Drug Immunoconjugates Research Articles

SY08-3 Recent advances in targeted therapy for B-cell malignancies

The introduction of rituximab has represented a major milestone in the treatment of B-cell malignancies. However, a proportion of patients eventually will relapse or will be primary refractory. In this clinical setting, targeting new surface antigens either using monospecific antibodies or drug immunoconjugates are appealing treatment approaches. Additionally, exploiting cytotoxic T-cells using bispecific antibodies has been considered as a disruptive therapeutic strategy for lymphomas.

On Site Manufacture of AntiCD19 CAR-T Cells. Responses in Subjects with Rapidly Progressive Refractory Lymphomas.

<h3>Background</h3> Patients (pts) with rapidly progressive lymphoma and urgent need for therapy have worse prognosis and may not be able to receive CAR-T cells. Decreasing apheresis to infusion time can make CAR-T cells available to this patient population. We present the results of a phase I trial using rapid on-site CAR-T manufacture for relapsed/refractory B cell non Hodgkin lymphoma (NHL). <h3>Methods</h3> Adult pts with CD19+ B NHL who failed ≥ 2 lines of therapy were enrolled. Autologous T cells were transduced with a lentiviral vector (Lentigen Technology, Inc) encoding antiCD19 binding motif, CD8 linker and TNFRS19 transmembrane region, and 4-lBB/CD3z costimulatory domains. GMP compliant manufacture was done using CliniMACS Prodigy, in a 12 day culture. Dose escalation was done using 3+3 design, with 3 dose levels (0.5, 1 and 2 × 10⁶ CAR-T cells/kg). Lymphodepletion was done with cyclophosphamide (60mg/kg × 1) and fludarabine (25mg/m²/d × 3). <h3>Results</h3> As of September 30, 2019, 14 pts were enrolled and treated. Table 1 lists baseline characteristics. 12 pts had refractory NHL, 6 had bulky disease and 9 had symptomatic disease at the time of lymphocyte collection. CAR-T cell product manufacture was successful in all pts. Median transduction rate was 48% [range 29-62] with median culture expansion of 41-fold [range 30-79]. All pts received their infusion of antiCD19 CAR-T cells. CAR-T cell doses were 0.5 × 10⁶/kg (n = 4) and 1 × 10⁶/kg (n = 10). Median apheresis to infusion time was 13 days [range 13–20]. CAR-T persistence, based on vector sequence, peaked in peripheral blood MNCs between days 14-21. All evaluable subjects had persistent CAR-Ts on PCR measurements done on days 30, 60 and 90. CAR-T cell dose did not have an impact in the time to peak <i>in vivo</i> CAR-T cell expansion or in the rate of CAR-T cell persistence (fig 1). Six pts experienced CRS. Five pts had grade 1 – 2 CRS and 1 pt died on day 8 due to CRS. Two pts presented grade 4 CRES, resolved after corticosteroids. No other grade ≥3 non-hematologic toxicity was observed. The most common non – hematologic toxicity was fatigue (n=7). Hematologic toxicity was common, with grade ≥ 3 neutropenia observed in all pts. Among 12 evaluable pts, 8 have achieved complete response (CR) and two had partial response (PR). Two pts did not respond. The CR rate was 67% and overall response rate was 83%. Three pts have died, causes of death include progressive disease (n=2) and CRS (n=1). After a median follow up 4.5 months (range 1 – 14) all responding pts are alive; 1 pt relapsed 6 months after treatment with CD19+ disease and entered CR after antiCD19 antibody drug immunoconjugate. <h3>Conclusions</h3> AntiCD19 CAR-T cells with TNFRS19 transmembrane domain have potent clinical activity. The short manufacture times achieved by local CAR-T cell manufacture enables treatment of a very high risk NHL population that would otherwise not be able to receive CAR-T products due to rapidly progressive disease.

Immunological Responses against HER2-targeted Idarubicin-ZHER2 Conjugate in BALB/c Mice.

Targeting of cancerous cells with a high level of human epidermal growth factor receptor 2 (HER2) expressions by drug immunoconjugates is a new approach for specific delivery of chemotherapeutic agents. Our previous work indicated that idarubicin-ZHER2 affibody conjugate has a great potential for the treatment of HER2-overexpressing malignant cell lines but possible induced immune response against constructed conjugate was not addressed. In the current study, the possibility of induction of humoral and cellular immune responses against idarubicin-ZHER2 affibody conjugate in BALB/c mice was investigated. For assessment of the induced immune response, prepared and qualified idarubicin-ZHER2 affibody conjugate was administrated intravenously to BALB/c mice and the induced cellular immune response was evaluated by measuring secretion levels of interferon gamma (IFN-γ) and interleukin 10 (IL-10) cytokines by the splenocytes. Humoral response of treated mice was also assessed by measuring total immunoglobulin G (IgG) titer in mice sera. The obtained results showed that idarubicin-ZHER2 affibody conjugate at any examined concentrations could not induce secretion of IFN-γ as a pro-inflammatory cytokine. A mild increase in the level of regulatory IL-10 cytokine was seen in the treated mice although no dose dependency in the level of IL-10 production was observed. Furthermore, results showed that idarubicin-ZHER2 conjugate could not induce IgG production in the treated mice. Based on these findings, the idarubicin-ZHER2 conjugate can be considered as a candidate for the development of new therapeutics against HER2-overexpressing cancers although further in vivo studies are needed.

Phase 1 Study of on Site Manufactured Anti-CD19 CAR-T Cells: Responses in Subjects with Rapidly Progressive Refractory Lymphomas

Phase 1 Study of on Site Manufactured Anti-CD19 CAR-T Cells: Responses in Subjects with Rapidly Progressive Refractory Lymphomas

Tyrosine Kinase Inhibitors and Therapeutic Antibodies in Advanced Eosinophilic Disorders and Systemic Mastocytosis

World Health Organization-defined myeloproliferative neoplasms share a common pathobiologic theme of constitutive activation of tyrosine kinases (TKs). While myeloid/lymphoid neoplasms with eosinophilia and rearrangement of PDGFRA or PDGFRB exhibit exquisite responsiveness to imatinib, other eosinophilic disorders such as chronic eosinophilic leukemia--not otherwise specified (CEL-NOS) and idiopathic hypereosinophilic syndrome (HES) lack recurrent gene mutations or known druggable targets. In systemic mastocytosis (SM), KIT D816V is identified in ∼ 90% of patients, but demonstrates imatinib resistance. Recently, the multikinase/KIT inhibitor midostaurin (PKC412) has demonstrated encouraging activity in patients with advanced SM, and selective KIT D816V inhibitors are entering clinical development. Pre-clinical rationale also exists for use of small molecule inhibitors of TK-linked pathways (e.g., BTK, JAK-STAT, PI3K/AKT, and FGFR1) that are implicated in normal or dysregulated signaling in eosinophils or mast cells. A complementary therapeutic approach is the use of naked antibody (e.g., mepolizumab and alemtuzumab) or antibody-based drug immunoconjugates (brentuximab vedotin) against targets expressed on the surface of eosinophils or mastocytes that can block proliferation and/or induce apoptosis of these cells. Ultimately, biologic and molecular characterization of eosinophilia and SM cases will help to optimize selection of TK inhibitors or therapeutic antibodies for individual patients.

Targeted Delivery of Dendritic Polyglycerol–Doxorubicin Conjugates by scFv-SNAP Fusion Protein Suppresses EGFR+ Cancer Cell Growth

Development of effective polymer-based nanocarriers for the successful application in cancer therapy still remains a great challenge in current research. In the present study we present a dendritic polyglycerol-based multifunctional drug immunoconjugate that specifically targets and kills cancer cell lines expressing epidermal growth factor receptor (EGFR). The nanocarrier was provided with a dendritic core as a multifunctional anchoring point, doxorubicin (Doxo) coupled through a pH-sensitive linker, a fluorescence marker, poly(ethylene glycol), as solubilizing and shielding moiety, and a scFv antibody conjugated through the SNAP-Tag technology. The study provides the proof of principle that SNAP-tag technology can be used to generate drug-carrying nanoparticles efficiently modified with single-chain antibodies to specifically target and destroy cancer cells.

Technology Insight: cytotoxic drug immunoconjugates for cancer therapy

The successful introduction of genetically engineered human and chimeric immunoglobulin proteins has established monoclonal antibodies (mAbs) as a validated approach for treating malignancies. The unique properties of mAb therapies including their high affinity and specificity, and the differential expression of target antigen in tumor cells versus normal cells make them attractive agents for cancer immunotherapy. The field of immunoconjugate development attempts to combine the specificity of mAb therapies with cytotoxic and radionuclide molecules, thereby combining the best characteristics of these two different modalities. Two radiolabeled mAbs, (90)Y-ibritumomab tiuxetan and (131)I-tositumomab, and one drug conjugate, gemtuzumab ozogamicin have been approved for the treatment of malignancies. Other conjugates carrying toxic payloads of calicheamicin, geldanamycin, maytansinoids and taxoids as well as peptide exotoxins are undergoing preclinical and clinical development. Nevertheless, several obstacles have limited robust antitumor activity and broad application of imunoconjugates, including the optimization of three structural components of the immunoconjugate (i.e. mAb and target specificity, chemical linker design, and the cytotoxin), as well as issues common to mAb therapy such as heterogeneous antigen expression, which can limit uniform antibody delivery. This Review examines optimal design, the lessons learned from clinical immunoconjugate development, and the promising agents in early preclinical/clinical development for the treatment of cancer.

Introduction to the Tenth Conference on Cancer Therapy with Antibodies and Immunoconjugates

Cancer immunotherapy has not only come of age, but has more ongoing clinical trials than any other area of cancer research. Already nine antibody products are approved as cancer therapeutics in the United States, six as naked monoclonal antibodies (mAb), one as a drug immunoconjugate, and two as

Anti-CD74 Antibody-Doxorubicin Conjugate, IMMU-110, in a Human Multiple Myeloma Xenograft and in Monkeys

IMMU-110 is a drug immunoconjugate composed of doxorubicin conjugated to the humanized anti-CD74 monoclonal antibody, hLL1, at a doxorubicin/monoclonal antibody ratio of approximately 8:1 (mol/mol). CD74 is a rapidly internalizing molecule associated with HLA-DR, which has high expression by several tumor types. Here, we describe safety evaluations of IMMU-110 in mice and monkeys as well as efficacy studies in a xenograft model of the human multiple myeloma cell line, MC/CAR. In vitro binding of IMMU-110 was determined by a cell-based ELISA and cytotoxicity of IMMU-110 assayed with a tetrazolium assay. Pharmacokinetics and biodistribution of radiolabeled IMMU-110 were examined in tumor-free BALB/c mice, and the therapeutic effectiveness was evaluated in severe combined immunodeficient mice bearing MC/CAR cells. Acute toxicity of IMMU-110 was studied in CD74-positive cynomolgus monkeys (Macaca fascicularis). In vitro, IMMU-110 specifically binds to CD74 and is cytotoxic against MC/CAR cells. In vivo, IMMU-110 displayed a pharmacokinetic and biodistribution profile identical to that of unconjugated hLL1 monoclonal antibody, except for higher kidney uptake. Treatment with a single dose of IMMU-110 as low as 50 microg antibody/mouse (or 1.4 microg doxorubicin/mouse), 5 days postinjection of the multiple myeloma cells, resulted in cure of most mice. In mice, no host toxicity of IMMU-110 was observed at the highest protein dose tested (125 mg/kg). In cynomolgus monkeys, bone marrow toxicity was observed at 30 and 90 mg/kg doses. The excellent safety and efficacy profile of IMMU-110 supports clinical testing of this immunoconjugate in the treatment of CD74-positive B-cell malignancies.

In Vitro and In Vivo Targeting and Therapy of an Antibody-Drug Conjugate (IMMU-110) in B-Cell Malignancies.

IMMU-110 is a drug immunoconjugate comprised of doxorubicin (DOX) conjugated to the humanized anti-CD74 monoclonal antibody (mAb), hLL1, at a DOX:mAb (mol/mol) ratio of 8:1. CD74 is a rapidly internalizing type-II transmembrane chaperone molecule associated with HLA-DR, and has high expression on human non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM) clinical specimens and cell lines. Here, we investigated the in vitro and in vivo efficacy of IMMU-110 in xenograft models of human NHL (Raji, Daudi) and MM (MC/CAR). In vitro cell binding of IMMU-110 with the CD74-positive cells was significantly higher than that of a non-specific isotype-matched mAb-DOX conjugate (DOX conjugated to a mAb against epithelial glycoprotein-1; DOX-hRS7), and was similar to that of naked hLL1. Both IMMU-110 and naked hLL1 bound CD74 with subnanomolar affinity. The in vitro cytotoxicity of IMMU-110 was significantly higher than non-specific antibody-DOX conjugate, DOX-hRS7, and was similar to free DOX in MC/CAR, Raji or Daudi human Burkitt's lymphoma cells. In CD74-negative cell lines, IMMU-110 was significantly less toxic than free DOX, having similar cytotoxicity to DOX-hRS7. In vivo, IMMU-110 displayed a pharmacokinetic and biodistribution profile almost identical to that of hLL1 mAb. Both hLL1 mAb and IMMU-110 had a biphasic clearance from the circulation; the α and β half-life (t1/2) of IMMU-110 were 4.6 h and 157.9 h, respectively, and those of hLL1 were 5.4 h and 151.5 h, respectively. In biodistribution studies, no significant difference was observed between IMMU-110 and naked hLL1 with regards to normal tissue uptake. Neither IMMU-110 nor naked hLL1 mAb had a significant association with any normal body tissue. In therapy experiments, a single i.v. protein dose of 350 μg IMMU-110, injected 5 days after implantation of MC/CAR cells in SCID mice, resulted in curing 70% of the animals. Similar cure rates were observed when treatment with IMMU-110 was given 10 days after transplantation of MC/CAR cells. In the Raji xenograft model, 100% of animals were cured with a single protein dose of 120 μg IMMU-110, injected 5 days after implantation of cells. In survival studies, the efficacy of IMMU-110 was significantly better than naked hLL1, the combination of naked hLL1 and free DOX, or of a non-specific antibody-DOX conjugate, DOX-hRS7. In a tolerability study in SCID mice, no toxic effect of IMMU-110 was observed even at the highest dose tested (2.5 mg /mouse). In conclusion, treatment of B-cell lymphoma and myeloma xenograft models with single injections of IMMU-110 resulted in high levels of response and long-term survivors. IMMU-110 is being further developed as a potential therapeutic for the treatment of CD74-positive tumors.

Therapeutic activity of a new antibody-drug immunoconjugate, IMMU-110, in preclinical studies targeted against multiple myeloma

6535 Background: CD74, a cell-surface-expressed epitope of the HLA class II-associated invariant chain [Ii], is a unique target for antibody-drug immunoconjugate therapy, being a transmembrane chaperone molecule that actively directs transport from the cell surface to an endosomal compartment. IMMU-110 is a drug immunoconjugate comprised of doxorubicin (dox) conjugated to the humanized anti-CD74 monoclonal antibody (MAb), hLL1, at 6–8 drug molecules per IgG molecule. The dox is linked to partially-reduced antibody inter-chain disulfide groups using 4-[N-maleimidomethyl] cyclohexane-1 carboxylhydrazide. We recently reported that IMMU-110 has potent therapeutic effects in a non-Hodgkin's lymphoma-SCID mouse model. Methods: Here we examine the efficacy of IMMU-110 for therapy of multiple myeloma (MM), based on the high prevalence of CD74 expression on MM specimens, using in vitro assays on MM cell lines and in vivo studies in a SCID mouse model of disseminated MM. Results: In vitro cytotoxicity assays demonstrated that in CD74+ cells IMMU-110 yields IC50 values similar to those of free dox, whereas a control MAb-dox conjugate is markedly less toxic. In contrast, activity of IMMU-110 is similar to that of the control conjugate in CD74- cell lines. For example, IMMU-110 was ∼7-fold more potent than the control conjugate on CAG cells. Untreated mice given an i.v. injection of 1x107 MC/CAR cells die of disseminated disease with a median survival time (MST) of 32 days post-injection. A single i.v. protein dose of IMMU-110 of 350 μg, given 5 days after injection of the MC/CAR cells, resulted in extension of MST to >150 days. Unconjugated hLL1 and dox mixed with hLL1 yield MSTs of 61.5 and 68 days, respectively. No significant effects were seen with free dox given at an equivalent dose, or a control conjugate. Similar survival extensions were observed when treatment with IMMU-110 was given 10 days after transplantation of MM cells, and a significant extension of MST was even observed 14 days post-inoculation (from 28 to 35 days P<0.002). Conclusions: IMMU-110 is a drug immunoconjugate showing considerable promise as a new therapeutic for MM. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Immunomedics, Inc. Immunomedics, Inc. Immunomedics Inc.

Monoclonal antibody drug immunoconjugates for targeted treatment of cancer.

Monoclonal antibodies (mAb) directed to tumor-associated antigens (TAA) or antigens differentially expressed on the tumor vasculature have been covalently linked to drugs that have different mechanisms of action and various levels of potency. The use of these mAb immunoconjugates to selectively deliver drugs to tumors has the potential to both improve antitumor efficacy and reduce the systemic toxicity of therapy. Several immunoconjugates, particularly those that incorporate internalizing antibodies and tumor-selective linkers, have demonstrated impressive activity in preclinical models. Immunoconjugates that deliver doxorubicin, maytansine and calicheamicin are currently being evaluated in clinical trials. The feasibility of using immunoconjugates as cancer therapeutics has been clearly demonstrated. Gemtuzumab ozogamicin, a calicheamicin conjugate that targets CD33, has recently been approved by the Food and Drug Administration (FDA) for treatment of acute myelogenous leukemia (AML). This review concentrates on the properties of the tumor and the characteristics of the mAb, linker, and drugs that influence the efficacy, potency, and selectivity of immunconjugates selected for cancer treatment.

Tumour eradication with high-dose idarubicin-anti-Ly-2.1 with murine tumour necrosis factor-alpha in mice.

The antitumour efficacy of Ida-anti-Ly-2.1 immunoconjugate in combination with murine TNF-alpha (mTNF-alpha) has been evaluated in a disseminated murine thymic lymphoma model E3. This tumour model shows characteristics similar to lymphoma in humans with widely disseminated tumour in major organs. In vitro both idarubicin (Ida) and Ida-anti-Ly-2.1 showed additive and synergistic cytotoxic effects, respectively, with mTNF-alpha when tested using the isobolagram method. Similar synergistic effects were also seen in vivo. Tumour-bearing mice were treated with various doses of mTNF-alpha and Ida alone, however, neither showed a therapeutic response in mice. When mice were treated with Ida-anti-Ly-2.1 'early' the median survival time (MST) was increased by 8 days, however, 'late' treatment was ineffective. Combination therapy of mTNF-alpha and Ida-anti-Ly-2.1 conjugate (42 micrograms) given 'early' was capable of curing 50% of mice. However, when tumour-bearing mice were given a high dose (234 micrograms) of Ida-anti-Ly-2.1 together with mTNF-alpha 100% of mice survived disease free. Such an effect was not observed when free mTNF-alpha, Ida-anti-Ly-2.1, anti-Ly-2.1 or a mixture of anti-Ly-2.1+mTNF-alpha was given at the same dose. These results show that a combination of very toxic drug immunoconjugates and TNF could lead to the eradication of disseminated tumour in mice and may be relevant for the treatment of minimal residual disease in cancer patients.

Novel trityl linked drug immunoconjugates for cancer therapy

Trityl linkers were utilized in the preparation of acid labile nucleoside monoclonal antibody conjugates, COL1-N6-Trityl-207702 (1a-f). Conjugation led to high levels of drug incorporation into the MoAb with retention of good immunoreactivity. A strong correlation was found between the cytotoxicity of the constructs and substituents R and R′ on the aromatic rings of the trityl linker. The synthesis, characterization and biological activity of a series of COL1-N6-Trityl-207702 conjugates is described. 1

Drug immunoconjugates for refractory cancers

Drug immunoconjugates for refractory cancers

Individually Specified Drug Immunoconjugates in Cancer Treatment

Forty-three patients with disseminated refractory malignancies each received an individually-specified combination of either Adriamycin (24 patients) or mitomycin-C (19 patients) conjugated murine monoclonal antibodies. Tumors were typed using a panel of antibodies with both immunohistochemistry and flow cytometry. Cocktails of up to six antibodies were selected based on binding greater than 80% of the malignant cells in the biopsy specimen. These monoclonal antibody cocktails were drug conjugated and administered intravenously. Seventeen out of twenty-four patients had reactions to the administration of Adriamycin immunoconjugates, but these were tolerable in all but two patients. Fever, chills, pruritis and skin rash were by far the most common transitory reactions. All were well controlled with premedication. In several patients it was demonstrated that there was limited antigenic drift among various biopsies within the same patient over time. Up to 1 gram of Adriamycin and up to 5 grams of monoclonal antibody were administered. The limiting factor appeared to be a variable dissociation of active Adriamycin from the antibody which unpredictably caused hemopoietic depression. Similar findings were noted in 19 patients with mitomycin-C conjugates. Thrombocytopenia at a 60mg dose of mitomycin-C in this schedule was dose limiting. Preliminary serological evidence suggests that the development of an IgM antibody which is specific against the mouse monoclonal antibody has the specificity and sensitivity to predict clinical reactions. These antibodies were quantitatively less in mitomycin-C patients. Selected patients were re-treated. One patient with chronic lymphocytic leukemia had re-treatment on three occasions and demonstrated regression of peripheral lymph nodes. Two patients with breast carcinoma had definite improvement in ulcerating skin lesions and two patients with tongue carcinoma had shrinkage of their lesions. No responses were seen with mitomycin-C conjugates but binding was noted to tumors and colon with likely drug induced colitis seen after colon binding. This study demonstrates the feasibility and illustrates technical considerations in preparing drug immunoconjugate cocktails for patients with refractory malignancies. Cocktail formulation and antibody delivery was accomplished. The major technical hurdle appears to be the selection of effective conjugation methods that can be used to optimally bind drugs to monoclonal antibodies for targeted cancer therapy.

Preparation and In Vitro Evaluation of Antibiotic Drug Immunoconjugates

The aim of the present study was to develop drug immunoconjugates (DIC) of antibiotics like Ampicillin trihydrate, Amoxycillin trihydrate, Streptomycin sulphate, Rifampicin and doxorubicin with Anti-D serum using different coupling methods. Protein content in Anti- D serum was estimated by Lowry’s method. The conjugates were purified from unbound drug by dialysis. The drugs to antibody ratios were estimated by determining the quantity of antibacterial drugs coupled during coupling reactions by turbidimetric or colorimetric assay. The conjugates were tested for both antibody and antibacterial activities by simple in vitro methods. Slide agglutination test was used to know the retention of activity of the antibody qualitatively. The antibacterial activity was observed by reduction in turbidity due to growth inhibition. When glutaraldehyde method was used to couple the drugs the resulted DICs of Ampicillin trihydrate, Amoxycillin trihydrate and Streptomycin sulphate retained both antibody and antibacterial activities, while a direct linkage of Streptomycin sulphate to antibody resulted in the loss of antibacterial activity. Rifampicin was also linked to antibody by two methods. Upon direct linkage of Rifampicin to antibody retained both activities, but when linked by dicyclohexyl carbodiimide method, an antibody inactive preparation was resulted. Doxorubicin an anticancer antibiotic was also linked to antibody by glutaraldehyde method which retained antibody activity. Taken together, the results of our studies would be useful in the design of drug immunoconjugates with antibiotic drugs to treat specific infections in chemotherapy.