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D2C7-(scdsFv)-PE38KDEL (D2C7-IT) is a novel recombinant Pseudomonas exotoxin A-based immunotoxin (IT), targeting both wild-type epidermal growth factor receptor (EGFRwt) and mutant EGFR variant III (EGFRvIII) proteins overexpressed in glioblastomas. Initial pre-clinical testing demonstrated the anti-tumor efficacy of D2C7-IT against orthotopic glioblastoma xenograft models expressing EGFRwt, EGFRvIII, or both EGFRwt and EGFRvIII. A good laboratory practice (GLP) manufacturing process was developed to produce sufficient material for a phase I/II clinical trial. D2C7-IT was expressed under the control of the T7 promoter in Escherichia coli BLR (λ DE3). D2C7-IT was produced by a 10-L batch fermentation process and was then purified from inclusion bodies using anion exchange, size exclusion, and an endotoxin removal process that achieved a yield of over 300 mg of purified protein. The final vialed batch of D2C7-IT for clinical testing was at a concentration of 0.12 ± 0.1 mg/mL, the pH was at 7.4 ± 0.4, and endotoxin levels were below the detection limit of 10 EU/mL (1.26 EU/mL). The stability of the vialed D2C7-IT has been monitored over a period of 42 months through protein concentration, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing, size exclusion chromatography, cytotoxicity, sterility, and pH measurements. The vialed D2C7-IT is currently being tested in a phase I/II clinical trial by intratumoral convection-enhanced delivery for 72 h in patients with recurrent glioblastoma (NCT02303678, D2C7 for Adult Patients with Recurrent Malignant Glioma; clinicaltrials.gov ).

Immunotoxins are cytolytic fusion proteins developed for cancer therapy, composed of an antibody fragment that binds to a cancer cell and a protein toxin fragment that kills the cell. exotoxin A (PE) is a potent toxin that is used for the killing moiety in many immunotoxins. Moxetumomab Pasudotox (Lumoxiti) contains an anti-CD22 Fv and a 38 kDa portion of PE. Lumoxiti was discovered in the Laboratory of Molecular Biology at the U.S. National Cancer Institute and co-developed with Medimmune/AstraZeneca to treat hairy cell leukemia. In 2018 Lumoxiti was approved by the US Food and Drug Administration for the treatment of drug-resistant Hairy Cell Leukemia. Due to the bacterial origin of the killing moiety, immunotoxins containing PE are highly immunogenic in patients with normal immune systems, but less immunogenic in patients with hematologic malignancies, whose immune systems are often compromised. LMB-100 is a de-immunized variant of the toxin with a humanized antibody that targets mesothelin and a PE toxin that was rationally designed for diminished reactivity with antibodies and B cell receptors. It is now being evaluated in clinical trials for the treatment of mesothelioma and pancreatic cancer and is showing somewhat diminished immunogenicity compared to its un modified parental counterpart. Here we review the immunogenicity of the original and de-immunized PE immunotoxins in mice and patients, the development of anti-drug antibodies (ADAs), their impact on drug availability and their effect on clinical efficacy. Efforts to mitigate the immunogenicity of immunotoxins and its impact on immunogenicity will be described including rational design to identify, remove, or suppress B cell or T cell epitopes, and combination of immunotoxins with immune modulating drugs.

Recombinant immunotoxins (RITs) are hybrid proteins used to treat cancer. These proteins are composed of an Fv that reacts with cancer cells joined to a portion of Pseudomonas exotoxin A, which kills the cell. Because the toxin is a foreign protein, it can induce neutralizing antibodies and thereby limit the number of doses a patient can receive. We previously identified seven major mouse B-cell epitopes in the toxin, and subsequently silenced them using point mutations that converted large hydrophilic amino acids to alanine, yet retained full antitumor activity. Here we present results in which we identify and silence human B-cell epitopes in the RIT HA22. We obtained B cells from patients with antibodies to RITs, isolated the corresponding variable fragments (Fvs), and constructed a phage-display library containing Fvs that bind to the RITs. We then used alanine scanning mutagenesis to locate the epitopes. We found that human and mouse epitopes frequently overlap but are not identical. Most mutations that remove mouse epitopes did not remove human epitopes. Using the epitope information, we constructed a variant immunotoxin, HA22-LR-LO10, which has low reactivity with human antisera, yet has high cytotoxic and antitumor activity and can be given to mice at high doses without excess toxicity. The toxin portion of this RIT (LR-LO10) can be used with Fvs targeting other cancer antigens and is suitable for clinical development.

LMB-100 is a recombinant immunotoxin composed of a Fab linked to a toxin. It kills cells expressing human mesothelin (hMSLN), which is highly expressed on the surface of mesothelioma and many other cancer cells. Clinically, we observed some patients had delayed responses to an anti-hMSLN immunotoxin treatment, suggesting the induction of anti-tumor immunity. We aimed to develop a mouse model to investigate whether immunotoxin alone can induce anti-tumor immunity and to study the mechanism of this immunity. An immunocompetent transgenic mouse was used to grow mouse mesothelioma AB1 cells expressing hMSLN in the peritoneal cavity. Mice were treated with LMB-100, and mice with complete responses (CRs) were rechallenged with tumor cells to determine whether anti-tumor immunity developed. Changes in gene expression profiles were evaluated by Nanostring, and changes in cytokines and chemokines were checked by protein arrays. The distribution of various immune cells was assessed by immunohistochemistry. Our results show that the mice with tumor reached CRs and developed anti-tumor immunity after LMB-100 treatment alone. The primary response requires CD8⁺ T cells, CD4⁺ T cells, and B cells. Transcriptional profiling shows that LMB-100 treatment reshapes the tumor immune microenvironment by upregulating chemotaxis signals. LMB-100 treatment upregulates genes associated with tertiary lymphoid structures (TLS) development and induces TLS formation in tumors. In sum, immunotoxin-mediated cell death induces anti-tumor immunity and the development of TLS, which provides insights into how immunotoxins cause tumor regressions.

The application of proteinaceous toxins for cell ablation is limited by their high on- and off-target toxicity, severe side effects, and a narrow therapeutic window. The selectivity of targeting can be improved by intein-based toxin reconstitution from two dysfunctional fragments provided their cytoplasmic delivery via independent, selective pathways. While the reconstitution of proteins from genetically encoded elements has been explored, exploiting cell-surface receptors for boosting selectivity has not been attained.We designed a robust splitting algorithm and achieved reliable cytoplasmic reconstitution of functional diphtheria toxin from engineered inteinflanked fragments upon receptor-mediated delivery of one of them to the cells expressing the counterpart. Retargeting the delivery machinery toward different receptors overexpressed in cancer cells enables selective ablation of specific subpopulations in mixed cell cultures. In a mouse model, the transmembrane delivery of a split-toxin construct potently inhibits the growth of xenograft tumors expressing the split counterpart. Receptor-mediated delivery of engineered split proteins provides a platform for precise therapeutic and experimental ablation of tumors or desired cell populations while also greatly expanding the applicability of the intein-based protein transsplicing.

Recombinant immunotoxins (RITs) are chimeric proteins composed of an Fv and a protein toxin being developed for cancer treatment. The Fv brings the toxin to the cancer cell, but most of the RITs do not reach the tumor and are removed by other organs. To identify cells responsible for RIT removal, and the pathway by which RITs reach these cells, we studied SS1P, a 63-kDa RIT that targets mesothelin-expressing tumors and has a short serum half-life. The major organs that remove RIT were identified by live mouse imaging of RIT labeled with FNIR-Z-759. Cells responsible for SS1P removal were identified by immunohistochemistry and intravital two-photon microscopy of kidneys of rats. The primary organ of SS1P removal is kidney followed by liver. In the kidney, SS1P passes through the glomerulus, is taken up by proximal tubular cells, and transferred to lysosomes. In the liver, macrophages are involved in removal. The short half-life of SS1P is due to its very rapid filtration by the kidney followed by degradation in proximal tubular cells of the kidney. In mice treated with SS1P, proximal tubular cells are damaged and albumin in the urine is increased. SS1P uptake by kidney is reduced by coadministration of L-lysine. Our data suggests that L-lysine administration to humans might prevent SS1P-mediated kidney damage, reduce albumin loss in urine, and alleviate capillary leak syndrome.

Immunotoxins are chimeric molecules with high potential as therapeutic candidates that combine antibody specificity to recognize and bind tumor-associated antigens and the cytotoxic potency of the enzymatic activity of a toxin, leading to the selective death of target cells. The use of immunotoxins as therapeutic tools remains limited by various issues, such as selecting the appropriate tumor-associated antigen (TAA), penetration difficulties in solid tumors, low renal clearance, and low toxic payload. For this purpose, in this work we have designed a novel trimeric immunotoxin (IMTXTriA33αS) against colorectal cancer, combining the scFv against GPA33 as a targeting domain and the fungal ribotoxin α-sarcin (αS) as the toxic fragment, linked by a trimerization domain (TIEXVIII). Our results demonstrate that IMTXTriA33αS has greater avidity and toxic load, showing a very significant increase in its in vitro and in vivo antitumor efficacy, due to its trimeric structure.

Cutaneous T‐cell lymphoma (CTCL) encompasses two main subtypes: mycosis fungoides and Sezary syndrome. Global response rates for the systemic treatment of mycosis fungoides and Sezary syndrome are approximately 30%, and none of these treatments are thought to be curative. C–C chemokine receptor type 4 (CCR4) and CD25 are encouraging targets for the treatment of CTCL and are individually targeted by mogamulizumab and denileukin diftitox, respectively. We developed a novel CCR4‐IL2 bispecific immunotoxin (CCR4‐IL2 IT) targeting both CCR4 and CD25. CCR4‐IL2 IT demonstrated superior efficacy against CCR4+CD25+CD30+ CTCL in an immunodeficient NSG mouse tumor model. Investigative New Drug‐enabling studies of CCR4–IL2 IT are ongoing, including Good Manufacturing Practice production and toxicology studies. In this study, we compared the in vivo efficacy of CCR4‐IL2 IT versus the US Food and Drug Administration–approved drug, brentuximab, using an immunodeficient mouse CTCL model. We demonstrated that CCR4–IL2 IT was significantly more effective in prolonging survival than brentuximab, and combination treatment of CCR4–IL2 IT and brentuximab was more effective than brentuximab or CCR4–IL2 IT alone in an immunodeficient NSG mouse CTCL model. Thus, CCR4–IL2 IT is a promising novel therapeutic drug candidate for CTCL treatment. We have compared the in vivo efficacy of CCR4‐IL2 bispecific immunotoxin (CCR4‐IL2 IT) versus the FDA‐approved brentuximab in an immunodeficient mouse CTCL model. We demonstrated that CCR4–IL2 IT was significantly more effective in prolonging survival than brentuximab, and combination treatment of CCR4–IL2 IT and brentuximab was more effective than brentuximab or CCR4–IL2 IT alone.

Nonhuman proteins have valuable therapeutic properties, but their efficacy is limited by neutralizing antibodies. Recombinant immunotoxins (RITs) are potent anticancer agents that have produced many complete remissions in leukemia, but immunogenicity limits the number of doses that can be given to patients with normal immune systems. Using human cells, we identified eight helper T-cell epitopes in PE38, a portion of the bacterial protein Pseudomonas exotoxin A which consists of the toxin moiety of the RIT, and used this information to make LMB-T18 in which three epitopes were deleted and five others diminished by point mutations in key residues. LMB-T18 has high cytotoxic and antitumor activity and is very resistant to thermal denaturation. The new immunotoxin has a 93% decrease in T-cell epitopes and should have improved efficacy in patients because more treatment cycles can be given. Furthermore, the deimmunized toxin can be used to make RITs targeting other antigens, and the approach we describe can be used to deimmunize other therapeutically useful nonhuman proteins.

The HER2-targeted antibody–drug conjugate trastuzumab emtansine (T-DM1) is approved for the treatment of metastatic, HER2-positive breast cancer after prior trastuzumab and taxane therapy, and has also demonstrated efficacy in the adjuvant setting in incomplete responders to neoadjuvant therapy. Despite its objective activity, intrinsic and acquired resistance to T-DM1 remains a major clinical challenge. T-DM1 mediates its activity in a number of ways, encompassing HER2 signalling blockade, Fc-mediated immune response and payload-mediated microtubule poisoning. Resistance mechanisms relating to each of these features have been demonstrated, and we outline the findings of these studies in this review. In our overview of the substantial literature on T-DM1 activity and resistance, we conclude that the T-DM1 resistance mechanisms most strongly supported by the experimental data relate to dysfunctional intracellular metabolism of the construct and subversion of DM1-mediated cell killing. Loss of dependence on signalling initiated by HER2–HER2 homodimers is not substantiated as a resistance mechanism by clinical or experimental studies, and the impact of EGFR expression and tumour immunological status requires further investigation. These findings are instructive with respect to strategies that might overcome T-DM1 resistance, including the use of second-generation anti-HER2 antibody–drug conjugates that deploy alternative linker-payload chemistries.