibody or a peptide.

91. The kit of claim 90, wherein the antibody comprises an immunoglobulin G (IgG), a Fab fragment, an Fv fragment, a single chain variable fragments (scFv), a single domain antibody (sdAb), a camelid heavy chain antibody, an immunoglobulin new antigen receptor (IgNAR), or a picobody.

92. The kit of claim 90, wherein the peptide comprises a miniprotein or a peptide aptamer.

93. The kit of claim 90, wherein the cancer marker binding domain comprises an sdAb and the immune cell activating epitope binding domain comprises an sdAb.

94. The kit of claim 90, wherein the cancer marker binding domain comprises an sdAb, the immune cell activating epitope binding domain comprises an sdAb, and the cancer supporter binding domain comprises a miniprotein.

95. The kit of claim 78, wherein the first polypeptide further comprises a linker, a signal peptide, and/or a tag.

96. The kit of claim 78, wherein the second polypeptide further comprises a linker, a signal peptide, and/or a tag.

97. The kit of claim 52, further comprising a pharmaceutically acceptable carrier.

98. The kit of claim 52, further comprising a delivery vehicle.

99. The kit of claim 98, wherein the delivery vehicle comprises a syringe.

100. A composition comprising a molecule of claim 5 and a pharmaceutically acceptable carrier.

101. A method of killing cancer antigen-positive and cancer antigen-negative cancer cells within a tumor, the method comprising administering a therapeutically effective amount of a composition of claim 100 to the tumor for a therapeutically effective duration, thereby killing cancer antigen-positive and cancer antigen-negative cancer cells within the tumor.

102. The method of claim 101 , wherein the tumor is within a subject.

103. The method of claim 101 , wherein the method reduces or reverses checkpoint inhibition at the tumor.

104. The method of claim 101 , wherein cancer marker-positive cancer cells comprise less than 50%, less than 40%, less than 30%, less than 20%, less, less than 10%, or less than 5% of the total cancer cells

105. The method of claim 101 , wherein the administering is intratumoral, intravenous, intradermal, intraarterial, intranodal, intravesicular, intrathecal, intraperitoneal, intraparenteral, intranasal, intralesional, intramuscular, or subcutaneous administering.

106. The method of claim 101 , wherein the administering is intratumoral administering.

107. The method of claim 101 , further comprising identifying a cancer marker expressed by the tumor.

108. The method of claim 107, further comprising selecting a molecule of claim 5, wherein the cancer marker binding domain binds the identified cancer marker.

109. The method of claim 107, wherein the identified cancer marker comprises receptor tyrosine kinase like orphan receptor 1 (ROR1), prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), mesothelin, CD19, CD20, CD33, Wilms tumor protein (WT1), CD123, CD22, CD133, B-cell maturation antigen (BCMA), human epidermal growth factor receptor 2 (HER2), carbonic Anhydrase IX (CAIX), carcinoembryonic antigen (CEA), or GD2.

110. The method of claim 107, wherein the identified cancer marker comprises ROR1 , PSMA, PSCA, mesothelin, CD19, CD20, CD33, WT1, or CD123.

111. The method of claim 107, wherein the identified cancer marker comprises a ROR1 binding domain.

112. The method of claim 107, wherein the identified cancer marker comprises ROR1 and the cancer marker binding domain comprises a sequence having SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, or SEQ ID NO: 23 or at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, or SEQ ID NO: 23.

113. The method of claim 101 , further comprising selecting an immune cell to activate.

114. The method of claim 113, further comprising selecting a molecule of claim 5, wherein the immune cell activating epitope binding domain binds an immune cell activating epitope on the selected immune cell.

115. The method of claim 113, wherein the selected immune cell is a T cell, an NK cell, or a macrophage.

116. The method of claim 113, wherein the selected immune cell is a T cell, an NK cell, or a macrophage and the immune cell activating epitope comprises CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, CD83, 4-1 BB, 0X40, lymphocyte function-associated antigen-1 (LFA- 1), LIGHT, NKG2C, or B7-H3.

117. The method of claim 113, wherein the selected immune cell is a T cell and the immune cell activating epitope comprises CD3, CD28, 4-1BB, or CD8.

118. The method of claim 113, wherein the selected immune cell is an NK cell and the immune cell activating epitope comprises NKG2D, CD8, CD16, KIR2DL4, KIR2DS1, KIR2DS2, KIR3DS1, NKG2C, NKG2E, or NKG2D.

119. The method of claim 113, wherein the selected immune cell is a macrophage and the immune cell activating epitope comprises CD11b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4/80, IFGR2 Toll-like receptors (TLRs) 1-9, IL-4Ra, or MARCO.

120. The method of claim 113, wherein the selected immune cell is a T cell, and the immune cell activating epitope binding domain binds CD3 and comprises a sequence as set forth in SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 63 or a sequence having at least 95% sequence identity to SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 63.

121. The method of claim 101 , further comprising identifying a cancer supporter that is or will be expressed by the tumor.

122. The method of claim 121 , further comprising selecting a molecule of claim 5, wherein the cancer supporter binding domain binds the identified cancer supporter.

123. The method of claim 122, wherein the cancer supporter binding domain binds a molecule that cancer cells upregulate or select for to promote their survival.

124. The method of claim 122, wherein the cancer supporter binding domain binds a molecule that down-regulates immune system activity against a cancer cell that expresses the cancer supporter.

125. The method of claim 122, wherein the cancer supporter binding domain binds a molecule that provides treatment resistance for a cancer cell that expresses the cancer supporter.

126. The method of claim 121 , wherein the cancer supporter comprises PD-L1 , ALCAM/CD166, C10orf54, CEACAM1 , CEACAM20, CMKLR1 , HLA-E, IFITM1 , IFITM2, IFITM3, IL31RA, LST1, MEFV, OSMR, PDL2, PIM1, PIM2, PLAAT3, PRAME, orSDCBP.

127. The method of claim 121, wherein the cancer supporter comprises OCSTAMP, UNC5C, KCNF1, TMEM45A, RXFP1, MGAM2, PTGES, TMEM92, CLEC9A, CAV2, LAMA3, RIMS2, BCAR1, PLEKHS1, SSTR2, KCNE5, ITGA7, GPR31, IL31RA, SCUBE1, HPN, GJA4, P2RY14, BPI, S1PR3, FCGR1A, KREMEN1, FLT1, DCSTAMP, GRIN3A, MUC1, DEFB1, CALHM6, KCNJ10, CPNE6, FFAR2, SLC6A12, HCAR3, F3, XIRP1, CLEC6A, FPR2, HCAR2, FRMD3, VAMP5, TMTC1, CD274, NPFFR1, GLDN, SMO, CD38, TNFSF10, GPR33, SLAMF7, NRN1, CYSLTR2, ATP1B2, PLSCR4, RRAD, KCNJ15, CCRL2, TMEM255A, CSPG4, FPR1, JAG1, CDCP1, GPR146, MYOF, PDGFRA, MY01A, ABCC11, LAG3, CAVIN1, PPFIA4, CR1L, GPC4, P2RX7, RARRES3, PLEKHA6, TMEM132A, SMCO2, SMCO4, MMP25, KCNJ2, RALB, TIE1, SCG3, PRRT2, SORT1, VCAM1, FAM241A, PTPRU, CLEC4E, NCF1, SLC12A3, IL15RA, SECTM1, LRRN2, OTOF, GEM, ENTHD1, OSBPL6, SCARF1, SUCNR1, SLC2A5, PSTPIP2, GPR84, GOLM1, GJB2, SLITRK4, SLC2A6, CLEC4D, SSPN, SORBS1, GAREM1, CLDN7, LRRC4, ASPHD2, STX11, EREG, RAPGEF2, MYO1B, CLEC5A, SYT17, LYPD5, PDCD1LG2, PHLDA2, CTTN, SLC31A2, BEGAIN, RIPK2, CSF2RB, DGKG, FRRS1, TNFSF13B, VRK2, DOCK7, DAPP1, PRRG4, CACNA1A, ITGB7, PRAME, MFSD2A, CEACAM4, TNF, SLC1A5, CDK14, FAS, ATP1B1, FFAR4, HVCN1, PROCR, CD80, IFITM1, TENM4, PLAUR, NETO2, SCIMP, PAK1, CAMK2D, C19orf38, CD83, RHOU, CD40, PLEK, ITPRIPL2, TREML2, EPB41L5, LILRA2, SLC1A4, LDLR, IFNGR2, SFT2D2, SLAMF8, TLR7, GPBAR1, SIRPB1, SIRPB1, NYNRIN, C3orf14, TJP2, LMTK2, PAQR6, FZD2, DIXDC1, STX1B, CNIH4, SLC8A1, CD33, TMEM131, NOD2, PDE4B, SH2B2, CLMP, SLC1A3, MCOLN2, IL12RB2, TLR1, CSF1, orTLR8.

128. The method of claim 121, wherein the cancer supporter is an immune checkpoint molecule, immune checkpoint ligand, or a drug efflux pump.

129. The method of claim 121, wherein the cancer supporter comprises PD-L1, 2B4, A2aR, B7HI, B7H3, B7H4, BTLA, CCR7, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276, CTLA-4, DR3, GAL9, GITR, HAVCR2, HVEM, ID01, ID02, ICOS, KIR, LAG3, LAIRI, LIGHT, MARCO, PD-1, PD-L2, PD-L3, PD-L4, PS, OX-40, SLAM, TIGHT, TIGIT, TIM3, VISTA, orVTCNI.

130. The method of claim 121, wherein the cancer supporter comprises PD-L1.

131. The method of claim 121, wherein the cancer supporter comprises PD-L1 and the cancer supporter binding domain comprises a sequence as set forth in SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97 or a sequence having at least 95% sequence identity to SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97.

132. The method of claim 128, wherein the immune checkpoint molecule comprises CD27, CD28, CD137, CEACAM1 , CTLA4, GITR, ICOS, LAG3, PD-1, OX-40, TIGIT, TIM3, or VISTA.

133. The method of claim 128, wherein the immune checkpoint ligand comprises CD70, CD80, CD86, CD137L, CD155/CD112, CEACAM1 , ICOSLG, Galectin 9, GITRL, lectins, OC40L, PD-L1 , PD-L2, or VSIG-3.

134. The method of claim 128, wherein the drug efflux pump comprises P-glycoprotein (P-gP or MDR1/ABCB1), monocarboxylate transporter (MCT), ATP-binding cassette transporters (ABC), peptide transporters (PEPTs), Na+ phosphate transporters (NPTs), MDR-associated protein (MRP1/ABCC1), breast cancer resistance protein (MXR or BCRP/ABCG2), MRP/ABCC- 2, MRP/ABCC-3, MRP/ABCC-4, MRP/ABCC-5, MRP/ABCC-6, or MRP/ABCC-7.

135. A method of selecting cancer supporter therapeutic targets, the method comprising: exposing healthy cells and cancer cells to an immune cell activation indicator; measuring gene expression in the healthy cells and the cancer cells after the exposing; and creating a set of genes with upregulated expression in the cancer cells but not the healthy cells after the exposing; and selecting from the set of genes those that encode a cell-surface expressed protein, thereby selecting cancer supporter therapeutic targets.

136. The method of claim 135, wherein the healthy cells comprise peripheral blood mononuclear cells (PBMCs).

137. The method of claim 135, wherein the cancer cells comprise diffuse midline glioma (DMG) primary tumor cells.

138. The method of claim 135, wherein the immune cell activation indicator comprises I FNy, TNFa, IL-2, IL-7, or IL-15.

139. The method of claim 135, wherein the immune cell activation indicator comprises supernatant from co-culture of activated T cells.

140. A method of designing a multi-specific binding molecule, the method comprising: selecting a cancer marker binding domain; selecting an immune cell activating epitope binding domain; exposing healthy cells and cancer cells to an immune cell activation indicator; measuring gene expression in the healthy cells and the cancer cells after the exposing; and creating a set of genes with upregulated expression in the cancer cells but not the healthy cells after the exposing; selecting a gene from the set of genes that encodes a cell-surface expressed protein, and selecting a binding domain that binds the cell-surface expressed protein thereby designing a multi-specific molecule.

141. The method of claim 140, wherein the healthy cells comprise peripheral blood mononuclear cells (PBMCs).

142. The method of claim 140, wherein the cancer cells comprise diffuse midline glioma (DMG) primary tumor cells.

143. The method of claim 140, wherein the immune cell activation indicator comprises I FNy, TNFa, IL-2, IL-7, or IL-15.

144. The method of claim 140, wherein the immune cell activation indicator comprises supernatant from co-culture of activated T cells.

145. The method of claim 140, further comprising selecting a multimerization domain.

146. The method of claim 145, wherein the multimerization domain comprises an lgG1 Fc knob and lgG1 Fc hole.