A single-day polychemotherapy regimen with proteasome inhibitor combinations for relapsed/refractory myeloma in the era of novel therapies.

PCAB (prednisone, cyclophosphamide, doxorubicin, carmustine) is a single-day regimen previously used for induction and now in relapsed/refractory multiple myeloma (RRMM). We retrospectively analysed the outcomes of 85 patients from five Australian centres. These included 30 patients (35.3%) who received PCAB with one additional agent (bortezomib most frequently). Median age of the patients was 65 years (37-80), with a median of four (1-8) prior lines of therapy. ORR was 37% (CR 4.9%). Median progression free survival and overall survival were 4.4 months (95% CI 3.5-6.7) and 7.4 months (95% CI 6.4-10.2), respectively. Extramedullary disease (EMD) was associated with shorter survival. Grade 3 or 4 cytopenia and febrile neutropenia occurred in 76.2% and 39.1%, respectively, with six (7.1%) treatment-related mortalities. Median inpatient stay was 3.3 days/28-day cycle (IQR 0.6-13), and for patients who died, a median of 20.2% of days alive were spent inpatient (IQR 6.4-39.1%). Three patients were successfully bridged to CAR T-cell therapy using PCAB, despite being penta-exposed and having EMD. PCAB may be considered as a useful salvage therapy amongst other polychemotherapy regimens in late relapse. Further studies is warranted to investigate and define its role as a bridging therapy to novel therapeutics.

PET/CT in the Evaluation of CAR-T Cell Immunotherapy in Hematological Malignancies.

Chimeric antigen receptor (CAR)-T cell-based immunotherapy has emerged as a path-breaking strategy for certain hematological malignancies. Assessment of the response to CAR-T therapy using quantitative imaging techniques such as positron emission tomography/computed tomography (PET/CT) has been broadly investigated. However, the definitive role of PET/CT in CAR-T therapy remains to be established. [18F]FDG PET/CT has demonstrated high sensitivity and specificity for differentiating patients with a partial and complete response after CAR-T therapy in lymphoma. The early therapeutic response and immune-related adverse effects such as cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome can also be detected on [18F]FDG PET images. In otherwise asymptomatic lymphoma patients with partial response following CAR-T therapy, the only positive findings could be abnormal PET/CT results. In multiple myeloma, a negative [18F]FDG PET/CT after receiving B-cell maturation antigen-directed CAR-T therapy has been associated with a favorable prognosis. In leukemia, [18F]FDG PET/CT can detect extramedullary metastases and treatment responses after therapy. Hence, PET/CT is a valuable imaging tool for patients undergoing CAR-T therapy for pretreatment evaluation, monitoring treatment response, assessing safety, and guiding therapeutic strategies. Developing guidelines with standardized cutoff values for various PET parameters and tumor cell-specific tracers may improve the efficacy and safety of CAR-T therapy.

Broadening anticancer spectrum by preprocessing and treatment of T- lymphocytes expressed FcγRI and monoclonal antibodies for refractory cancers.

Background: Chimeric antigen receptor T (CAR-T) cell therapies have achieved remarkable success in the treatment of hematological tumors. However, given the distinct features of solid tumors, particularly heterogeneity, metabolic aggressiveness, and fewer immune cells in tumor microenvironment (TME), the practical utility of CAR-T cells for solid tumors remains as a challenging issue. Meanwhile, although anti-PD-1 monoclonal antibody (mAb) has shown clinical efficacy, most mAbs also show limited clinical benefits for solid tumors due mainly to the issues associated with the lack of immune cells in TME. Thus, the infiltration of targeted immunological active cells into TME could generate synergistic efficacy for mAbs. Methods: We present a combinational strategy for solid tumor treatment, which combines armored-T cells to express Fc-gamma receptor I (FcγRI) fragment on the surfaces for targeting various tumors with therapeutically useful mAbs. Choosing CD20 and HER-2 as the targets, we characterized the in vitro and in vivo efficacy and latent mechanism of the combination drug by using flow cytometry, ELISA and other methods. Results: The combination and preprocessing of armored T-cells with corresponding antibody of Rituximab and Pertuzumab exerted profound anti-tumor effects, which is demonstrated to be mediated by synergistically produced antibody-dependent cellular cytotoxicity (ADCC) effects. Meanwhile, mAb was able to carry armored-T cell by preprocessing for the infiltration to TME in cell derived xenograft (CDX) model. Conclusions: This combination strategy showed a significant increase of safety profiles from the reduction of antibody doses. More importantly, the present strategy could be a versatile tool for a broad spectrum of cancer treatment, with a simple pairing of engineered T cells and a conventional antibody.

Clinical and Genomic Profile of Primary Cranial Neurolymphomatosis.

Primary cranial neurolymphomatosis (PCNL) is a rare subtype of primary CNS lymphoma (PCNSL) in which infiltrative lymphomatous involvement is confined to cranial nerves. Here, we report a case of PCNL with successful genomic profiling. A 57-year-old male had a lengthy prediagnostic phase spanning approximately 30 months, characterized by multiple episodes of cranial neuropathies managed by steroids. At the time of diagnosis, the patient had right-sided cranial neuropathies involving cranial nerves (CN) V, VI, and VII. Pathological findings of the right cavernous lesion biopsy were consistent with large B-cell lymphoma-infiltrating nerve fibers. The clinical course was aggressive and refractory, characterized by relentless progression with the development of cervical spinal neurolymphomatosis, cerebrospinal fluid involvement, and ependymal and intraparenchymal cerebral involvement, despite multiple lines of therapy, including chemoimmunotherapy, Bruton's tyrosine kinase inhibitor, radiation, autologous stem cell transplant, chimeric antigen receptor T-cell therapy (CAR-T), and whole-brain radiation. The patient survived for 22 months from the time of the initial diagnosis and 52 months after the first episode of cranial neuropathy. Next-generation sequencing identified mutations (MYD88, CD79b, and PIM1) that are frequently observed in PCNSL. The unusual findings included a total of 22 mutations involving PIM1, indicating a highly active aberrant somatic hypermutation and two missense CXCR4 mutations. CXCR4 mutations have never been described in PCNSL and may have implications for disease biology and therapeutic interventions. We provide a literature review to further elucidate PCNL.

[Novel therapies for multiple myeloma].

B-cell maturation antigen (BCMA)-targeting therapy is the most common approach to immunotherapy and cellular therapy for multiple myeloma (MM). Three major agents, CAR-T cells, bispecific antibodies, and ADC have been developed as novel therapeutic agents. CAR-T therapy showed favorable efficacy in the treatment of relapsed and refractory MM (RR MM) and was tried in early lines of therapy. Similarly, bispecific antibodies targeting BCMA or other targets have also shown promising effects in treatment of RR MM, and have been now tested in combination with other agents. Although issues such as poor fitness or exhaustion of T cells and increased susceptibility to viral infection remain to be fully resolved, novel immunotherapies and cellular therapies should further improve the prognosis of patients with RR MM.

Age-appropriate BMI cut-offs for malnutrition among older adults in India.

With the increasing prevalence of obesity in India, body mass index (BMI) has garnered importance as a disease predictor. The current World Health Organization (WHO) body mass index (BMI) cut-offs may not accurately portray these health risks in older adults aged 60 years and above. This study aims to define age-appropriate cut-offs for older adults (60-74 years and 75 years and above) and compare the performance of these cut-offs with the WHO BMI cut-offs using cardio-metabolic conditions as outcomes. Using baseline data from the Longitudinal Ageing Study in India (LASI), classification and regression tree (CART) cross-sectional analysis was conducted to obtain age-appropriate BMI cut-offs based on cardio-metabolic conditions as outcomes. Logistic regression models were estimated to compare the association of the two sets of cut-offs with cardio-metabolic outcomes. The area under the receiver operating characteristic curve (AUC), sensitivity and specificity were estimated. Agreement with waist circumference, an alternate measure of adiposity, was conducted. For older adults aged 60-74 years and 75 years and above, the cut-off for underweight reduced from < 18.5 to < 17.4 and < 13.3 respectively. The thresholds for overweight and obese increased for older adults aged 60-74 years old from > = 25 to > 28.8 and > = 30 to > 33.7 respectively. For older adults aged 75 years and above, the thresholds decreased for both categories. The largest improvement in AUC was observed in older adults aged 75 years and above. The newly derived cut-offs also demonstrated higher sensitivity and specificity among all age-sex stratifications. There is a need to adopt greater rigidity in defining overweight/obesity among older adults aged 75 years and above, as opposed to older adults aged 60-74 years old among whom the thresholds need to be less conservative. Further stratification in the low risk category could also improve BMI classification among older adults. These age-specific thresholds may act as improved alternatives of the current WHO BMI thresholds and improve classification among older adults in India.

Multiple myeloma and infections in the era of novel treatment modalities.

Infections are major cause of morbidity and mortality in patients with multiple myeloma. Current treatment landscape of newly-diagnosed multiple myeloma includes different classes of drugs, such as proteasome inhibitors, immunomodulatory drugs, and monoclonal antibodies, all of which are characterized by specific risk and pattern of infectious complications. Additionally, autologous and allogeneic hematopoietic cell transplantation, widely used in the treatment of multiple myeloma, are complex procedures, carrying a significant risk of complications, and mainly infections. Finally, novel treatment modalities such as bispecific T-cell engagers and chimeric antigen receptor T-lymphocytes have been changing the paradigm of myeloma treatment in relapsed-refractory setting. These agents due to unique mechanism of action carry distinct pattern of infectious complications. In this review, an attempt has been made to summarize the incidence, risk factors, and patterns of infections during different stages of myeloma treatment including novel treatment modalities, and to provide evidence underlying the current concept of infectious disease prophylaxis in this category of patients.

FDG-PET/CT Imaging in Chimeric Antigen Receptor-Engineered T-Cell Treatment in Patients with B-Cell Lymphoma: Current Evidence.

The Food and Drug Administration and the European Medicines Agency have recently approved chimeric antigen receptor-engineered (CAR) T cells to treat several refractory/relapsed B-cell lymphomas. This comprehensive review aims to demonstrate the pivotal role that [18F]-FDG PET/computed tomographic (CT) imaging can play to enhance the care of patients treated with CAR T-cell therapy. To this end, this review deciphers evidence showing the diagnostic, prognostic, predictive, and theragnostic value of [18F]-FDG PET/CT-derived parameters.

In vitro CAR-T cell killing: validation of the potency assay.

For advanced therapy medicinal products, the development and validation of potency assays are required, in accordance with international guidelines, to characterise the product and obtain reliable and consistent data. Our purpose was to validate the killing assay for the evaluation of autologous anti-CD19 chimeric antigen receptor (CAR) T potency. We used CD4 + and CD8 + lymphocytes or anti-CD19 CAR-T cells as effector cells and REH (CD19 +) or MOLM-13 (CD19 -) cell lines as target cells. After co-culturing target and effector cells (1:1 ratio) for 24 h, samples were labelled with 7-AAD, anti-CD3 and anti-CD19 antibodies and the frequency of CD19 + dead cells was evaluated by flow cytometry. In order to verify the CAR-T specificity for the CD19 + target, the co-culture between CAR-T and REH or MOLM-13 at different effector-to-target ratios was scheduled. Moreover, not transduced CD4 + and CD8 + lymphocytes were tested in comparison with CAR-T from the same donor to demonstrate the assay specificity. Linearity and accuracy were evaluated, and established acceptance criteria were compiled for both parameters (r2 ≥ 0.97 for linearity and average relative error ≤ 10% for accuracy). Furthermore, the method was considered robust when performed between 23 and 25 h of co-culture, and the intra-assay, inter-assay and inter-day precision was obtained. Finally, in order to verify the inter-analyst precision, the test was executed by three different operators and the intra-class correlation coefficient was > 0.4 in both cases. In conclusion, we consider this CAR-T potency assay as validated and usable in all steps of product development and quality control.

Toxic epidermal necrolysis-like cutaneous toxicity following chimeric antigen receptor T-cell therapy in recurrent large B-cell lymphoma.

Chimeric antigen receptor (CAR) T-cell therapy has demonstrated remarkable success in treating various B-cell malignancies, redirecting T-cell cytotoxicity toward cancer cells. Despite its efficacy, CAR-T therapy is associated with potential risks, including cytokine release syndrome (CRS) and cytopenia. We present a case of a 69-year-old man with diffuse large B-cell lymphoma treated with axicabtagene-ciloleucel CAR-T therapy, who developed a rare and severe cutaneous toxicity resembling toxic epidermal necrolysis (TEN). The patient exhibited persistent fevers, CRS, and subsequent development of a widespread erythematous macular eruption, progressing to vesiculation with bullae. Notably, allopurinol-induced TEN was considered with the patient's recent exposure to allopurinol, although the onset and minimal mucosal involvement did not align with typical presentations of allopurinol-induced cases. The cutaneous reaction, distinct from typical SJS/TEN, showed minimal mucosal involvement and coincided with the cytokine release storm, differing from allopurinol-induced TEN. Despite the absence of guidelines, the patient was managed with systemic steroids, achieving significant improvement. This case expands the spectrum of CAR-T therapy-related cutaneous toxicities, highlighting the need for early recognition of histopathology and tailored management by dermatologists. Further understanding of these reactions is crucial for optimizing the safety profile of this groundbreaking immunotherapy.

Adverse events associated with chimeric antigen receptor T-cell therapy in ophthalmology: a narrative review.

Chimeric antigen receptors are synthetically produced receptors engineered to engage with target cells with high specificity. These cells are created by inserting an artificial T-cell receptor into an immunoglobulin's antigen-binding region, allowing the cells to combine and target specific antigens. The use of chimeric antigen receptor (CAR) T-cell therapy has been a remarkable achievement in the field of immunotherapy, particularly in the treatment of ophthalmic tumors like retinoblastoma and uveal melanoma. However, there are some documented side effects, such as cytokine release syndrome (CRS) and immunological effector cell-associated neurotoxicity syndrome (ICANS). Additionally, ocular side effects such as blurred vision, vision impairment, and intraocular infections are also concerning and require further evaluation. This review highlights the advances made in chimeric antigen receptor (CAR) immunotherapy, including its structure and manufacture, as well as relevant clinical discoveries and associated adverse effects. By identifying the gaps in current research, this analysis provides insights into potential strategies and solutions for addressing some of the most severe side effects.

Endogenous bystander killing mechanisms enhance the activity of novel FAP-specific CAR-T cells against glioblastoma.

Objectives: CAR-T cells are being investigated as a novel immunotherapy for glioblastoma, but clinical success has been limited. We recently described fibroblast activation protein (FAP) as an ideal target antigen for glioblastoma immunotherapy, with expression on both tumor cells and tumor blood vessels. However, CAR-T cells targeting FAP have never been investigated as a therapy for glioblastoma. Methods: We generated a novel FAP targeting CAR with CD3ζ and CD28 signalling domains and tested the resulting CAR-T cells for their lytic activity and cytokine secretion function in vitro (using real-time impedance, flow cytometry, imaging and bead-based cytokine assays), and in vivo (using a xenograft mimicking the natural heterogeneity of human glioblastoma). Results: FAP-CAR-T cells exhibited target specificity against model cell lines and potent cytotoxicity against patient-derived glioma neural stem cells, even when only a subpopulation expressed FAP, indicating a bystander killing mechanism. Using co-culture assays, we confirmed FAP-CAR-T cells mediate bystander killing of antigen-negative tumor cells, but only after activation by FAP-positive target cells. This bystander killing was at least partially mediated by soluble factors and amplified by IL-2 which activated the non-transduced fraction of the CAR-T product. Finally, a low dose of intravenously administered FAP-CAR-T cells controlled, without overt toxicity, the growth of subcutaneous tumors created using a mixture of antigen-negative and antigen-positive glioblastoma cells. Conclusions: Our findings advance FAP as a leading candidate for clinical CAR-T therapy of glioblastoma and highlight under-recognised antigen nonspecific mechanisms that may contribute meaningfully to the antitumor activity of CAR-T cells.

Riding the storm: managing cytokine-related toxicities in CAR-T cell therapy.

The advent of chimeric antigen receptor T cells (CAR-T) has been a paradigm shift in cancer immunotherapeutics, with remarkable outcomes reported for a growing catalog of malignancies. While CAR-T are highly effective in multiple diseases, salvaging patients who were considered incurable, they have unique toxicities which can be life-threatening. Understanding the biology and risk factors for these toxicities has led to targeted treatment approaches which can mitigate them successfully. The three toxicities of particular interest are cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and immune effector cell-associated hemophagocytic lymphohistiocytosis (HLH)-like syndrome (IEC-HS). Each of these is characterized by cytokine storm and hyperinflammation; however, they differ mechanistically with regard to the cytokines and immune cells that drive the pathophysiology. We summarize the current state of the field of CAR-T-associated toxicities, focusing on underlying biology and how this informs toxicity management and prevention. We also highlight several emerging agents showing promise in preclinical models and the clinic. Many of these established and emerging agents do not appear to impact the anti-tumor function of CAR-T, opening the door to additional and wider CAR-T applications.

[Screening of the lncRNA SNHG3 regulating CART expression in the bovine hypothalamus].

This study aimed to screen for the long non-coding RNA (lncRNA) small nucleolar RNA host gene 3 (SNHG3) capable of regulating the expression of cocaine- and amphetamine-regulated transcriptional peptide (CART) in the bovine hypothalamus and elucidate the underlying mechanism. StarBase v2.0, NCBI, and DIANA tools were used to predict the lncRNAs targeting miR-381 and miR-491, which were responsible for inhibiting CART expression. The binding sites were analyzed, and the endogenous expression of the selected lncRNAs was determined by semi-quantitative RT-PCR of the hypothalamus tissue from three healthy adult Simmental cows. The dual-luciferase reporter gene assay was employed to detect the targeted binding relationship between miR-381/491 and lncRNAs. The over-expression vectors of lncRNAs, CART, and miR-381/491 mimics were constructed and transfected into 293T cells to reveal the mechanism of lncRNAs in regulating the CART expression. Animal experiments were conducted to analyze the regulatory function of the strongest lncRNA at the cellular level. The results showed that lncRNAs TUG1, SNHG3, H19, SNHG12, and DANCR were expressed in the bovine hypothalamus. The lncRNAs TUG1 and SNHG3 had binding sites for miR-381, and H19, SNHG12, and DANCR had binding sites for miR-491. The dual-luciferase reporter gene assay showed that miR-381 inhibited the relative luciferase activities of TUG1-WT (P < 0.05) and SNHG3-WT (P < 0.01), and miR-491 inhibited the luciferase activities of DANCR-WT (P < 0.05), H19-WT (P < 0.05), and SNHG12-WT (P < 0.01). SNHG3 and SNHG12 up-regulated the CART expression by specifically binding to miR-381 (P < 0.001) and miR-491 (P < 0.01), respectively, and SNHG3 had the strongest effect of regulating CART expression. The results from animal experiments showed that SNHG3 significantly up-regulated the mRNA and protein levels of CART by specifically binding to miR-381. This study confirmed that the lncRNA SNHG3, acting as a competing endogenous RNA of miR-381, significantly up-regulated CART expression at the transcriptional and post-transcriptional levels, laying a foundation for deciphering the mechanism of the molecular network regulation of CART in the bovine hypothalamus.

Palliating hematologic oncologic emergencies with radiation in the age of targeted systemic therapies: three illustrative cases.

Hematologic oncologic emergencies with an urgent indication for radiation are a relatively routine occurrence for the radiation oncologist. As patients are living longer and have multiple treatment options for their relapsed or refractory diseases, it is important that palliative treatments avoid precluding patients from or delaying next-line potentially curative treatments wherever possible. We highlight the following experiences from our clinical practice: newly diagnosed plasma cell disease causing cord compression; life threatening cutaneous lymphoma with tumors covering the majority of the body surface area; and relapsed/refractory diffuse large B-cell lymphoma (DLBCL) requiring bridging radiation to a mass impinging on the brachial plexus combined with chimeric antigen receptor (CAR)-T cell therapy. In each case, urgent palliative radiation was utilized, but the approaches were nuanced, with careful consideration of subsequent potential therapies and how the current course of radiation should be tailored for the best interplay with the overall treatment course and trajectory of the disease. With the rapid development of new therapies, it can be difficult to stay up to date on the most recent practice guidelines. Drawing on hematologic-specific guidelines, such as those provided by the International Lymphoma Radiation Oncology Group, and disease site experts can aid in ensuring patients are appropriately palliated and eligible for future therapies.

Hu8F4-CAR T cells with mutated Fc spacer segment improve target specificity and mediate anti-leukemia activity in vivo.

BACKGROUND AIMS: Hu8F4 is a T-cell receptor-like antibody with high affinity for the leukemia-associated antigen PR1/HLA-A2 epitope. Adapted into a chimeric antigen receptor (CAR) format, Hu8F4-CAR is composed of the Hu8F4 single-chain variable fragment, the human IgG1 CH2CH3 extracellular spacer domain, a human CD28 costimulatory domain and the human CD3ζ signaling domain. We have demonstrated high efficacy of Hu8F4-CAR-T cells against PR1/HLA-A2-expressing cell lines and leukemic blasts from patients with acute myeloid leukemia in vitro. Previous studies have shown that modification of the Fc domains of IgG4 CH2CH3 spacer regions can eliminate activation-induced cell death and off-target killing mediated by mouse Fc gamma receptor-expressing cells. METHODS: We generated Hu8F4-CAR(PQ) with mutated Fc receptor binding sites on the CH2 domain of Hu8F4-CAR to prevent unwanted interactions with Fc gamma receptor-expressing cells in vivo. RESULTS: The primary human T cells transduced with Hu8F4-CAR(PQ) can specifically lyse HLA-A2+ PR1-expressing leukemia cell lines in vitro. Furthermore, both adult donor-derived and cord blood-derived Hu8F4-CAR(PQ)-T cells are active and can eliminate U937 leukemia cells in NSG mice. CONCLUSIONS: Herein, we demonstrate that modification of the IgG1-based spacer can eliminate Fc receptor binding-induced adverse effects and Hu8F4-CAR(PQ)-T cells can kill leukemia in vivo.

Rapidly-manufactured CD276 CAR-T cells exhibit enhanced persistence and efficacy in pancreatic cancer.

BACKGROUND: Pancreatic cancer is one of the most lethal malignancies and the lack of treatment options makes it more deadly. Chimeric Antigen Receptor T-cell (CAR-T) immunotherapy has revolutionized cancer treatment and made great breakthroughs in treating hematological malignancies, however its success in treating solid cancers remains limited mainly due to the lack of tumor-specific antigens. On the other hand, the prolonged traditional manufacturing process poses challenges, taking 2 to 6 weeks and impacting patient outcomes. CD276 has recently emerged as a potential therapeutic target for anti-solid cancer therapy. Here, we investigated the efficacy of CD276 CAR-T and rapidly-manufactured CAR-T against pancreatic cancer. METHODS: In the present study, CD276 CAR-T was prepared by CAR structure carrying 376.96 scFv sequence, CD8 hinge and transmembrane domain, 4-1BB and CD3ζ intracellular domains. Additionally, CD276 rapidly-manufactured CAR-T (named CD276 Dash CAR-T) was innovatively developed by shortening the duration of ex vitro culture to reduce CAR-T manufacturing time. We evaluated the anti-tumor efficacy of CD276 CAR-T and further compared the functional assessment of Dash CAR-T and conventional CAR-T in vitro and in vivo by detecting the immunophenotypes, killing ability, expansion capacity and tumor-eradicating effect of CAR-T. RESULTS: We found that CD276 was strongly expressed in multiple solid cancer cell lines and that CD276 CAR-T could efficiently kill these solid cancer cells. Moreover, Dash CAR-T was successfully manufactured within 48-72 h and the functional validation was carried out subsequently. In vitro, CD276 Dash CAR-T possessed a less-differentiated phenotype and robust proliferative ability compared to conventional CAR-T. In vivo xenograft mouse model, CD276 Dash CAR-T showed enhanced anti-pancreatic cancer efficacy and T cell expansion. Besides, except for the high-dose group, the body weight of mice was maintained stable, and the state of mice was normal. CONCLUSIONS: In this study, we proved CD276 CAR-T exhibited powerful activity against pancreatic cancer cells in vitro and in vivo. More importantly, we demonstrated the manufacturing feasibility, acceptable safety and superior anti-tumor efficacy of CD276 Dash CAR-T generated with reduced time. The results of the above studies indicated that CD276 Dash CAR-T immunotherapy might be a novel and promising strategy for pancreatic cancer treatment.

Extracellular vesicle isolation methods identify distinct HIV-1 particles released from chronically infected T-cells.

The current study analyzed the intersecting biophysical, biochemical, and functional properties of extracellular particles (EPs) with the human immunodeficiency virus type-1 (HIV-1) beyond the currently accepted size range for HIV-1. We isolated five fractions (Frac-A through Frac-E) from HIV-infected cells by sequential differential ultracentrifugation (DUC). All fractions showed a heterogeneous size distribution with median particle sizes greater than 100 nm for Frac-A through Frac-D but not for Frac-E, which contained small EPs with an average size well below 50 nm. Synchronized and released cultures contained large infectious EPs in Frac-A, with markers of amphisomes and viral components. Additionally, Frac-E uniquely contained EPs positive for CD63, HSP70, and HIV-1 proteins. Despite its small average size, Frac-E contained membrane-protected viral integrase, detectable only after SDS treatment, indicating that it is enclosed in vesicles. Single particle analysis with dSTORM further supported these findings as CD63, HIV-1 integrase, and the viral surface envelope (Env) glycoprotein (gp) colocalized on the same Frac-E particles. Surprisingly, Frac-E EPs were infectious, and infectivity was significantly reduced by immunodepleting Frac-E with anti-CD63, indicating the presence of this protein on the surface of infectious small EPs in Frac-E. To our knowledge, this is the first time that extracellular vesicle (EV) isolation methods have identified infectious small HIV-1 particles (smHIV-1) that are under 50 nm. Collectively, our data indicate that the crossroads between EPs and HIV-1 potentially extend beyond the currently accepted biophysical properties of HIV-1, which may have further implications for viral pathogenesis.